ATM-kinase deficiency triggers early multi-compartment remodeling of the cerebellar microenvironment

ATM-kinase deficiency triggers early multi-compartment remodeling of the cerebellar microenvironment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article ATM-kinase deficiency triggers early multi-compartment remodeling of the cerebellar microenvironment Francesca Montarolo, Luna Berrino, Anita Maria Rominto, Matilde Loddo, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8832557/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Ataxia Telangiectasia (A-T) is a neurodegenerative disorder characterized by early onset, cerebellar ataxia and progressive motor decline. The causative gene, ATM (A-T Mutated), encodes a Ser/Thr kinase, that belongs to the phosphoinositide 3-kinase-related protein kinase family and is crucial for the response to DNA double-strand breaks. While ATM is classically known for its role in the DNA damage response, increasing evidence points to its critical function in maintaining cellular homeostasis, particularly in the central nervous system (CNS). Yet the mechanisms linking ATM-kinase deficiency to cerebellar circuit dysfunction remain poorly defined. Using a CNS Nestin-Cre-restricted mouse model carrying a kinase-dead Atm allele combined with a null allele ( Atm KDF/CNS−KO ) , we integrated proteomics, structural and ultrastructural analyses, electrophysiology, and behavioral testing to understand how the loss of Atm kinase activity influence the cerebellar microenvironment from the earliest stages of the disease. Proteomic profiling revealed alterations across four major pathways in Atm KDF/CNS−KO mice, such as disorganization of the extracellular matrix, astrocytosis, downregulation of myelin and oligodendrocyte lineage proteins and changes in neuronal excitability. Histological and electron microscopy analyses confirmed astrocytosis and reduction of myelin content without axonal or oligodendrocyte loss, consistent with impaired myelination at early stages. These microenvironmental changes were associated to Purkinje cell dark cell degeneration and increased intrinsic excitability, demonstrating early circuit dysfunction in the absence of overt neuronal loss. Consistently, the compromised cerebellar output is confirmed by the progressive motor impairment developed by Atm KDF/CNS−KO mice. Our findings indicate that Atm kinase deficiency disrupts cerebellar homeostasis through interconnected and bidirectional mechanisms involving ECM remodeling, astrocytic activation, impaired oligodendrocyte maturation and altered intrinsic excitability, reflecting a network level destabilization of the cerebellar microenvironment. Ataxia Telangiectasia (A-T) cerebellar atrophy motor deficit Purkinje cell degeneration excitability Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 INTRODUCTION Ataxia Telangiectasia (A-T) is a rare autosomal recessive disorder that is characterized by early onset, progressive cerebellar ataxia, oculocutaneous telangiectasia, immunodeficiency, and increased risk of developing cancer [ 2 ]. A-T is caused by biallelic mutations in the Ataxia Telangiectasia Mutated ( ATM ) gene (Chr 11q22.3-23.1) [ 12 ]. The protein encoded by the ATM gene is a 370 kDa Ser/Thr kinase member of the phosphoinositide 3-kinase-related protein kinase (PIKK) family, whose members are stress-responsive kinases. ATM is activated by DNA damage, and phosphorylates its downstream targets that coordinate the DNA damage response and regulates the activation of cell cycle checkpoints in order to preserve genome integrity [ 14 ]. Besides these well-known roles, Atm plays an important role in the central nervous system (CNS) by modulating glutamatergic and GABAergic synapses [ 25 , 40 , 53 ], and maintaining proper homeostasis of mitochondria and peroxisomes, acting thus as a sensor for changes in the ROS levels to prevent oxidative damage [ 7 , 60 ]. Neuropathologically, A-T is characterized by a severe and progressive degeneration of the cerebellar cortex, with a massive loss of Purkinje cells (PCs), granule cells, and a reduction of the molecular layer thickness [ 13 ]. While cerebellar cortical degeneration has long been considered the primary substrate of the neurological phenotype, increasing evidence indicates that white matter abnormalities are a consistent and clinically relevant component of A-T pathology. Neuroimaging studies in patients have reported marked cerebellar and white matter atrophy [ 8 , 10 , 42 – 44 ], along with degeneration of cortical-projecting tracts, including corticomotor, corticospinal, and somatosensory pathways [ 43 , 44 ]. Consistently, myelin disruption has also been observed in non-human primate model of A-T [ 55 ], further supporting the notion that impaired myelin homeostasis may represent a conserved and early feature of the disease. Despite extensive research, the mechanisms linking ATM kinase deficiency to myelin defects and cerebellar circuit dysfunction remain poorly understood. Importantly, ATM deficiency affects multiple cellular processes across neurons and glial cells, raising the possibility that neurodegeneration in A-T does not arise solely from intrinsic neuronal defects [ 32 ]. Instead, accumulating evidence points toward cooperative mechanisms, [ 20 , 22 , 33 , 38 , 50 , 50 ], in which disrupted interactions among neurons, astrocytes, oligodendrocytes, and extracellular matrix (ECM), progressively compromise cerebellar circuit integrity and contribute to the neurological decline characteristic in A-T. Oxidative stress and DNA damage, hallmarks of ATM deficiency, are known to profoundly alter ECM composition and mechanics [ 18 ], which in turn regulate glial differentiation, myelin maintenance, and neuronal excitability [ 21 , 30 ]. However, how these microenvironmental changes integrate with myelin pathology and neuronal dysfunction in A-T remains unresolved. To investigate how the loss of Atm kinase activity impacts the cerebellar circuitry microenvironment, from the earliest stages of the disease, we employed a Nestin-Cre–restricted mouse model carrying a kinase-dead Atm allele in combination with a null allele, resulting in selective loss of Atm kinase activity in the CNS [ 48 , 57 ]. By combining unbiased proteomic profiling with structural, ultrastructural, electrophysiological, and behavioral analyses, we examined how Atm kinase deficiency reshapes the cerebellar tissue environment, with particular focus on myelin integrity, glial responses, and PC function from early stages of the disease. Our findings reveal that Atm kinase loss triggers an early, coordinated disruption of white matter homeostasis and cerebellar microenvironment stability, preceding neuronal loss and contributing to circuit dysfunction and motor impairment. MATERIALS AD METHODS Animals All experimental procedures have been carried out at the Neuroscience Institute Cavalieri Ottolenghi (NICO), in accordance with the European Communities Parliament and Council Directives of 24 November 1986 (86/609/EEC) and 22 September 2010 (2010/63/EU), approved by the Ethical Committee of the University of Torino and authorized by the Italian Ministry of Health (authorization number: 466/2021-PR). Mice were housed with a 12 h light/dark cycle and had free access to food/water. Adequate measures were taken to minimize pain and discomfort. Female and male mice showed the same phenotype; hence they were pooled together for all the experimental paradigms. Animal description Generation of the Atm KDF allele The Atm KDF allele carrying knock-in D2880A/N2885K mutations (corresponding to D2870A/N2875K in human ATM) was generated using a strategy similar to that used for the original KD Atm allele [ 57 ]. The D2870A/N2875K double mutation was selected because it has been extensively characterized and shown to support normal ATM protein expression while completely abolishing kinase activity [ 1 , 4 ]. Briefly, the targeting construct was designed to insert a neomycin resistance (NeoR) cassette, oriented opposite to the endogenous Atm promoter, into intron 57 adjacent to the D2880A/N2885K mutations in exon 58. The NeoR cassette was flanked by a pair of FRT sites. A 3.5-kb 5′ homology arm and a 5.1-kb 3′ homology arm were PCR-amplified using high-fidelity polymerase, cloned into shuttle vectors, and fully sequenced. The 5′ arm was subcloned directly into the pEMC targeting vector [ 31 ] in the desired orientation. The D2880A/N2885K mutations were introduced into the 3′ arm by site-directed mutagenesis and confirmed by sequencing prior to subcloning into pEMC. The finalized targeting construct was electroporated into CSL3 ES cells (129 strain), and correctly targeted clones were identified by Southern blot analysis. Initial screening was performed using KpnI and EcoRV double digestion with a 5′ genomic probe ( Supplementary Fig. 1B-C ), yielding a ~ 13.1-kb germline band and a ~ 4.7-kb targeted band due to the introduction of an additional EcoRV site. Correct targeting was further confirmed using a 3′ probe following KpnI and EcoRV digestion, with expected band sizes of ~ 13.1 kb for the germline allele and ~ 10 kb for the targeted allele (Supplementary Fig. 1B-C). More than 12 independently targeted ES cell clones were identified, eight of which were sequenced across exon 58; three clones were confirmed to carry the correct D2880A/N2885K mutations (Supplementary Fig. 1D). Two validated clones were injected to obtain germline transmission. Resulting Atm +/KDFN chimeras were crossed with Rosa26a FLIP/FLIP mice (Jackson Laboratory, Cat. 003946) [ 9 ] to induce FLP-mediated excision of the Neoᴿ cassette, generating the Atm KDF allele. In the resulting Atm +/KDF mice, the kinase-dead ATM protein is expressed from the endogenous Atm promoter. A single FRT site remains in intron 57 and does not affect ATM expression. Generation of the Atm KDF murine model Atm +/KDF mice were crossed with 'floxed' Atm alleles ( Atm fl/fl ) mice, containing loxP sites flanking exons 57–58 of the gene (Jackson Laboratory, U.S.A). To inactivate floxed alleles only in the CNS, the Atm KDF/fl mice were crossed with Nestin-Cre mice (Cre recombinase expressed from a transgene driven by the Nestin promoter) (Jackson Laboratory, U.S.A). Using these crossings, we have developed Atm KDF/KO Nestin CRE positive mice, which are kinase-dead Atm in one allele and knocked out in the other allele only in Nestin positive cells of the CNS, thus preventing premature mortality from cancer predisposition ( Atm KDF/CNS−KO ). Atm KDF/fl Cre negative were used as control mice. Proteomic analysis 2 months old mice of wild type (n = 6) and Atm KDF/CNS−KO (n = 6) mice were anesthetized with isoflurane (Isoflurane-Vet, Merial, Italy) and decapitated. The cerebella were removed, frozen in ice-cold isopentane, and stored at -80°C until use. Cerebella were omogenized by a blender using EasyPep MS Sample Kit (Thermo Scientific Pierce). Peptides were solubilized in 0.1% formic acid, and they were quantified through the quantitative colorimetric peptide assay (Thermo Fisher Scientific). 1.9 micrograms of each sample was injected into an UltiMate 3000 RSLC nano system coupled to the Exploris 240 mass spectrometer (Thermo Fisher Scientific) and resolved by Easy-Spray Pepmap RSLC 18 (2µm, 75cm × 75µm) at a flow rate of 200nL/min with a gradient of phase B (80% acetonitrile/0.1% formic acid, solvent A was 0.1% formic acid in water) from 2% to 40% in 250 min. Then (B) was changed to 95% in 30 min, kept for 5 min, and then the column was re-equilibrated for 15 min. Data was acquired in a positive mode and data dependent manner. For MS1 m/z range was set to 350– 1500 at 120,000 resolution (at m/z 200), AGC target 3e6, and auto maximum injection time. MS2 switch when ions intensity was above 5e3, with m/z range in auto mode, normalized HCD energy 30%, AGC target 7.5e4, and maximum injection time 40ms. The resolution was set to 15,000 at m/z 200 and the internal calibrant for employed in run start mode. Analysis was performed by five technical replicates for each sample. Raw data generated by Xcalibur 4.2 software (Thermo Fisher Scientific) were analyzed using Proteome Discoverer 2.5 (Thermo Fisher Scientific), by using SEQUEST algorithm. carbamidomethylation of Cysteines was considered as fixed modification, while as variable serine, threonine or tyrosine phosphorylation. Also, variables oxidation (M) and deamidation (N, Q) were counted. The false discovery rate was evaluated by a target-decoy strategy in concatenated q-value manner. FDR (strict) was set as 0.01, while FDR (relaxed) was set as 0.05. Differentially expressed master proteins (|log 2 FC| ≥ 0.5, p < 0.05) were selected and individually evaluated. Fold changes, adjusted p and accension number were submitted to iPathwayGuide software using Advaita’s proprietary Impact Analysis method (Advaita Corporation Ann Arbor, MI). Histological procedures and image analysis 2 months-old animals of both genotypes were anesthetized using a cocktail of zoletil (100 mg/kg body weight) and xylazine (5 mg/kg body weight) via intraperitoneal injection. The mice were intracardially perfused initially with a physiological solution (NaCl 0.9%) and then with 4% paraformaldehyde in 0.12 M phosphate buffer, pH 7.2–7.4. Following perfusion, the brains were removed and stored at 4°C for 24 hours immersed in the same fixative. The brains were then transferred to a cryoprotectant solution made of 30% sucrose in 0.12 M phosphate buffer for few days. For each mouse, the cerebellum was separated from the telencephalon and embedded in optimal cutting temperature compound, frozen in ice-cold isopentane. Samples were stored at -80°C until sectioning. Cerebella and telencephalons were serially cut by a cryostat in 30 µm-thick sagittal and coronal slices respectively and collected in phosphate buffered saline (PBS). Histological procedures were performed on sagittal cerebellar slices of vermis and/or coronal telencephalic sections including the corpus callosum of Atm KDF/CNS−KO and wild type mice. At least 3 slices/animal and 3 animals/time point were analyzed. All the measurements were done blind to the mouse genotypes. Cresyl Violet Staining (Nissl Staining) was performed on sagittal cerebellar slices of the vermis and on coronal telencephalic sections as previously described [ 18 ], to measure the thickness of layers [ 17 ], and slice and white matter area. Images were acquired by means of ZEISS Axioscan 7 microscope slide scanner (Oberkochen, Germany) with a Plan Apochromat 20X/0.8 M27 objective. Quantitative evaluations were performed on images with ImageJ software ( http://rsbweb.nih.gov/ij/ index. html). The area of the slice and white matter was obtained by drawing the outline. The density of PCs was measured in anti-calbindin immunostained sections using Neurolucida software (MicroBrightField, Colchester, VT, USA) connected to an E-800 Nikon microscope under a 20x objective. Cerebellar slices were incubated overnight at 4°C with the polyclonal anti-rabbit calbindin (CB38a, Swant, Switzerland) antibody diluted 1:1000 in PBS with 1% TritonX-100 and 1.5% normal donkey serum. Immunohistochemical reactions were performed by the avidin–biotin–peroxidase method (Vectastain ABC Elite kit; Vector Laboratories, Burlingame, CA, USA) and revealed using 3,3′-diaminobenzidine (3% in Tris–HCl) as chromogen as reported in [ 35 ]. After processing, sections were mounted on microscope slides with Neo Mount (1.09016, Merck, Darmstadt, Germany). The density of PCs (expressed as number of PCs/mm) was obtained by drawing the outline of PC layer and marking the position of every labelled cell, using the Neurolucida software (MicroBrightField, Colchester, VT, USA) connected to an E-800 Nikon microscope under a 20x objective. Silver nitrate Gallyas staining to detect myelin was performed on sagittal cerebellar slices of vermis and on coronal telencephalic sections as previously described [ 11 , 39 ]. Images were acquired by means of ZEISS Axioscan 7 microscope slide scanner (Oberkochen, Germany) with a Plan Apochromat 20X/0.8 M27 objective. Quantitative evaluations were performed on images with ImageJ software ( http://rsbweb.nih.gov/ij/ index. html). To quantify the Gallyas staining, was considered the fractioned area, defined as the percentage of positive pixels relative to the total area analyzed. To detect astrocytes and oligodendrocytes , immunofluorescence staining was performed as follows. Cerebellar and/or telencephalic sections were incubated overnight at 4°C with primary antibodies against glial fibrillary acidic protein (GFAP; rabbit polyclonal, Z033429-2, Dako, Agilent, Santa Clara, CA, USA; 1:500) and SRY-box transcription factor 10 (Sox10; rabbit polyclonal, HPA068898, Sigma-Aldrich, Darmstadt, Germany; 1:1000) to label astrocytes and oligodendrocytes, respectively. Antibodies were diluted in PBS containingf 1% Triton-X-100 and 1.5% normal donkey serum. Sections were then incubated for 2 h at room temperature with secondary anti-rabbit 647- and Cy3-conjugated antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). 4,6-diamidino-2- phenylindole (DAPI, Fluka, Saint Louis, USA) was used to counterstain cell nuclei. Finally, sections were mounted on microscope slides with Tris-glycerol mounting medium supplemented with 10% Mowiol (Calbiochem, La Jolla, CA). GFAP-immunostained images were acquired by means of ZEISS Axioscan 7 microscope slide scanner (Oberkochen, Germany) with a Plan Apochromat 20X/0.8 M27 objective. To quantify the astroglial reactivity, the expression level of GFAP+ astrocytes in the cerebellar vermis was assessed by measuring the GFAP+ fractioned area, defined as the percentage of positive pixels relative to the total area analyzed. Images of Sox10-immunostained white matter regions from the cerebellum and corpus callosum were acquired using a Leica TCS SP5 confocal microscope. Confocal images were captured as z-stacked focal planes through the thickness of the slice (30 µm) at 1-µm optical steps with an oil-immersed Plan-Apochromat 40X/1.25 objective, zoom 1.0, and resolution of 1024/1024 pixels and 100 Hz (1 pixel = 0.38 µm). The density of Sox10 + oligodendrocytes was calculated as the number of positive cells per mm 2 . Adobe Photoshop 6.0 (Adobe Systems, San Jose, CA, RRID:SCR_014199) was used to assemble the final figure panels. High resolution light microscopy and transmission electron microscopy High resolution light and transmission electron microscopy (TEM) were carried out as reported in [ 16 , 27 ]. 2 to 4 months-old mice were anaesthetized by intraperitoneal injection with zoletil (100 mg/kg body weight) and xylazine (5 mg/kg body weight) and were perfused intracardially with 0.12 M phosphate buffer, pH 7.2–7.4 followed by 2% paraformaldehyde and 2% glutaraldehyde in phosphate buffer. Brains were post-fixed overnight at 4°C in the same fixative. Vibratome sections (300 µm thick) were cut, and post-fixed with 1% osmium tetroxide for 1 h at 4°C, then stained with uranyl acetate replacement stain (Electron Microscopy Sciences, USA). After dehydration in ethanol, samples were cleared in propylene oxide and embedded in Araldite (Fluka, Saint Louis, USA). Semithin sections (1 µm thick) were obtained using an ultramicrotome (Ultracut UCT, Leica, Wetzlar, Germany), stained with 1% toluidine blue and 2% borate in distilled water, and then observed under a light microscope to ensure accurate localization of the corpus callosum. For each sample, bright-field images of the medial corpus callosum were acquired at 100x magnification a Nikon Eclipse 80i microscope. Axon density was quantified using ImageJ software. Each image was overlaid with a grid (via the Grid tool), and eight non-overlapping regions were randomly selected. Axons were manually counted within each square, and axon density was calculated as the number of axons µm 2 . For each sample, the mean axon density was obtained by averaging values from the eight 8 squares. The obtained ultrathin sections (70–100 nm) were examined by TEM and STEM. The G-ratio (inner perimeter/outer perimeter), axon diameter, and myelin thickness were determined by using a JEOL, JEM-1400Flash, (Tokyo, Japan) operated at 80 kV, and equipped with a Mega-View-III digital camera and a Soft-Imaging-System (SIS, Münster, Germany). The recorded images were elaborated and measured by using the ImageJ software [ 45 ]. The quantification of G-ratio and axon diameter was performed on at least 50 axons/animal and on 4–5 mice per genotype. Protein analysis Protein analysis was performed as previously described [ 16 ]. Mice were anesthetized with isoflurane (Isoflurane-Vet, Merial, Italy) and decapitated. The cerebella were removed, frozen in ice-cold isopentane, and stored at -80°C until use. Cerebella from wild type and Atm KDF/CNS−KO mice were resuspended in 20% (w/v) RIPA buffer (25 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, 0.5 mM PMSF, 10 µg/ml Aprotinine, 10 µg/ml Leupeptine, 2 mM sodium orthovanadate), and homogenized with a tissue lyser. The lysates were centrifuged at 10,000 g for 20 min at 4°C and the supernatant was collected and stored at − 80°C until use. Twenty micrograms of proteins were separated by using a 4–12% Bis-Tris precast gel (Life Technologies) and transferred onto nitrocellulose membrane (GE‐Healthcare). Membranes were than blocked with 50 g/L (5%) nonfat dry milk (Bio‐Rad) in 50 mM Tris–HCl pH7.4, containing 200 mM NaCl and 0.5 mM Tween‐20 and then incubated overnight at 4°C with primary antibodies. The following primary antibodies were used: myelin basic protein (MBP) (1:1000, Covance, Cat# SMI‐99P‐500, RRID: AB_10120130), and Gapdh (1:1000, Abcam, Cat# ab181602, RRID: AB_2630358). HRP‐conjugated goat anti‐mouse (1:5000, Bio‐Rad, Cat# 170–6516, RRID: AB_11125547) and goat anti‐rabbit (1:5000, Bio‐Rad, 170–6515, RRID: AB_11125142) immunoglobulins, in Tris‐buffered saline Tween containing 20 g/L non‐fat dry milk, were used for detection with Luminata Forte Western substrate (WBLUF0100, Millipore, Darmstadt, Germany). Densitometric values were normalized to gapdh. Images were acquired by Chemidoc (Bio‐Rad) and quantified by ImageLab software (RRID: SCR_014210, Bio‐Rad). The protein extracts were run at least three times to check reproducibility. Purkinje cell dark cell degeneration assessment 2 to 4 months-old mice were anaesthetized by intraperitoneal injection with zoletil (100 mg/kg body weight) and xylazine (5 mg/kg body weight). They were subsequently fixed by transcardiac perfusion with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3). Sagittal sections of the cerebellum (500 µm thick) were cut using a vibratome and post-fixed by incubation for 2 hours in 1% (wt/vol) OsO4 supplemented with 1.5% (wt/vol) potassium ferrocyanide. The sections were then dehydrated in a graded ethanol series (30% to 100%, 5 minutes per step), followed by two 10-minute passages in propylene oxide and 1 hour in a 1:1 mixture of propylene oxide and Epon resin. Finally, the samples were embedded in resin. Semithin sections (1 µm thick) were prepared using an ultramicrotome (Ultracut UCT, Leica, Wetzlar, Germany) and stained with 1% toluidine blue and 2% borate in distilled water and mounted onto glass cover slips for morphological assessment and quantitation via standard light microscopy [ 28 ]. Dark cell degeneration (DCD) was verified by the appearance of a minimum of two of the following features: cytoplasmic darkening, soma shrinkage, and nuclear darkening/chromatin aggregation [ 28 ]. Electrophysiology Slices preparation Cerebellar slices were prepared as previously described [ 19 , 34 ]. The animals were anesthetized with isoflurane (Isoflurane-Vet, Merial, Italy) and decapitated. The cerebellar vermis was removed and transferred to an ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM); 125 NaCl, 2.5 KCl, 2 CaCl 2 , 1 MgCl 2 , 1.25 NaH 2 PO 4 , 26 NaHCO 3 , 20 glucose, which was bubbled with 95% O 2 /5% CO 2 (pH 7.4). Parasagittal cerebellar slices (200 µm thickness) were obtained using a vibratome (Leica Microsystems GmbH, Wetzlar, Germany) and kept for 1 h at 35°C and then at 31°C. Single slices were placed in the recording chamber, which was perfused at a rate of 2–3 ml/min with ACSF bubbled with the 95% O 2 /5% CO 2 . All recordings were performed at 31 ± 1°C temperature. Electrophysiological recordings Recordings from PCs were performed by an EPC-10 patch‐clamp amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany). PCs were held at – 70 mV, and data were filtered at 9.1 kHz and sampled at 20 kHz. For current clamp recordings, patch pipettes were filled with a K-gluconate-based internal solution containing (in mM): 140 K-gluconate, 10 HEPES, 0.5 EGTA, 4 MgCl 2 , 4 Na 2 ATP, 0.4 Na 3 GTP and the pH was adjusted to 7.3 with KOH and filtered at 0.2 µm. Hyperpolarizing (form − 400pA to -100 pA) and depolarizing (from + 100 to + 1000 pA) current steps in increments of 100 pA, each lasting 1000 ms and a step interval of 10 s were delivered to PCs. For cell-attached recordings the pipette was filled with NaCl 0.9% solution filtered at 0.2 µm. Gabazine (SR 95531, 20 µM), DAP5 (50 µM) and NBQX (10 µM) were added to the recording chamber to inhibit the GABAA and ionotropic glutamate receptors of PCs, respectively. Data were analyzed using Axograph software (AxoGraph Scientific, Sydney, Australia) and graphs were designed using Igor Pro (Wavemetrics, Lake Oswego, Oregon, USA). Data have been derived from at least three animals per genotype. Drugs All drugs were purchased from HelloBio (Bristol, UK) and were applied via the chamber perfusion line. Motor Tests We performed a series of motor tests to evaluate the presence of motor impairment on Atm KDF/CNS−KO mice. All the experiments were conducted in the morning and with the same light conditions for all animals. Balance Beam test We performed balance beam test to evaluate balance and motor coordination on 2, 12, and 24 months old Atm KDF/CNS−KO and wild type mice [ 17 ]. We used a metal beam 1 cm wide, and 100 cm long suspended 12 cm above the bench. For 24-months old animals, a wider beam (1.5 cm) was used. The mice had to cross the beam to reach a cage enriched with toys. The day of the test was preceded by three days of habituation to allow the mice to familiarize with the experimental apparatus. On the day of the test, the animals were placed to acclimate in the behavioral room at least 15 minutes before the experiment. The test consisted of three bar crossings for three consecutive days. We recorded the crossings using a video camera and analyzed offline by an operator blind to the genotype. The performance was assessed by measuring the latency required to cross the beam as well as the number of slips. The data are presented as violin plot (median and 25th to 75th percentiles are shown). Footprint Analysis The footprinting test was used to analyze gait parameters. The test was performed as previously described [ 17 , 19 ]. We used a transparent Plexiglass walkway (20 cm high, 67 cm long and 4 cm wide) elevated 70 cm from the floor. A digital camera was placed underneath the clear platform and video recordings of the mice walking were collected. The mice had to walk at least for three consecutive steps per crossing. Still-frames from the recordings were extracted and analyzed offline using the ImageJ software in order to obtain data concerning stride length (the distance of the same paw in consecutive steps), width (the distance between the center of the two hind or fore paws), the distance between ipsilateral fore paw (FP) and hind paw (HP) placements and the fore and hind stance. Accelerated rotarod test We assessed the locomotor function using the accelerated rotarod test as previously described [ 17 ]. We tested mice for five consecutive days and then again 5 days later (10th day). This protocol was repeated at different ages, at 2, 12, and 24 months, to evaluate long term retention of the motor improvements. In each day, after 1 minute training session at constant speed (4 rpm), mice received three test sessions (with a minute interval between sessions) in which the rod (Mouse Rota-Rod, Ugo Basile Biological Research Apparatus, Comerio, Italy) accelerated continuously from 4 to 65 rpm with an acceleration of 5.5 rpm. The latency to fall off the rod was recorded. The cutoff in this experiment was set to 300 seconds. Statistical analysis All data are presented as mean ± SEM. The Shapiro-Wilk test was used to check whether the data followed a normal distribution. For the data sets that passed the normality test, comparisons were done with unpaired two tailed Student’s t -test or by one-way or two-way analyses of variance (ANOVA), followed by the appropriate post hoc correction. Data for which the normality test failed, were compared by the Mann-Whitney u-test or by Kruskal-Wallis test. P values lesser than 0.05 was accepted as significant. Correlation analysis between proteomic protein expression levels and motor performance was performed using SPSS (IBM SPSS Statistics, version 7). Spearman’s rank correlation coefficient was applied to identify proteins significantly associated with motor outcomes. Proteins showing significant correlations were further analyzed using STRING for protein–protein interaction network construction and functional enrichment analysis. Network outputs were subsequently redrawn and customized using in-house Python scripts. RESULTS Atm KDF murine model Our initial Atm KD allele with knocking in D2880A/N2885K (corresponding to D2870A/N2875K in humans) double-mutation into the conserved catalytic loop also contains a single loxP site after the Cre-mediated deletion of the Neo resistance cassette (NeoR) [ 57 ]. Using this KD allele, we successfully demonstrated accelerated oncogenesis using an early and robust Vav-Cre allele, leading to aggressive immature T cell lymphomas [ 56 ]. But in the slow progressive B cell lymphoma model or in neuronal models as described here, a rare recombination between the residual loxP site in the KD allele and the loxP sites in the Atm conditional/null allele [ 59 ] leads to inactivation (null) of both alleles (Supplementary. Figure 1 A ) and effectively creates a null model. To avoid this, we have generated a new Atm KDF allele (termed KDF for FRT), in which the LoxP sites flanked NeoR were replaced by a pair of FRT sites ( Supplementary Fig. 1B ). Therefore, after the neo-deletion, only an FRT is left (not a loxP) that would not recombine with the loxP on the conditional allele ( Supplementary Fig. 1A-B ). Southern blot analyses and the Sanger sequencing validate the correct targeting and the presence of the double mutations ( Supplementary Fig. 1C-D ). Using the Vav-Cre that expresses in all hematopoietic lineages, we confirmed that the newly generated Atm KDF allele conferred no ATM kinase activity, evidenced by reduced surface TCRβ-positive mature thymocytes and decreased Immunoglobulin class switch recombination at levels comparable to the Atm −/− and Atm KD alleles [ 3 , 26 , 41 , 57 ] ( Supplementary Fig. 1E) . Differentially expressed proteins in the cerebellum of Atm KDF/CNS−KO mice To identify molecular alterations induced by Atm kinase deficiency, we performed proteomic analysis of whole-cerebellum lysates from Atm KDF/CNS−KO mice and wild type littermates. A total of 8302 master proteins were identified, revealing a profound disruption of cerebellar protein homeostasis in Atm KDF/CNS−KO mice. Global differential expression analysis demonstrated a widespread molecular phenotype associated with Atm kinase deficiency, as illustrated by the volcano plot (Fig. 1 a). We identified 299 differentially expressed proteins: 165 proteins were upregulated, and 134 downregulated in Atm KDF/CNS−KO mice compared to wild type littermates, with |log2(Fold-change)| ≥0.5 and statistical significance set at p < 0.05, revealing a coordinated disruption of multiple cellular compartments (Table S1 ). Hierarchical clustering analysis (Fig. 1 b) based on the Z-score distribution demonstrates a clear-cut proteomic signature that distinguishes Atm KDF/CNS−KO replicates from the wild type littermates, indicating high reproducibility of the molecular phenotype and supporting the existence of a consistent cerebellar proteomic remodeling induced by Atm deficiency. KEGG pathway enrichment analysis revealed a significant enrichment of cellular processes, metabolic, and signaling pathways (Fig. 1 c). Importantly, KEGG enrichment analyses identified extracellular matrix (ECM)-receptor interaction and focal adhesion pathways, indicating early disruptions in the ECM stability and matrix-cell signaling in the cerebellum of Atm KDF/CNS−KO mice (Fig. 1 c). These findings highlight a broad disruption of the cellular homeostasis in the cerebellum of Atm KDF/CNS−KO mice. Consistently, Gene Ontology (GO) analysis of the upregulated proteome revealed a strong enrichment of basement membrane and interstitial ECM components together with astrocyte- and immune-related processes (Table S1 ) (Fig. 1 d, top ). In contrast, the downregulated proteome was dominated by pathways related to myelination, oligodendrocyte differentiation, axon ensheathment, and neuronal communication (Table S1 ) (Fig. 1 d, bottom ), suggesting a coordinated impairment of white matter integrity and axon-glia interactions. Atm KDF/CNS−KO mice display myelination defects Given the strong enrichment of ECM, glial and myelin-related pathways, we next asked whether these molecular changes corresponded to structural and ultrastructural alterations. We examined whether the cerebellar white matter was affected in Atm KDF/CNS−KO mice. Despite the normal foliation, overall cytoarchitecture, and preserved thickness of the molecular and granular layers ( Supplementary Fig. 2 ), Atm KDF/CNS−KO mice showed a significant reduction in the cerebellar slice area compared to wild type controls (p < 0.05, Unpaired Student’s t-test: t (9) = 2.40) (Fig. 2 a-c). This reduction was primarily driven by with a significant shrinkage of the cerebellar white matter area (p < 0.01, Mann-Whitney test) (Fig. 2 d). Moreover, the white matter are/slice area ratio was significantly reduced in Atm KDF/CNS−KO mice (p < 0.01, Mann-Whitney test) (Fig. 2 e). To directly assess myelin integrity, we performed Gallyas staining on cerebellar sections (Fig. 3 a, b). The analysis revealed a marked reduction of myelin staining in Atm KDF/CNS−KO mice compared to control animal (p < 0.01, Unpaired Student’s t-test: t (12) = 3.73; Fig. 3 c). In parallel with the structural analyses, we assessed the expression level of MBP, the most abundantly expressed myelin protein in the CNS, by performing western blotting of cerebellar extracts. Quantitative analyses revealed a significant reduction in both isoforms of Mbp in Atm KDF/CNS−KO mice compared to wild type littermates (p < 0.05, Unpaired Student’s t-test; Fig. 3 d), supporting the presence of an early hypomyelinating phenotype. To determine whether myelin abnormalities were restricted to the cerebellum, we analyzed the corpus callosum (CC), the largest white matter tract in the brain. Atm KDF/CNS−KO mice showed a significant reduction in CC thickness compared to wild type littermates (7.56 ± 0.37 vs 6.67 ± 0.32 µm, respectively, p < 0.05, Unpaired Student’s t-test: t (7) = 3.03). Gallyas staining (Fig. 3 e, f ) revealed a significant reduction of the myelin content within the CC (cortex: p < 0.001, Mann-Whitney test; Fig. 3 g; cortex+corpus callosum: p < 0.0001, Unpaired Student’s t-test: t (12) = 6.17; Fig. 3 h), together with a patchy appearance of the myelin in the overlying cortical regions (Fig. 3 f, g), indicating that white matter pathology in Atm KDF/CNS−KO mice is not confined to the cerebellum. To determine whether reduced myelin staining reflected axonal loss or primary myelin defects, we analyzed semithin and ultrastructural sections of the corpus callosum. Quantification of toluidine blue-stained CC sections revealed comparable axonal densities between genotypes, indicating that Atm kinase deficiency does not affect axon number at this stage (p > 0.05, Unpaired Student’s t-test; Fig. 4 a-c). TEM revealed several abnormalities in myelin ultrastructure in Atm KDF/CNS−KO mice ( Fig. 4 d, e). We evaluated myelin thickness by calculating the g-ratio (myelin thickness relative to axon diameter), defined as the ratio between the inner axonal diameter and the total diameter of the myelinated fiber. Cumulative frequency analysis revealed a significant rightward shift in the g-ratio distribution in Atm KDF/CNS−KO mice, consistent with thinner myelin sheaths (p < 0.001, Mann-Whitney test; Fig. 4 f). Two-way ANOVA analysis of g-ratio as a function of axonal diameter showed a significant main effect of genotype (p < 0.0001, Two-way ANOVA: genotype effect, F (1, 472) = 21.38; Fig. 4 g), with Sidak’s post hoc multiple comparisons indicating a selective increase in g-ratio in medium-caliber axons in Atm KDF/CNS−KO mice compared to wild type littermates (p < 0.01; Fig. 4 g ) . Consistent with these findings, direct measurements of myelin thickness confirmed a significant reduction in Atm KDF/CNS−KO mice (p < 0.05, Mann-Whitney test; Fig. 4 h). Linear regression analysis of myelin thickness across all axon diameters revealed a significant reduction of the intercept in Atm KDF/CNS−KO mice (p < 0.0001) without a significant change in slope, indicating a global reduction in myelin thickness rather than defective scaling with axon caliber (Fig. 4 i ). To assess whether the reduction in myelin resulted from oligodendrocyte depletion, we quantified cells expressing the transcription factor Sox10, a marker of the oligodendrocyte lineage across all stages of their development. Notably, the density of Sox10-positive cells in both the cerebellum and corpus callosum was comparable between genotypes (Supplementary Fig. 3) , indicating that myelin defects occur in the absence of oligodendrocyte loss and suggesting impaired myelin maintenance rather than reduced cell number. Atm KDF/CNS−KO mice show cerebellar astroglial reactivity Given the prominent white matter abnormalities, we next investigated whether Atm kinase deficiency was associated with astroglial activation, a hallmark of disrupted neuron–glia interactions in cerebellar degeneration [ 5 , 58 ]. Immunofluorescence of cerebellar slices with GFAP, a canonical indicator of the astrocyte activation, revealed a robust increase in astrogliosis in Atm KDF/CNS−KO mice across all cerebellar regions examined (Fig. 5 a-d). Specifically, the percentage area covered by GFAP was higher in the molecular layer (p < 0.001, Mann-Whitney test), granular layer (p < 0.001, Mann-Whitney test), and white matter area (p < 0.01, Mann-Whitney test). Similarly, GFAP mean intensity was significantly increased in the molecular layer (p < 0.001, Mann-Whitney test), granular layer (p < 0.01, Unpaired Student’s t-test: t (14) = 8.94), and white matter (p < 0.01, Mann-Whitney test) (Fig. 5 e-g). These findings indicate a widespread astrocytes activation affecting both gray and white matter regions of the cerebellum. Atm KDF/CNS−KO mice show early signs of Purkinje cell degeneration White matter disruption and astroglial reactivity are known to alter ionic homeostasis and axonal conduction, potentially increasing the vulnerability of neuronal cells [ 37 , 46 , 54 ]. To assess whether PCs were affected at early stages of the disease, we first quantified PC density on calbindin-stained cerebellar sections. At 2 months of age, PC density was comparable between genotypes, indicating the absence of neuronal cell loss at this stage (wild type: 28.69 ± 1.35 cells/mm vs Atm KDF/CNS−KO : 28.26 ± 1.14 cells/mm, p > 0.05, Unpaired Student’s t-test: t (16) = 0.24). We next investigated whether PCs exhibited early degenerative changes by assessing DCD [ 47 ]. DCD is a type of neuronal death characterized by a shrunken, electron-dense appearance of the cell body and nucleus [ 47 ]. While no DCD PCs were detected in wild type mice ( Fig. 6 a-b ) , Atm KDF/CNS−KO mice displayed multiple PCs with DCD-like morphology ( Fig. 6 c-f ). The presence of DCD PCs in the absence of any exogenous insult suggests an increased intrinsic vulnerability of Atm KDF/CNS−KO PCs to degeneration compared to wild type ones. Purkinje cells exhibit early hyperexcitable phenotype in Atm KDF/CNS−KO mice To determine whether increased vulnerability of PCs was associated with altered intrinsic excitability, we performed electrophysiological recordings from PCs in acute cerebellar slices. Analysis of spontaneous firing recorded in cell-attached mode revealed comparable interspike interval and coefficient of variation between genotypes (Supplementary Fig. 4) . In contrast, analysis of evoked firing, recorded in whole-cell current clamp configuration, revealed marked differences between Atm KDF/CNS−KO and wild type PCs. We performed the analysis of evoked action potential discharge by applying a fixed depolarizing current step (600 pA) and found a significant shift to the left in the distribution of interspike intervals (p < 0.0001, K-S test) ( Fig. 7 a-c ) , an increase of the instantaneous frequency (p 0.05, Unpaired Student’s t-test) ( Fig. 7 d ) . When PCs were challenged with progressively increasing depolarizing current steps, Atm KDF/CNS−KO PCs displayed a significant higher number of action potentials compared to wild type mice across stimulus intensities (p < 0.01, Two-way ANOVA: genotype effect, F (1, 21) = 10.34) (Fig. 8 a, b). Consistent with a hyperexcitable phenotype, the latency to the first action potential was significantly shorter for Atm KDF/CNS−KO PCs compared to wild type littermates (p < 0.05, Unpaired Student’s t-test: t (17) = 2.22) (Fig. 8 d). The analysis of the features of the first action potential (Fig. 8 c) revealed a significant reduction of the threshold (p < 0.05, Unpaired Student’s t-test: t (19) = 2.44) (Fig. 8 d) and of the after hyperpolarization (AHP) (p < 0.01, Unpaired Student’s t-test: t (19) = 3.04) (Fig. 8 f). Overall, our analysis demonstrates that Atm KDF/CNS−KO PCs present an early increase in intrinsic excitability compared to wild type mice. Early motor coordination impairment and gait disturbance in Atm KDF/CNS−KO mice To determine whether the structural and functional cerebellar alterations at early stages translated into motor impairment, we assessed motor coordination and gait across disease progression, starting at 2 months of age, an early phase of the disease, and repeating the analysis at 12 and 24 months to monitoring the progressive worsening of the phenotype over time. The balance beam test is very effective in detecting very slight deficits in both motor coordination and balance ( Fig. 9 a ) . At 2 months of age, Atm KDF/CNS−KO mice already displayed impaired motor performance in the balance beam task, characterized by increased crossing latency (p < 0.0001; Two-way ANOVA: genotype effect, F (1, 114) = 61.49); for 2 months old mice: (wild type: 4.27 ± 0.27; Atm KDF/CNS−KO : 7.15 ± 0.55), for 12 months old: (wild type: 5.20 ± 0.39; Atm KDF/CNS−KO : 6.63 ± 0.60), and for 24 months old: (wild type: 8.10 ± 0.77; Atm KDF/CNS−KO : 15.77 ± 1.37) ( Fig. 9 b ) . The number of slips was higher compared to controls (p < 0.0001; Two-way ANOVA: genotype effect, F (1, 185) = 134.4); for 2 months old mice (wild type: 0.25 ± 0.05; Atm KDF/CNS−KO : 1.80 ± 0.29), for 12 months old: (wild type: 0.46 ± 0.08; Atm KDF/CNS−KO : 1.59 ± 0.32) and for 24 months old: (wild type: 1.08 ± 0.35; Atm KDF/CNS−KO : 5.89 ± 0.70) ( Fig. 9 c ) . These deficits progressively worsened with age, indicating a gradual deterioration of motor coordination (genotype per age interaction effect: for latency, F (2, 72) = 9.97; for number of slips, F (2, 185) = 20.53). We performed the footprinting test to analyze the gait characteristics in mice of both genotypes at 2 months of age. Our analysis indicated a significant reduction of the FP-HP placement (wild type: 1.17 ± 0.04 cm and Atm KDF/CNS−KO : 0.91 ± 0.04 cm; p < 0.0001, Unpaired Student’s t-test) as well as an increased coefficient of variation of FP-HP placement (wild type: 0.48 ± 0.02 and Atm KDF/CNS−KO : 0.62 ± 0.03; p < 0.0001, Unpaired Student’s t-test) ( Fig. 9 d-f ). No significant changes between genotypes were observed for the other gait parameters and their coefficient of variation (for stride length: wild type: 5.9 ± 0.06 cm and Atm KDF/CNS−KO : 6.02 ± 0.09; for fore limb width: wild type: 3.40 ± 0.03 cm and Atm KDF/CNS−KO : 3.47 ± 0.05; for hind limb width: wild type: 3.71 ± 0.03 cm and Atm KDF/CNS−KO : 3.67 ± 0.05 cm; for each gait parameter: p > 0.05, Unpaired Student’s t-test). By means of accelerated rotarod test we evaluated the motor learning and memory in Atm KDF/CNS−KO and wild type mice. Two-way ANOVA repeated measures revealed a significant time x genotype interaction effect (P < 0.05; F(5, 335) = 2.72) suggesting a different response of genotypes over time. Indeed, the learning over days of Atm KDF/CNS−KO mice was delayed compared to wild type mice (2nd day vs 1st day; one way ANOVA repeated measures, P > 0.05 Tukey’s Post hoc ). Moreover, the 10th day Atm KDF/CNS−KO mice did not have the retention of the memory (10th day vs 1st day; one-way ANOVA repeated measures, P > 0.05 Tukey’s Post hoc ) compared to wild type littermates ( Fig. 9 g, h ) . At 12 months of age Atm KDF/CNS−KO mice could not learn the task over days compared to wild type littermates and they reached a plateau from day 2 to 10 (one way ANOVA repeated measures, P > 0.05) ( Fig. 9 h ) . This indicates a progressive deficit of motor learning in Atm KDF/CNS−KO mice. The absolute time on the rotating rod is influenced by the body mouse weight while the motor learning is not dependent on it. We found a significant reduction in body weight of Atm KDF/CNS−KO compared to wild type mice at each age point under analysis: for 2 months old mice: wild type (23.86 ± 0.40 g) and Atm KDF/CNS−KO (20.10 ± 0.46 g); p < 0.0001, Mann-Whitney test), for 12 months old: wild type (37.67 ± 1.26 g) and Atm KDF/CNS−KO (31.01 ± 1.40 g); p < 0.001, Unpaired Student’s t-test), and for 24 months old: wild type (34.34 ± 1.33 g) and Atm KDF/CNS−KO (29.23 ± 1.73 g); p < 0.05, Unpaired Student’s t-test) which might explain the slightly higher time on the rotating rod. DISCUSSION In this study, we show that loss of Atm kinase activity in the CNS induces an early and coordinated remodeling of the cerebellar microenvironment without overt neuronal loss. By combining proteomic, structural, ultrastructural, electrophysiological and behavioral analysis, we demonstrate that Atm kinase deficiency is associated with ECM alterations, astroglial reactivity, impaired white matter homeostasis, increased PC vulnerability, and early circuit dysfunction. Together, these data indicate that cerebellar pathology in Atm KDF/CNS-KO mice emerges from a destabilization of the tissue environment that compromises circuit integrity at early stages of the disease. Our proteomic profiling revealed broad alteration across four major pathways in Atm KDF/CNS-KO mice: i) disorganization of the ECM, ii) astrocytosis, iii) downregulation of myelin/oligodendroglial lineage proteins and iv) alteration in neuronal excitability and synaptic organization. These findings indicate a global destabilization of cerebellar homeostasis rather than isolated molecular changes. A central finding of our study is an early and extensive remodeling of the ECM in the cerebellum of Atm KDF/CNS-KO mice. The ECM remodeling is a well-recognized consequence of oxidative stress and DNA damage, hallmark features of A-T, through their effects on ECM synthesis, turnover, and stabilization [ 30 ]. The upregulation of the components of the basement membrane and the interstitial matrix suggests that ECM could be biochemically remodeled toward greater stiffness and rigidity. Such changes are known to aggravate oxidative stress and DNA damage [ 6 , 30 ], stressors particularly detrimental in the absence of ATM. A properly regulated ECM is required throughout development and remains crucial in adulthood. It preserves network function by regulating cell survival, proliferation, migration and differentiation. Furthermore, it modulates synaptic plasticity and provides structural anchorage for both neuronal and glial processes [ 23 ]. Although ECM is secreted by both neurons and glial cells, astrocytes and microglia are the primary regulators of ECM remodeling [ 49 ]. Accordingly, we observed increased expression of astrocytic and astrocyte endfoot markers, indicative of reactive astrogliosis and vascular endfoot remodeling, consistent with the profound ECM reorganization. The diffused astrocytosis in the cerebellar cortex of Atm KDF/CNS-KO mice likely contributes to ECM remodeling, although ECM alterations can themselves activate astrocytes [ 23 , 30 ]. This reciprocal influence suggests a self-reinforcing cycle in which mechanical and biochemical changes of the ECM and astrocytic reactivity perpetuate each other, progressively destabilizing the cerebellar microenvironment. The proteomic profile also revealed a coordinated downregulation of multiple myelin and oligodendrocyte lineage proteins in the cerebellum of Atm KDF/CNS-KO mice. This pattern is consistent with a failure of the oligodendrocyte lineage to mature and sustain myelination. Structural and ultrastructural analyses confirmed a marked reduction of myelination in cerebellum and corpus callosum despite preserved axon and oligodendrocyte numbers, supporting a defect in myelin maintenance rather than cell loss. Oxidative stress and DNA damage are known to impair the oligodendrocyte lineage and Atm deficiency may exacerbate this vulnerability [ 51 ]. However, the myelin deficits occur in the context of a profoundly remodeled and stiffened ECM and reactive astrocytosis, both conditions that are known to create a non-permissive environment for the correct oligodendrocyte lineage development and myelination [ 23 , 29 , 49 , 52 ]. Thus, the myelin phenotype likely reflects both cell-autonomous vulnerability and non-cell-autonomous constraints imposed by the remodeled microenvironment. The combination of stiffened ECM, astrogliosis and defective myelination in Atm KDF/CNS-KO mice is associated with PCs exhibiting DCD and increased intrinsic excitability, indicating that these neurons are highly vulnerable in early stages of the disease. The influence of astrocytes, ECM and oligodendrocytes on neuronal excitability and synaptic function is well established [ 24 , 46 ]. Moreover, the relationship between myelin and excitability is bidirectional: neuronal activity regulates myelination and myelin integrity modulates neuronal firing properties [ 36 ]. Thus, the altered excitability of PCs likely reflects the cumulative impact of microenvironment instability. Although most ataxias are characterized by reduced PC excitability [ 15 ], the increased activity observed in Atm KDF/CNS-KO mice likely represents an early and potentially transient compensatory response aimed at maintaining cerebellar output in the face of widespread microenvironmental disruption. Such compensation is likely unsustainable, ultimately contributing to circuit failure and motor impairment even in the absence of PC loss. In conclusion, although our data provide a comprehensive view of the molecular, structural, and functional alterations in the cerebellum of Atm KDF/CNS−KO mice, they do not allow us to infer a temporal sequence or to identify a single primary causal event. Instead, our findings support a coordinated disruption across multiple cellular compartments, both intra- and extracellular, suggesting that Atm kinase deficiency alters cerebellar homeostasis through a network level mechanism rather than a single pathway. The simultaneous upregulation of basement-membrane and interstitial ECM proteins, the downregulation of myelin and oligodendrocyte maturation markers, the activation of astrocytic pathways and the increased excitability of PCs point to a multifaceted microenvironmental imbalance in which these processes likely interact bidirectionally. In this context, ECM remodeling may impair oligodendrocyte maturation; dysfunctional oligodendrocytes may trigger astrocytic activation; reactive astrocytes may further reshape the ECM; or early neuronal alterations may secondarily affect myelination and glial reactivity. Thus, while causality cannot be assigned to any single component, the convergence of these alterations strongly supports a model in which ATM is a master regulator of cerebellar microenvironment stability, and identify ECM-glia-neuron interaction as early therapeutic target in A-T. Declarations Ethics approval and consent to participate All experimental procedures have been carried out in accordance with the European Communities Parliament and Council Directives of 24 November 1986 (86/609/EEC) and 22 September 2010 (2010/63/EU), approved by the Ethical Committee of the University of Torino and authorized by the Italian Ministry of Health (authorization number: 466/2021-PR). Consent for publication Not applicable Availability of data and material: All data supporting the findings of this study are available either within the paper or from the corresponding author on reasonable request. Competing interests: All other authors declare they have no competing interests. Funding: The research was funded by Associazione Nazionale Atassia Telangiectasia (ANAT), Fondazione Cassa di Risparmio di Torino (CRT Foundation), Banca d’Italia, a local grant of the University of Torino (2021), and the Department of Excellence funding from the Ministry of University and Research (MUR) for 2023–2027, awarded to the Department of Neuroscience “Rita Levi Montalcini” (University of Turin). Authors contribution: FM and EH performed and supervised the experiments and data analysis and wrote the manuscript. FM, LB, AMR, ML, IB, AR, GPS, GM, RP, KY, and EH performed the experiments and data analysis. FM, MM, FT, RV, AB, SZ and EH contributed to the conception of the work and to the discussion of the results. All authors approved the final version of the manuscript. Acknowledgments Not applicable References Bakkenist CJ, Kastan MB (2003) DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 421:499–506. doi: 10.1038/nature01368 Boder E, Sedgwick RP (1958) Ataxia-telangiectasia; a familial syndrome of progressive cerebellar ataxia, oculocutaneous telangiectasia and frequent pulmonary infection. Pediatrics 21:526–554 Borghesani PR, Alt FW, Bottaro A, Davidson L, Aksoy S, Rathbun GA, Roberts TM, Swat W, Segal RA, Gu Y (2000) Abnormal development of Purkinje cells and lymphocytes in Atm mutant mice. Proc Natl Acad Sci USA 97:3336–3341. doi: 10.1073/pnas.97.7.3336 Canman CE, Lim D-S, Cimprich KA, Taya Y, Tamai K, Sakaguchi K, Appella E, Kastan MB, Siliciano JD (1998) Activation of the ATM Kinase by Ionizing Radiation and Phosphorylation of p53. Science 281:1677–1679. doi: 10.1126/science.281.5383.1677 Cerrato V (2020) Cerebellar Astrocytes: Much More Than Passive Bystanders In Ataxia Pathophysiology. JCM 9:757. doi: 10.3390/jcm9030757 Cho S, Vashisth M, Abbas A, Majkut S, Vogel K, Xia Y, Ivanovska IL, Irianto J, Tewari M, Zhu K, Tichy ED, Mourkioti F, Tang H-Y, Greenberg RA, Prosser BL, Discher DE (2019) Mechanosensing by the Lamina Protects against Nuclear Rupture, DNA Damage, and Cell-Cycle Arrest. Developmental Cell 49:920-935.e5. doi: 10.1016/j.devcel.2019.04.020 Chow H-M, Cheng A, Song X, Swerdel MR, Hart RP, Herrup K (2019) ATM is activated by ATP depletion and modulates mitochondrial function through NRF1. Journal of Cell Biology 218:909–928. doi: 10.1083/jcb.201806197 Chung EO, Bodensteiner JB, Noorani PA, Schochet SS (1994) Cerebral White-Matter Changes Suggesting Leukodystrophy in Ataxia Telangiectasia. J Child Neurol 9:31–35. doi: 10.1177/088307389400900106 Farley FW, Soriano P, Steffen LS, Dymecki SM (2000) Widespread recombinase expression using FLPeR (flipper) mice. Genesis 28:106–110 Fırat AK, Karakaş HM, Fırat Y, Yakıncı C (2005) Quantitative evaluation of brain involvement in ataxia telangiectasia by diffusion weighted MR imaging. European Journal of Radiology 56:192–196. doi: 10.1016/j.ejrad.2005.04.009 Gallyas F (1979) Silver Staining of Myelin by Means of Physical Development. Neurological Research 1:203–209. doi: 10.1080/01616412.1979.11739553 Gatti RA, Berkel I, Boder E, Braedt G, Charmley P, Concannon P, Ersoy F, Foroud T, Jaspers NGJ, Lange K, Lathrop GM, Leppert M, Nakamura Y, O’Connell P, Paterson M, Salser W, Sanal O, Silver J, Sparkes RS, Susi E, Weeks DE, Wei S, White R, Yoder F (1988) Localization of an ataxia-telangiectasia gene to chromosome 11q22–23. Nature 336:577–580. doi: 10.1038/336577a0 Gatti RA, Vinters HV (1985) Cerebellar pathology in ataxia-telangiectasia: the significance of basket cells. Kroc Found Ser 19:225–232 Guo Z, Deshpande R, Paull TT (2010) ATM activation in the presence of oxidative stress. Cell Cycle 9:4805–4811. doi: 10.4161/cc.9.24.14323 Hoxha E, Balbo I, Miniaci MC, Tempia F (2018) Purkinje Cell Signaling Deficits in Animal Models of Ataxia. Front Synaptic Neurosci 10:6. doi: 10.3389/fnsyn.2018.00006 Hoxha E, Balbo I, Parolisi R, Audano M, Montarolo F, Ravera F, Guglielmotto M, Muratori L, Raimondo S, DiGregorio E, Buffo A, Brusco A, Borroni B, Mitro N, Caruso D, Tempia F (2021) Elovl5 is required for proper action potential conduction along peripheral myelinated fibers. Glia 69:2419–2428. doi: 10.1002/glia.24048 Hoxha E, Gabriele RMC, Balbo I, Ravera F, Masante L, Zambelli V, Albergo C, Mitro N, Caruso D, Di Gregorio E, Brusco A, Borroni B, Tempia F (2017) Motor Deficits and Cerebellar Atrophy in Elovl5 Knock Out Mice. Front Cell Neurosci 11:343. doi: 10.3389/fncel.2017.00343 Hoxha E, Marcinnò A, Montarolo F, Masante L, Balbo I, Ravera F, Laezza F, Tempia F (2019) Emerging roles of Fgf14 in behavioral control. Behavioural Brain Research 356:257–265. doi: 10.1016/j.bbr.2018.08.034 Hoxha E, Tonini R, Montarolo F, Croci L, Consalez GG, Tempia F (2013) Motor dysfunction and cerebellar Purkinje cell firing impairment in Ebf2 null mice. Molecular and Cellular Neuroscience 52:51–61. doi: 10.1016/j.mcn.2012.09.002 Kanner S, Goldin M, Galron R, Ben Jacob E, Bonifazi P, Barzilai A (2018) Astrocytes restore connectivity and synchronization in dysfunctional cerebellar networks. Proc Natl Acad Sci USA 115:8025–8030. doi: 10.1073/pnas.1718582115 Krishnaswamy VR, Benbenishty A, Blinder P, Sagi I (2019) Demystifying the extracellular matrix and its proteolytic remodeling in the brain: structural and functional insights. Cell Mol Life Sci 76:3229–3248. doi: 10.1007/s00018-019-03182-6 Lai J, Demirbas D, Kim J, Jeffries AM, Tolles A, Park J, Chittenden TW, Buckley PG, Yu TW, Lodato MA, Lee EA (2024) ATM-deficiency-induced microglial activation promotes neurodegeneration in ataxia-telangiectasia. Cell Reports 43:113622. doi: 10.1016/j.celrep.2023.113622 Lau LW, Cua R, Keough MB, Haylock-Jacobs S, Yong VW (2013) Pathophysiology of the brain extracellular matrix: a new target for remyelination. Nat Rev Neurosci 14:722–729. doi: 10.1038/nrn3550 Lawrence JM, Schardien K, Wigdahl B, Nonnemacher MR (2023) Roles of neuropathology-associated reactive astrocytes: a systematic review. acta neuropathol commun 11:42. doi: 10.1186/s40478-023-01526-9 Li J, Han YR, Plummer MR, Herrup K (2009) Cytoplasmic ATM in Neurons Modulates Synaptic Function. Current Biology 19:2091–2096. doi: 10.1016/j.cub.2009.10.039 Lumsden JM, McCarty T, Petiniot LK, Shen R, Barlow C, Wynn TA, Morse HC, Gearhart PJ, Wynshaw-Boris A, Max EE, Hodes RJ (2004) Immunoglobulin Class Switch Recombination Is Impaired in Atm -deficient Mice. The Journal of Experimental Medicine 200:1111–1121. doi: 10.1084/jem.20041074 Mancini C, Hoxha E, Iommarini L, Brussino A, Richter U, Montarolo F, Cagnoli C, Parolisi R, Gondor Morosini DI, Nicolò V, Maltecca F, Muratori L, Ronchi G, Geuna S, Arnaboldi F, Donetti E, Giorgio E, Cavalieri S, Di Gregorio E, Pozzi E, Ferrero M, Riberi E, Casari G, Altruda F, Turco E, Gasparre G, Battersby BJ, Porcelli AM, Ferrero E, Brusco A, Tempia F (2019) Mice harbouring a SCA28 patient mutation in AFG3L2 develop late-onset ataxia associated with enhanced mitochondrial proteotoxicity. Neurobiology of Disease 124:14–28. doi: 10.1016/j.nbd.2018.10.018 Mansouri B, Henne WM, Oomman SK, Bliss R, Attridge J, Finckbone V, Zeitouni T, Hoffman T, Bahr BA, Strahlendorf HK, Strahlendorf JC (2007) Involvement of calpain in AMPA-induced toxicity to rat cerebellar Purkinje neurons. European Journal of Pharmacology 557:106–114. doi: 10.1016/j.ejphar.2006.11.032 Marangon D, Boccazzi M, Lecca D, Fumagalli M (2020) Regulation of Oligodendrocyte Functions: Targeting Lipid Metabolism and Extracellular Matrix for Myelin Repair. JCM 9:470. doi: 10.3390/jcm9020470 Martins SG, Zilhão R, Thorsteinsdóttir S, Carlos AR (2021) Linking Oxidative Stress and DNA Damage to Changes in the Expression of Extracellular Matrix Components. Front Genet 12:673002. doi: 10.3389/fgene.2021.673002 Menolfi D, Lee BJ, Zhang H, Jiang W, Bowen NE, Wang Y, Zhao J, Holmes A, Gershik S, Rabadan R, Kim B, Zha S (2023) ATR kinase supports normal proliferation in the early S phase by preventing replication resource exhaustion. Nat Commun 14:3618. doi: 10.1038/s41467-023-39332-5 Mitiagin Y, Barzilai A (2023) Ataxia-telangiectasia mutated plays an important role in cerebellar integrity and functionality. Neural Regen Res 18:497. doi: 10.4103/1673-5374.350194 Mitiagin Y, Barzilai A (2023) Ataxia-telangiectasia mutated plays an important role in cerebellar integrity and functionality. Neural Regen Res 18:497. doi: 10.4103/1673-5374.350194 Montarolo F, Gao F, Loddo M, Milek J, Zawadzka M, Miniaci MC, Brusco A, Balbo I, Dziembowska M, Tempia F, Skowronska-Krawczyk D, Hoxha E (2025) Impact of Elovl5 deficiency on cerebellar excitatory synaptic transmission in mice. J Neurosci e1529242025. doi: 10.1523/JNEUROSCI.1529-24.2025 Montarolo F, Thielens S, Bove M, Bertolotto A, Tempia F, Hoxha E (2025) NURR1 Deficiency Is Associated to Altered Microglial Phenotype in Male Mice. Mol Neurobiol 62:8887–8899. doi: 10.1007/s12035-025-04787-8 Pease-Raissi SE, Chan JR (2021) Building a (w)rapport between neurons and oligodendroglia: Reciprocal interactions underlying adaptive myelination. Neuron 109:1258–1273. doi: 10.1016/j.neuron.2021.02.003 Pepper RE, Pitman KA, Cullen CL, Young KM (2018) How Do Cells of the Oligodendrocyte Lineage Affect Neuronal Circuits to Influence Motor Function, Memory and Mood? Front Cell Neurosci 12:399. doi: 10.3389/fncel.2018.00399 Petersen AJ, Rimkus SA, Wassarman DA (2012) ATM kinase inhibition in glial cells activates the innate immune response and causes neurodegeneration in Drosophila . Proc Natl Acad Sci USA 109. doi: 10.1073/pnas.1110470109 Pistorio AL, Hendry SH, Wang X (2006) A modified technique for high-resolution staining of myelin. Journal of Neuroscience Methods 153:135–146. doi: 10.1016/j.jneumeth.2005.10.014 Pizzamiglio L, Focchi E, Murru L, Tamborini M, Passafaro M, Menna E, Matteoli M, Antonucci F (2016) New Role of ATM in Controlling GABAergic Tone During Development. Cereb Cortex 26:3879–3888. doi: 10.1093/cercor/bhw125 Reina-San-Martin B, Chen HT, Nussenzweig A, Nussenzweig MC (2004) ATM is required for efficient recombination between immunoglobulin switch regions. J Exp Med 200:1103–1110. doi: 10.1084/jem.20041162 Rothblum-Oviatt C, Wright J, Lefton-Greif MA, McGrath-Morrow SA, Crawford TO, Lederman HM (2016) Ataxia telangiectasia: a review. Orphanet J Rare Dis 11:159. doi: 10.1186/s13023-016-0543-7 Sahama I, Sinclair K, Fiori S, Doecke J, Pannek K, Reid L, Lavin M, Rose S (2015) Motor pathway degeneration in young ataxia telangiectasia patients: A diffusion tractography study. NeuroImage: Clinical 9:206–215. doi: 10.1016/j.nicl.2015.08.007 Sahama I, Sinclair K, Pannek K, Lavin M, Rose S (2014) Radiological Imaging in Ataxia Telangiectasia: a Review. Cerebellum 13:521–530. doi: 10.1007/s12311-014-0557-4 Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez J-Y, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682. doi: 10.1038/nmeth.2019 Song I, Dityatev A (2018) Crosstalk between glia, extracellular matrix and neurons. Brain Research Bulletin 136:101–108. doi: 10.1016/j.brainresbull.2017.03.003 Strahlendorf J, Box C, Attridge J, Diertien J, Finckbone V, Henne WM, Medina MS, Miles R, Oomman S, Schneider M, Singh H, Veliyaparambil M, Strahlendorf H (2003) AMPA-induced dark cell degeneration of cerebellar Purkinje neurons involves activation of caspases and apparent mitochondrial dysfunction. Brain Research 994:146–159. doi: 10.1016/j.brainres.2003.09.048 Tal E, Alfo M, Zha S, Barzilai A, De Zeeuw CI, Ziv Y, Shiloh Y (2018) Inactive Atm abrogates DSB repair in mouse cerebellum more than does Atm loss, without causing a neurological phenotype. DNA Repair 72:10–17. doi: 10.1016/j.dnarep.2018.10.001 Tewari BP, Chaunsali L, Prim CE, Sontheimer H (2022) A glial perspective on the extracellular matrix and perineuronal net remodeling in the central nervous system. Front Cell Neurosci 16:1022754. doi: 10.3389/fncel.2022.1022754 Tse K-H, Cheng A, Yeung SH-S, Ng J-N, Cheng GW-Y, Wang Q, Zhu B, Cui Y, Jiang L, Kofler J, Herrup K (2021) Myelin pathology in ataxia-telangiectasia is the cell autonomous effect of ATM deficiency in oligodendrocytes Tse K-H, Herrup K (2017) DNA damage in the oligodendrocyte lineage and its role in brain aging. Mechanisms of Ageing and Development 161:37–50. doi: 10.1016/j.mad.2016.05.006 Urbanski MM, Kingsbury L, Moussouros D, Kassim I, Mehjabeen S, Paknejad N, Melendez-Vasquez CV (2016) Myelinating glia differentiation is regulated by extracellular matrix elasticity. Sci Rep 6:33751. doi: 10.1038/srep33751 Vail G, Cheng A, Han YR, Zhao T, Du S, Loy MMT, Herrup K, Plummer MR (2016) ATM protein is located on presynaptic vesicles and its deficit leads to failures in synaptic plasticity. Journal of Neurophysiology 116:201–209. doi: 10.1152/jn.00006.2016 Verhoog QP, Holtman L, Aronica E, van Vliet EA (2020) Astrocytes as Guardians of Neuronal Excitability: Mechanisms Underlying Epileptogenesis. Front Neurol 11:591690. doi: 10.3389/fneur.2020.591690 Xu K, Zhang Y, Chen Y, Zhu X, Li Y, Lv L, He X, Hu Z, Li Y, Ye M, Jiang D, He Z, Jin W, Li Y, Yu X, Zhang D-F, Herrup K, Zheng P, Yao Y-G, Wu D-D, Li J (2025) ATM deficiency drives phenotypic diversity and Purkinje cell degeneration in a macaque model of ataxia-telangiectasia. Cell Reports Medicine 6:102355. doi: 10.1016/j.xcrm.2025.102355 Yamamoto K, Wang J, Sprinzen L, Xu J, Haddock CJ, Li C, Lee BJ, Loredan DG, Jiang W, Vindigni A, Wang D, Rabadan R, Zha S (2016) Kinase-dead ATM protein is highly oncogenic and can be preferentially targeted by Topo-isomerase I inhibitors. eLife 5:e14709. doi: 10.7554/eLife.14709 Yamamoto K, Wang Y, Jiang W, Liu X, Dubois RL, Lin C-S, Ludwig T, Bakkenist CJ, Zha S (2012) Kinase-dead ATM protein causes genomic instability and early embryonic lethality in mice. Journal of Cell Biology 198:305–313. doi: 10.1083/jcb.201204098 Yang K, Dunn M, Torres-Ramirez G, Dobbs N, Shakkottai VG, Yan N (2025) Autonomous STING signaling in Purkinje cells drives neurodegeneration independent of type I interferon. Cell Reports 44:116480. doi: 10.1016/j.celrep.2025.116480 Zha S, Sekiguchi J, Brush JW, Bassing CH, Alt FW (2008) Complementary functions of ATM and H2AX in development and suppression of genomic instability. Proc Natl Acad Sci USA 105:9302–9306. doi: 10.1073/pnas.0803520105 Zhang J, Tripathi DN, Jing J, Alexander A, Kim J, Powell RT, Dere R, Tait-Mulder J, Lee J-H, Paull TT, Pandita RK, Charaka VK, Pandita TK, Kastan MB, Walker CL (2015) ATM functions at the peroxisome to induce pexophagy in response to ROS. Nat Cell Biol 17:1259–1269. doi: 10.1038/ncb3 Additional Declarations No competing interests reported. Supplementary Files MontaroloetalSupplTableS1.xlsx MontaroloetalSupplTable2.docx MontaroloetalSupplementarymaterial.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 01 Mar, 2026 Reviews received at journal 01 Mar, 2026 Reviews received at journal 25 Feb, 2026 Reviewers agreed at journal 19 Feb, 2026 Reviewers agreed at journal 18 Feb, 2026 Reviewers invited by journal 17 Feb, 2026 Editor assigned by journal 13 Feb, 2026 Submission checks completed at journal 11 Feb, 2026 First submitted to journal 09 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8832557","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":591148974,"identity":"4fa1c898-23b7-472e-906d-aac17c498f63","order_by":0,"name":"Francesca Montarolo","email":"","orcid":"","institution":"University of Torino","correspondingAuthor":false,"prefix":"","firstName":"Francesca","middleName":"","lastName":"Montarolo","suffix":""},{"id":591148975,"identity":"f4ead32c-e0af-4b76-bc24-ac5a0f9881af","order_by":1,"name":"Luna Berrino","email":"","orcid":"","institution":"University of Torino","correspondingAuthor":false,"prefix":"","firstName":"Luna","middleName":"","lastName":"Berrino","suffix":""},{"id":591148976,"identity":"fdf01297-5784-4033-9e05-4cbbc5c53f55","order_by":2,"name":"Anita Maria Rominto","email":"","orcid":"","institution":"University of Torino","correspondingAuthor":false,"prefix":"","firstName":"Anita","middleName":"Maria","lastName":"Rominto","suffix":""},{"id":591148977,"identity":"5fe7ccbe-4134-4ea8-a1b1-aa966687c07c","order_by":3,"name":"Matilde Loddo","email":"","orcid":"","institution":"Neuroscience Institute Cavalieri Ottolenghi (NICO) Regione","correspondingAuthor":false,"prefix":"","firstName":"Matilde","middleName":"","lastName":"Loddo","suffix":""},{"id":591148978,"identity":"95359cf3-ea0d-428c-a662-b76e28ee75b7","order_by":4,"name":"Ilaria Balbo","email":"","orcid":"","institution":"Columbia University","correspondingAuthor":false,"prefix":"","firstName":"Ilaria","middleName":"","lastName":"Balbo","suffix":""},{"id":591148979,"identity":"ebe6cb8d-6551-4ae8-81d5-37eabd416ac7","order_by":5,"name":"Anastasia Ricci","email":"","orcid":"","institution":"University of Urbino \"Carlo Bo\" Campus Scientifico Enrico Mattei","correspondingAuthor":false,"prefix":"","firstName":"Anastasia","middleName":"","lastName":"Ricci","suffix":""},{"id":591148980,"identity":"16e660f5-6a51-4980-88bb-21375fba2159","order_by":6,"name":"Giulia Pia Servetto","email":"","orcid":"","institution":"University of Turin","correspondingAuthor":false,"prefix":"","firstName":"Giulia","middleName":"Pia","lastName":"Servetto","suffix":""},{"id":591148981,"identity":"dd9f03fe-e0ff-4a0d-82f3-3c41179d7a0d","order_by":7,"name":"Ginevra Mango","email":"","orcid":"","institution":"University of Turin","correspondingAuthor":false,"prefix":"","firstName":"Ginevra","middleName":"","lastName":"Mango","suffix":""},{"id":591148982,"identity":"41cf82c3-e5c4-4ab8-a854-472473289332","order_by":8,"name":"Kenta Yamamoto","email":"","orcid":"","institution":"Columbia University","correspondingAuthor":false,"prefix":"","firstName":"Kenta","middleName":"","lastName":"Yamamoto","suffix":""},{"id":591148983,"identity":"0b32beed-022c-40ff-bada-4be50e9994ec","order_by":9,"name":"Roberta Parolisi","email":"","orcid":"","institution":"University of Torino","correspondingAuthor":false,"prefix":"","firstName":"Roberta","middleName":"","lastName":"Parolisi","suffix":""},{"id":591148984,"identity":"04e503f1-b7b9-44f9-89f4-4bd08ca4a25a","order_by":10,"name":"Antonio Bertolotto","email":"","orcid":"","institution":"Neuroscience Institute Cavalieri Ottolenghi (NICO) Regione","correspondingAuthor":false,"prefix":"","firstName":"Antonio","middleName":"","lastName":"Bertolotto","suffix":""},{"id":591148985,"identity":"be2090bc-e8a5-4531-90a0-a3196b0932c4","order_by":11,"name":"Ruggero Vigliaturo","email":"","orcid":"","institution":"University of Turin","correspondingAuthor":false,"prefix":"","firstName":"Ruggero","middleName":"","lastName":"Vigliaturo","suffix":""},{"id":591148986,"identity":"5a4c9562-52a0-4dfd-a3e6-39895afd8cc4","order_by":12,"name":"Michele Menotta","email":"","orcid":"","institution":"University of Urbino \"Carlo Bo\" Campus Scientifico Enrico Mattei","correspondingAuthor":false,"prefix":"","firstName":"Michele","middleName":"","lastName":"Menotta","suffix":""},{"id":591148987,"identity":"dfccfeef-d176-4e4b-8f64-58ca229a0012","order_by":13,"name":"Shan Zha","email":"","orcid":"","institution":"Columbia University","correspondingAuthor":false,"prefix":"","firstName":"Shan","middleName":"","lastName":"Zha","suffix":""},{"id":591148988,"identity":"26757fd3-3685-405d-ade0-518edfbf1200","order_by":14,"name":"Filippo Tempia","email":"","orcid":"","institution":"University of Torino","correspondingAuthor":false,"prefix":"","firstName":"Filippo","middleName":"","lastName":"Tempia","suffix":""},{"id":591148989,"identity":"2507c4ff-9e42-4664-b06b-ed4db3c442a6","order_by":15,"name":"Eriola Hoxha","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIiWNgGAWjYFACxgaGBwwMCQwMPAwMH8AkA8MBgloSoFoYZwBJkJ4DBPQwwLUwg5QTtIZf+nDjg4Qahjx+idyDn23b7sjYszcwHv6AR4tkX2KzQcIxhmLJnnPJ0rltz3h4eA7gd5jBGcY2iQQ2hsQNx3sMgFoO8/BIJODXYg/W8g+o5TCP8W9LYrQY8AC1JLaBbTGTZiRGi8QZxmaDxD6JxJk9Z8wse84BtZw52HDgDB4t/D3sDx98+GaT2C+RY3zjR9lhe/b25sMfKvBogVmGzAFG7igYBaNgFIwCygAABtZOrN5aSUwAAAAASUVORK5CYII=","orcid":"","institution":"University of Torino","correspondingAuthor":true,"prefix":"","firstName":"Eriola","middleName":"","lastName":"Hoxha","suffix":""}],"badges":[],"createdAt":"2026-02-09 16:16:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8832557/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8832557/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102849597,"identity":"e33db4c6-50ac-450d-98fb-673c4f35708c","added_by":"auto","created_at":"2026-02-17 14:00:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":37953413,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntegration of Quantitative Proteomics and Functional Enrichment in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAtm\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003eKDF/CNS-KO\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e mice\u003c/strong\u003e. Differential Expression Profile (Volcano Plot) \u003cstrong\u003e(a)\u003c/strong\u003e: The plot displays the distribution of differentially expressed proteins (DEPs). Highlighted data points indicate proteins that exceeded the statistical significance (-log10 p-value) and magnitude of change (fold-change) thresholds. Key pathological drivers are labeled to emphasize major biochemical shifts. Hierarchical Clustering Analysis (Heatmap)\u003cstrong\u003e (b)\u003c/strong\u003e: The heatmap illustrates protein expression patterns across wild type (WT) and \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e \u003c/strong\u003esamples. Hierarchical clustering reveals a clear separation of proteomic signatures, identifying distinct clusters of proteins that are consistently downregulated or upregulated in the disease model. KEGG Pathway Enrichment Analysis \u003cstrong\u003e(c)\u003c/strong\u003e: Bar graph showing the top enriched pathways based on -log10 p value. Bars are color-coded by functional category. Gene Ontology (GO) Enrichment of Up- and Down-regulated Proteins \u003cstrong\u003e(d)\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"MontaroloetalFig1.png","url":"https://assets-eu.researchsquare.com/files/rs-8832557/v1/b9f6cd1ca57570ddcc223226.png"},{"id":102849589,"identity":"eddbaf0d-d4f9-4367-b86f-1dfdd80fe840","added_by":"auto","created_at":"2026-02-17 14:00:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5983027,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAtm\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003eKDF/CNS-KO\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e mice show white matter area reduction\u003c/strong\u003e. Nissl-stained sagittal sections of the cerebellum from wild type \u003cstrong\u003e(a)\u003c/strong\u003e and \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e mice \u003cstrong\u003e(b)\u003c/strong\u003e at 2 months of age (wild type n = 6; \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e n = 5). Significant reduction of the slice area (\u003cstrong\u003ec)\u003c/strong\u003e, white matter area (\u003cstrong\u003ed), \u003c/strong\u003eand of the ratio white matter to slice area (\u003cstrong\u003ee) \u003c/strong\u003ein the cerebellum of \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e mice (*p\u0026lt;0.05; **p\u0026lt;0.01, Unpaired Student’s t-test). Results are reported as mean ± SEM. Scale bar 500 mm.\u003c/p\u003e","description":"","filename":"MontaroloetalFig2.png","url":"https://assets-eu.researchsquare.com/files/rs-8832557/v1/8ae4b6b984c433854452f8dd.png"},{"id":102849596,"identity":"6f72bfb8-8e65-4dee-84df-ace0fc8644ef","added_by":"auto","created_at":"2026-02-17 14:00:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":8029541,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAtm\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003eKDF/CNS-KO\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e \u003cstrong\u003emice display myelination defects. \u003c/strong\u003eSilver nitrate\u003cstrong\u003e \u003c/strong\u003eGallyas myelin staining (brown) in the cerebellum (\u003cstrong\u003ea, b\u003c/strong\u003e) and corpus callosum (\u003cstrong\u003ee, f\u003c/strong\u003e) regions in both \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e and wild type mice. Quantification of Gallyas myelin fractioned area in the cerebellar slices from both genotypes (\u003cstrong\u003ec)\u003c/strong\u003e (wild type n = 7; \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e n = 7). Representative western blots of cerebellar extracts from wild type and \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e mice and densitometric quantification of Mbp protein subunits (wild type n = 4; \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e n = 5). Gapdh served as loading control (\u003cstrong\u003ed). \u003c/strong\u003eQuantification of Gallyas myelin fractioned area in the dorsal cortex and corpus callosum from both genotypes \u003cstrong\u003e(g, h)\u003c/strong\u003e (wild type n = 6; \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e n = 8). Scale bars: for \u003cstrong\u003e(a)\u003c/strong\u003e and \u003cstrong\u003e(d)\u003c/strong\u003e 500 mm; for \u003cstrong\u003e(b)\u003c/strong\u003e and \u003cstrong\u003e(f)\u003c/strong\u003e 100 mm. **p\u0026lt;0.01; ***p\u0026lt;0.001; ****p\u0026lt;0.0001, Unpaired Student’s t-test. Results are reported as mean ± SEM. Abbreviations: cc, corpus callosum; ctx: cortex.\u003c/p\u003e","description":"","filename":"MontaroloetalFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-8832557/v1/d10e82c84da63e4c2beac590.png"},{"id":102849600,"identity":"fe850a80-d70a-410a-a0c3-6f8c90c3949e","added_by":"auto","created_at":"2026-02-17 14:00:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":8698627,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUltrastructural defects of myelin in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAtm\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003eKDF/CNS-KO\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e mice. \u003c/strong\u003eRepresentative\u003cstrong\u003e \u003c/strong\u003etoluidine blue staining of the corpus callosum on semithin sections of wild type (n = 3) (\u003cstrong\u003ea\u003c/strong\u003e) and \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e mice (n = 3) (\u003cstrong\u003eb\u003c/strong\u003e). Scale bar, 20 μm. Quantification of myelinated axon density in toluidine blue staining (\u003cstrong\u003ec\u003c/strong\u003e). Data are represented as mean ± SEM. Representative transmission electron microscopy image of corpus callosum from wild type (\u003cstrong\u003ed\u003c/strong\u003e) and \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e mice (\u003cstrong\u003ee\u003c/strong\u003e). Scale bar, 500 nm. \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e mice exhibit a significant rightward shift of the cumulative frequency in the g-ratio distribution (p\u0026lt;0.001, Mann-Whitney test) \u003cstrong\u003e(f)\u003c/strong\u003e and significant higher g‐ratios in medium-caliber axons compared to wild type\u003cem\u003e \u003c/em\u003emice \u003cstrong\u003e(g)\u003c/strong\u003e. Reduced mean myelin thickness in \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e mice (p\u0026lt;0.05, Mann-Whitney test) \u003cstrong\u003e(h). \u003c/strong\u003eScatter plot of myelin thickness values across all axon diameters for both genotypes \u003cstrong\u003e(i). \u003c/strong\u003eResults are reported as mean ± SEM. *p\u0026lt;0.05, **p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"MontaroloetalFig4.png","url":"https://assets-eu.researchsquare.com/files/rs-8832557/v1/3bcb45c3705e6729e99b4b9c.png"},{"id":102849599,"identity":"4a8d9aff-a09a-442e-ba5e-fc2270bbe458","added_by":"auto","created_at":"2026-02-17 14:00:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":10004119,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCerebellar astroglial reactivity in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAtm\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003eKDF/CNS-KO\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e mice\u003c/strong\u003e. Representative fluorescent images of the abundance of GFAP+ (white) astrocytes and Dapi (blue) stained cerebellar sections form wild type (\u003cstrong\u003ea\u003c/strong\u003e) and \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e mice (\u003cstrong\u003eb\u003c/strong\u003e). Panels (\u003cstrong\u003ec\u003c/strong\u003e) and (\u003cstrong\u003ed\u003c/strong\u003e) are the high-resolution confocal images of boxed regions in the upper panels. Quantification of GFAP+ fractioned area and mean fluorescence intensity of the signal in the molecular layer (\u003cstrong\u003ee\u003c/strong\u003e), granular layer (\u003cstrong\u003ef\u003c/strong\u003e), and white matter (\u003cstrong\u003eg\u003c/strong\u003e) area of the cerebellum. **p\u0026lt;0.01; ***p\u0026lt;0.001, (wild type n = 8; \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e n = 8). Unpaired Student’s t-test. Results are reported as mean ± SEM.\u003c/p\u003e","description":"","filename":"MontaroloetalFig5.png","url":"https://assets-eu.researchsquare.com/files/rs-8832557/v1/74c810e6f4e7d1e83237e71e.png"},{"id":102849590,"identity":"229eee6f-e57d-4800-8060-f3a254feccec","added_by":"auto","created_at":"2026-02-17 14:00:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":572500,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDark cell degeneration (DCD) of Purkinje cells in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAtm\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003eKDF/CNS-KO\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e mice\u003c/strong\u003e. Toluidine blue staining of sagittal cerebellar sections obtained from wild type (\u003cstrong\u003ea, b)\u003c/strong\u003e and \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e \u003cstrong\u003e(c, e, d, f)\u003c/strong\u003e mice. Arrows indicate DCD positive (darker) cells in the \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e which are absent in wild type slices. Calibration bar 50 μm \u003cstrong\u003e(a-f)\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"MontaroloetalFig6.png","url":"https://assets-eu.researchsquare.com/files/rs-8832557/v1/a18029cc7c105c35f42a01a1.png"},{"id":102849592,"identity":"702ec9ba-740c-47e4-b1fd-15a9b4dbe6e1","added_by":"auto","created_at":"2026-02-17 14:00:42","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":917268,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIncreased evoked firing frequency of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAtm\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003eKDF/CNS-KO\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e PCs. \u003c/strong\u003eRepresentative traces of action potential discharge evoked by the injection of a 600pA depolarizing step current in wild type (blue trace) and \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e PCs (red trace) \u003cstrong\u003e(a)\u003c/strong\u003e. Distribution histograms of interspike intervals (ISI) from the wild type and \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e PCs (\u003cstrong\u003eb\u003c/strong\u003e) and their cumulative distribution (inset in b) (p \u0026lt; 0.0001, K-S test). Increased mean instantaneous frequency in \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e PCs (\u003cstrong\u003ec\u003c/strong\u003e) (p \u0026lt; 0.01, Student’s t-test). Mean coefficient of variation of ISI (\u003cstrong\u003ed\u003c/strong\u003e). \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e PCs (cells: n = 9); wild type (cells: n= 12); **p\u0026lt;0.01; ****p\u0026lt;0.0001. Results are reported as mean ± SEM.\u003c/p\u003e","description":"","filename":"MontaroloetalFig7.png","url":"https://assets-eu.researchsquare.com/files/rs-8832557/v1/0b70337dad3303f6de605447.png"},{"id":102849595,"identity":"243b28e0-227c-441e-a601-e6e9427e8cb1","added_by":"auto","created_at":"2026-02-17 14:00:42","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1033537,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIncreased intrinsic excitability of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAtm\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003eKDF/CNS-KO\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e PCs. \u003c/strong\u003eRepresentative traces of action potential firing evoked by injection of increasing depolarizing currents (\u003cstrong\u003ea\u003c/strong\u003e). Evoked firing frequency as a function of stimulation intensity (\u003cstrong\u003eb\u003c/strong\u003e) (\u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDFCNS-KO\u003c/em\u003e\u003c/sup\u003e: \u003cem\u003en\u003c/em\u003e=13 cells; wild type: \u003cem\u003en\u003c/em\u003e=10 cells). Representative action potential waveforms from wild type (blue) and \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e PCs (red) (\u003cstrong\u003ec\u003c/strong\u003e). Mean latency (\u003cstrong\u003ed\u003c/strong\u003e) action potential threshold (\u003cstrong\u003ee\u003c/strong\u003e) and AHP (\u003cstrong\u003ef\u003c/strong\u003e) of wild type (\u003cem\u003en\u003c/em\u003e=12 cells) and \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDFCNS-KO\u003c/em\u003e\u003c/sup\u003e (\u003cem\u003en\u003c/em\u003e=9 cells) PCs. **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, two way-ANOVA followed by Sidak's \u003cem\u003epost hoc\u003c/em\u003e analysis in (\u003cstrong\u003ec\u003c/strong\u003e); *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, Student's unpaired \u003cem\u003et\u003c/em\u003e-test in (\u003cstrong\u003ed-f\u003c/strong\u003e). Results are reported as mean ± SEM.\u003c/p\u003e","description":"","filename":"MontaroloetalFig8.png","url":"https://assets-eu.researchsquare.com/files/rs-8832557/v1/8c693d8372f93eb7cd651e6b.png"},{"id":102849594,"identity":"814aeddb-33a2-45d6-8375-fe384cac7192","added_by":"auto","created_at":"2026-02-17 14:00:42","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1401304,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpaired motor performance in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAtm\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003eKDFCNS-KO\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e mice. \u003c/strong\u003eMotor coordination and gait were evaluated by means of balance beam test (\u003cstrong\u003ea-c\u003c/strong\u003e), rotarod test (\u003cstrong\u003eg-h\u003c/strong\u003e), and footprinting test (\u003cstrong\u003ed-f\u003c/strong\u003e) respectively.\u003cstrong\u003e \u003c/strong\u003eSchematic representation\u003cstrong\u003e \u003c/strong\u003eof balance beam test \u003cstrong\u003e(a). \u003c/strong\u003e2 months old \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e mice (n= 37) committed more errors than wild type littermates (n= 53) traversing the bar (\u003cstrong\u003ec\u003c/strong\u003e) and the time to cross the bar was higher for \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e mice (\u003cstrong\u003eb\u003c/strong\u003e). \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e mice (n= 29) of 12 months old continued to commit more errors than wild type (n= 40) and the latency was maintained higher (\u003cstrong\u003eb-c\u003c/strong\u003e). \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e mice 24 months old have a worse phenotype. The latency and the number of errors is the highest over months (\u003cstrong\u003eb-c\u003c/strong\u003e). (***p \u0026lt; 0.001; ****p \u0026lt; 0.0001, Student’s t-test). Schematic representation\u003cstrong\u003e \u003c/strong\u003eof the footprinting test performed at 2 months of age in 2 months old mice of both genotypes\u003cstrong\u003e (d).\u003c/strong\u003e Significant reduction of the FP-HP distance in \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e mice (n = 34) compared to wild type littermates (n = 45) (\u003cstrong\u003ee)\u003c/strong\u003e. Increased coefficient of variation of FP-HP in \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e mice. (****p \u0026lt; 0.0001, Student’s t-test). Schematic representation\u003cstrong\u003e \u003c/strong\u003eof rotarod test \u003cstrong\u003e(g).\u003c/strong\u003e Time on rotating rod for wild type mice at 2 months (n=43; all comparisons \u003cem\u003eversus\u003c/em\u003e 1\u003csup\u003est\u003c/sup\u003e day, One way ANOVA RM) and for \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e mice (n=24; all comparisons \u003cem\u003eversus\u003c/em\u003e 1\u003csup\u003est\u003c/sup\u003e day, One way ANOVA RM) \u003cstrong\u003e(h\u003c/strong\u003e). Time on rotating rod for wild type mice at 12 months (n=30; all comparisons \u003cem\u003eversus\u003c/em\u003e 1\u003csup\u003est\u003c/sup\u003e day, One way ANOVA RM) and for \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e mice (n=22; all comparisons \u003cem\u003eversus\u003c/em\u003e 1\u003csup\u003est\u003c/sup\u003e day). (*p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001; ****p \u0026lt; 0.0001, Tukey’s Post hoc). Values are reported as mean ± SEM. Schematic representations were created using BioRender.\u003c/p\u003e","description":"","filename":"MontaroloetalFig9.png","url":"https://assets-eu.researchsquare.com/files/rs-8832557/v1/239a9c425dfda24526cf9ef9.png"},{"id":107479653,"identity":"f843342d-60ba-42c9-9b92-80baf3a48222","added_by":"auto","created_at":"2026-04-22 01:42:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":74737278,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8832557/v1/90bb8597-1544-4556-9a33-da50a5ac2868.pdf"},{"id":102963413,"identity":"b874faea-dbfd-4360-94e7-2c46cfce60e9","added_by":"auto","created_at":"2026-02-19 04:17:46","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":42376,"visible":true,"origin":"","legend":"","description":"","filename":"MontaroloetalSupplTableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8832557/v1/0f6f45a234a720c23e27676d.xlsx"},{"id":103049379,"identity":"c35e69ac-0d48-4cca-bb28-211e53e34e8a","added_by":"auto","created_at":"2026-02-20 07:40:29","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15272,"visible":true,"origin":"","legend":"","description":"","filename":"MontaroloetalSupplTable2.docx","url":"https://assets-eu.researchsquare.com/files/rs-8832557/v1/f3503429787977e7c596049b.docx"},{"id":102849598,"identity":"ea782915-65b6-4a68-8d3b-a94e78329959","added_by":"auto","created_at":"2026-02-17 14:00:42","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":11053492,"visible":true,"origin":"","legend":"","description":"","filename":"MontaroloetalSupplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8832557/v1/6e018c5c47ea3a52f69edda3.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"ATM-kinase deficiency triggers early multi-compartment remodeling of the cerebellar microenvironment","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAtaxia Telangiectasia (A-T) is a rare autosomal recessive disorder that is characterized by early onset, progressive cerebellar ataxia, oculocutaneous telangiectasia, immunodeficiency, and increased risk of developing cancer [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. A-T is caused by biallelic mutations in the \u003cem\u003eAtaxia Telangiectasia Mutated\u003c/em\u003e (\u003cem\u003eATM\u003c/em\u003e) gene (Chr 11q22.3-23.1) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The protein encoded by the \u003cem\u003eATM\u003c/em\u003e gene is a 370 kDa Ser/Thr kinase member of the phosphoinositide 3-kinase-related protein kinase (PIKK) family, whose members are stress-responsive kinases. ATM is activated by DNA damage, and phosphorylates its downstream targets that coordinate the DNA damage response and regulates the activation of cell cycle checkpoints in order to preserve genome integrity [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Besides these well-known roles, Atm plays an important role in the central nervous system (CNS) by modulating glutamatergic and GABAergic synapses [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], and maintaining proper homeostasis of mitochondria and peroxisomes, acting thus as a sensor for changes in the ROS levels to prevent oxidative damage [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNeuropathologically, A-T is characterized by a severe and progressive degeneration of the cerebellar cortex, with a massive loss of Purkinje cells (PCs), granule cells, and a reduction of the molecular layer thickness [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. While cerebellar cortical degeneration has long been considered the primary substrate of the neurological phenotype, increasing evidence indicates that white matter abnormalities are a consistent and clinically relevant component of A-T pathology. Neuroimaging studies in patients have reported marked cerebellar and white matter atrophy [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], along with degeneration of cortical-projecting tracts, including corticomotor, corticospinal, and somatosensory pathways [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Consistently, myelin disruption has also been observed in non-human primate model of A-T [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], further supporting the notion that impaired myelin homeostasis may represent a conserved and early feature of the disease.\u003c/p\u003e \u003cp\u003eDespite extensive research, the mechanisms linking ATM kinase deficiency to myelin defects and cerebellar circuit dysfunction remain poorly understood. Importantly, ATM deficiency affects multiple cellular processes across neurons and glial cells, raising the possibility that neurodegeneration in A-T does not arise solely from intrinsic neuronal defects [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Instead, accumulating evidence points toward cooperative mechanisms, [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], in which disrupted interactions among neurons, astrocytes, oligodendrocytes, and extracellular matrix (ECM), progressively compromise cerebellar circuit integrity and contribute to the neurological decline characteristic in A-T. Oxidative stress and DNA damage, hallmarks of ATM deficiency, are known to profoundly alter ECM composition and mechanics [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], which in turn regulate glial differentiation, myelin maintenance, and neuronal excitability [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. However, how these microenvironmental changes integrate with myelin pathology and neuronal dysfunction in A-T remains unresolved.\u003c/p\u003e \u003cp\u003eTo investigate how the loss of Atm kinase activity impacts the cerebellar circuitry microenvironment, from the earliest stages of the disease, we employed a Nestin-Cre\u0026ndash;restricted mouse model carrying a kinase-dead \u003cem\u003eAtm\u003c/em\u003e allele in combination with a null allele, resulting in selective loss of Atm kinase activity in the CNS [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. By combining unbiased proteomic profiling with structural, ultrastructural, electrophysiological, and behavioral analyses, we examined how Atm kinase deficiency reshapes the cerebellar tissue environment, with particular focus on myelin integrity, glial responses, and PC function from early stages of the disease. Our findings reveal that Atm kinase loss triggers an early, coordinated disruption of white matter homeostasis and cerebellar microenvironment stability, preceding neuronal loss and contributing to circuit dysfunction and motor impairment.\u003c/p\u003e"},{"header":"MATERIALS AD METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003e All experimental procedures have been carried out at the Neuroscience Institute Cavalieri Ottolenghi (NICO), in accordance with the European Communities Parliament and Council Directives of 24 November 1986 (86/609/EEC) and 22 September 2010 (2010/63/EU), approved by the Ethical Committee of the University of Torino and authorized by the Italian Ministry of Health (authorization number: 466/2021-PR). Mice were housed with a 12 h light/dark cycle and had free access to food/water. Adequate measures were taken to minimize pain and discomfort. Female and male mice showed the same phenotype; hence they were pooled together for all the experimental paradigms.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimal description\u003c/h3\u003e\n\u003cp\u003e \u003cb\u003eGeneration of the\u003c/b\u003e \u003cb\u003eAtm\u003c/b\u003e\u003csup\u003e\u003cb\u003eKDF\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eallele\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF\u003c/em\u003e\u003c/sup\u003e allele carrying knock-in D2880A/N2885K mutations (corresponding to D2870A/N2875K in human ATM) was generated using a strategy similar to that used for the original KD Atm allele [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. The D2870A/N2875K double mutation was selected because it has been extensively characterized and shown to support normal ATM protein expression while completely abolishing kinase activity [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Briefly, the targeting construct was designed to insert a neomycin resistance (NeoR) cassette, oriented opposite to the endogenous Atm promoter, into intron 57 adjacent to the D2880A/N2885K mutations in exon 58. The NeoR cassette was flanked by a pair of FRT sites. A 3.5-kb 5\u0026prime; homology arm and a 5.1-kb 3\u0026prime; homology arm were PCR-amplified using high-fidelity polymerase, cloned into shuttle vectors, and fully sequenced. The 5\u0026prime; arm was subcloned directly into the pEMC targeting vector [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] in the desired orientation. The D2880A/N2885K mutations were introduced into the 3\u0026prime; arm by site-directed mutagenesis and confirmed by sequencing prior to subcloning into pEMC. The finalized targeting construct was electroporated into CSL3 ES cells (129 strain), and correctly targeted clones were identified by Southern blot analysis. Initial screening was performed using KpnI and EcoRV double digestion with a 5\u0026prime; genomic probe (\u003cb\u003eSupplementary Fig.\u0026nbsp;1B-C\u003c/b\u003e), yielding a\u0026thinsp;~\u0026thinsp;13.1-kb germline band and a\u0026thinsp;~\u0026thinsp;4.7-kb targeted band due to the introduction of an additional EcoRV site. Correct targeting was further confirmed using a 3\u0026prime; probe following KpnI and EcoRV digestion, with expected band sizes of ~\u0026thinsp;13.1 kb for the germline allele and ~\u0026thinsp;10 kb for the targeted allele \u003cb\u003e(Supplementary Fig.\u0026nbsp;1B-C).\u003c/b\u003e More than 12 independently targeted ES cell clones were identified, eight of which were sequenced across exon 58; three clones were confirmed to carry the correct D2880A/N2885K mutations \u003cb\u003e(Supplementary Fig.\u0026nbsp;1D).\u003c/b\u003e Two validated clones were injected to obtain germline transmission. Resulting \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e+/KDFN\u003c/sup\u003e chimeras were crossed with \u003cem\u003eRosa26a\u003c/em\u003e\u003csup\u003e\u003cem\u003eFLIP/FLIP\u003c/em\u003e\u003c/sup\u003e mice (Jackson Laboratory, Cat. 003946) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] to induce FLP-mediated excision of the Neoᴿ cassette, generating the \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003eKDF\u003c/sup\u003e allele. In the resulting \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/KDF\u003c/em\u003e\u003c/sup\u003e mice, the kinase-dead ATM protein is expressed from the endogenous Atm promoter. A single FRT site remains in intron 57 and does not affect ATM expression.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGeneration of the\u003c/b\u003e \u003cb\u003eAtm\u003c/b\u003e\u003csup\u003e\u003cb\u003eKDF\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emurine model\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eAtm\u003c/em\u003e \u003csup\u003e+/KDF\u003c/sup\u003e mice were crossed with 'floxed' Atm alleles (\u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e) mice, containing loxP sites flanking exons 57\u0026ndash;58 of the gene (Jackson Laboratory, U.S.A). To inactivate floxed alleles only in the CNS, the \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/fl\u003c/em\u003e\u003c/sup\u003e mice were crossed with Nestin-Cre mice (Cre recombinase expressed from a transgene driven by the Nestin promoter) (Jackson Laboratory, U.S.A). Using these crossings, we have developed \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/KO\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eNestin CRE\u003c/em\u003e positive mice, which are kinase-dead Atm in one allele and knocked out in the other allele only in Nestin positive cells of the CNS, thus preventing premature mortality from cancer predisposition (\u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e). \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/fl\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCre\u003c/em\u003e negative were used as control mice.\u003c/p\u003e\n\u003ch3\u003eProteomic analysis\u003c/h3\u003e\n\u003cp\u003e2 months old mice of wild type (n\u0026thinsp;=\u0026thinsp;6) and \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e (n\u0026thinsp;=\u0026thinsp;6) mice were anesthetized with isoflurane (Isoflurane-Vet, Merial, Italy) and decapitated. The cerebella were removed, frozen in ice-cold isopentane, and stored at -80\u0026deg;C until use. Cerebella were omogenized by a blender using EasyPep MS Sample Kit (Thermo Scientific Pierce). Peptides were solubilized in 0.1% formic acid, and they were quantified through the quantitative colorimetric peptide assay (Thermo Fisher Scientific). 1.9 micrograms of each sample was injected into an UltiMate 3000 RSLC nano system coupled to the Exploris 240 mass spectrometer (Thermo Fisher Scientific) and resolved by Easy-Spray Pepmap RSLC 18 (2\u0026micro;m, 75cm \u0026times; 75\u0026micro;m) at a flow rate of 200nL/min with a gradient of phase B (80% acetonitrile/0.1% formic acid, solvent A was 0.1% formic acid in water) from 2% to 40% in 250 min. Then (B) was changed to 95% in 30 min, kept for 5 min, and then the column was re-equilibrated for 15 min. Data was acquired in a positive mode and data dependent manner. For MS1 m/z range was set to 350\u0026ndash; 1500 at 120,000 resolution (at m/z 200), AGC target 3e6, and auto maximum injection time. MS2 switch when ions intensity was above 5e3, with m/z range in auto mode, normalized HCD energy 30%, AGC target 7.5e4, and maximum injection time 40ms. The resolution was set to 15,000 at m/z 200 and the internal calibrant for employed in run start mode. Analysis was performed by five technical replicates for each sample. Raw data generated by Xcalibur 4.2 software (Thermo Fisher Scientific) were analyzed using Proteome Discoverer 2.5 (Thermo Fisher Scientific), by using SEQUEST algorithm. carbamidomethylation of Cysteines was considered as fixed modification, while as variable serine, threonine or tyrosine phosphorylation. Also, variables oxidation (M) and deamidation (N, Q) were counted. The false discovery rate was evaluated by a target-decoy strategy in concatenated q-value manner. FDR (strict) was set as 0.01, while FDR (relaxed) was set as 0.05. Differentially expressed master proteins (|log 2 FC| \u0026ge; 0.5, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were selected and individually evaluated. Fold changes, adjusted p and accension number were submitted to iPathwayGuide software using Advaita\u0026rsquo;s proprietary Impact Analysis method (Advaita Corporation Ann Arbor, MI).\u003c/p\u003e\n\u003ch3\u003eHistological procedures and image analysis\u003c/h3\u003e\n\u003cp\u003e2 months-old animals of both genotypes were anesthetized using a cocktail of zoletil (100 mg/kg body weight) and xylazine (5 mg/kg body weight) via intraperitoneal injection. The mice were intracardially perfused initially with a physiological solution (NaCl 0.9%) and then with 4% paraformaldehyde in 0.12 M phosphate buffer, pH 7.2\u0026ndash;7.4. Following perfusion, the brains were removed and stored at 4\u0026deg;C for 24 hours immersed in the same fixative. The brains were then transferred to a cryoprotectant solution made of 30% sucrose in 0.12 M phosphate buffer for few days. For each mouse, the cerebellum was separated from the telencephalon and embedded in optimal cutting temperature compound, frozen in ice-cold isopentane. Samples were stored at -80\u0026deg;C until sectioning. Cerebella and telencephalons were serially cut by a cryostat in 30 \u0026micro;m-thick sagittal and coronal slices respectively and collected in phosphate buffered saline (PBS). Histological procedures were performed on sagittal cerebellar slices of vermis and/or coronal telencephalic sections including the corpus callosum of \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e and wild type mice. At least 3 slices/animal and 3 animals/time point were analyzed. All the measurements were done blind to the mouse genotypes.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCresyl Violet Staining (Nissl Staining)\u003c/em\u003e was performed on sagittal cerebellar slices of the vermis and on coronal telencephalic sections as previously described [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], to measure the thickness of layers [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and slice and white matter area. Images were acquired by means of ZEISS Axioscan 7 microscope slide scanner (Oberkochen, Germany) with a Plan Apochromat 20X/0.8 M27 objective. Quantitative evaluations were performed on images with ImageJ software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://rsbweb.nih.gov/ij/\u003c/span\u003e\u003cspan address=\"http://rsbweb.nih.gov/ij/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e index. html). The area of the slice and white matter was obtained by drawing the outline.\u003c/p\u003e \u003cp\u003e \u003cem\u003eThe density of PCs\u003c/em\u003e was measured in anti-calbindin immunostained sections using Neurolucida software (MicroBrightField, Colchester, VT, USA) connected to an E-800 Nikon microscope under a 20x objective. Cerebellar slices were incubated overnight at 4\u0026deg;C with the polyclonal anti-rabbit calbindin (CB38a, Swant, Switzerland) antibody diluted 1:1000 in PBS with 1% TritonX-100 and 1.5% normal donkey serum. Immunohistochemical reactions were performed by the avidin\u0026ndash;biotin\u0026ndash;peroxidase method (Vectastain ABC Elite kit; Vector Laboratories, Burlingame, CA, USA) and revealed using 3,3\u0026prime;-diaminobenzidine (3% in Tris\u0026ndash;HCl) as chromogen as reported in [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. After processing, sections were mounted on microscope slides with Neo Mount (1.09016, Merck, Darmstadt, Germany). The density of PCs (expressed as number of PCs/mm) was obtained by drawing the outline of PC layer and marking the position of every labelled cell, using the Neurolucida software (MicroBrightField, Colchester, VT, USA) connected to an E-800 Nikon microscope under a 20x objective.\u003c/p\u003e \u003cp\u003e \u003cem\u003eSilver nitrate Gallyas staining\u003c/em\u003e to detect myelin was performed on sagittal cerebellar slices of vermis and on coronal telencephalic sections as previously described [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Images were acquired by means of ZEISS Axioscan 7 microscope slide scanner (Oberkochen, Germany) with a Plan Apochromat 20X/0.8 M27 objective. Quantitative evaluations were performed on images with ImageJ software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://rsbweb.nih.gov/ij/\u003c/span\u003e\u003cspan address=\"http://rsbweb.nih.gov/ij/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e index. html). To quantify the Gallyas staining, was considered the fractioned area, defined as the percentage of positive pixels relative to the total area analyzed.\u003c/p\u003e \u003cp\u003e \u003cem\u003eTo detect astrocytes and oligodendrocytes\u003c/em\u003e, immunofluorescence staining was performed as follows. Cerebellar and/or telencephalic sections were incubated overnight at 4\u0026deg;C with primary antibodies against glial fibrillary acidic protein (GFAP; rabbit polyclonal, Z033429-2, Dako, Agilent, Santa Clara, CA, USA; 1:500) and SRY-box transcription factor 10 (Sox10; rabbit polyclonal, HPA068898, Sigma-Aldrich, Darmstadt, Germany; 1:1000) to label astrocytes and oligodendrocytes, respectively. Antibodies were diluted in PBS containingf 1% Triton-X-100 and 1.5% normal donkey serum. Sections were then incubated for 2 h at room temperature with secondary anti-rabbit 647- and Cy3-conjugated antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). 4,6-diamidino-2- phenylindole (DAPI, Fluka, Saint Louis, USA) was used to counterstain cell nuclei. Finally, sections were mounted on microscope slides with Tris-glycerol mounting medium supplemented with 10% Mowiol (Calbiochem, La Jolla, CA). GFAP-immunostained images were acquired by means of ZEISS Axioscan 7 microscope slide scanner (Oberkochen, Germany) with a Plan Apochromat 20X/0.8 M27 objective. To quantify the astroglial reactivity, the expression level of GFAP+ astrocytes in the cerebellar vermis was assessed by measuring the GFAP+ fractioned area, defined as the percentage of positive pixels relative to the total area analyzed. Images of Sox10-immunostained white matter regions from the cerebellum and corpus callosum were acquired using a Leica TCS SP5 confocal microscope. Confocal images were captured as z-stacked focal planes through the thickness of the slice (30 \u0026micro;m) at 1-\u0026micro;m optical steps with an oil-immersed Plan-Apochromat 40X/1.25 objective, zoom 1.0, and resolution of 1024/1024 pixels and 100 Hz (1 pixel\u0026thinsp;=\u0026thinsp;0.38 \u0026micro;m). The density of Sox10\u0026thinsp;+\u0026thinsp;oligodendrocytes was calculated as the number of positive cells per mm\u003csup\u003e2\u003c/sup\u003e. Adobe Photoshop 6.0 (Adobe Systems, San Jose, CA, RRID:SCR_014199) was used to assemble the final figure panels.\u003c/p\u003e\n\u003ch3\u003eHigh resolution light microscopy and transmission electron microscopy\u003c/h3\u003e\n\u003cp\u003eHigh resolution light and transmission electron microscopy (TEM) were carried out as reported in [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. 2 to 4 months-old mice were anaesthetized by intraperitoneal injection with zoletil (100 mg/kg body weight) and xylazine (5 mg/kg body weight) and were perfused intracardially with 0.12 M phosphate buffer, pH 7.2\u0026ndash;7.4 followed by 2% paraformaldehyde and 2% glutaraldehyde in phosphate buffer. Brains were post-fixed overnight at 4\u0026deg;C in the same fixative. Vibratome sections (300 \u0026micro;m thick) were cut, and post-fixed with 1% osmium tetroxide for 1 h at 4\u0026deg;C, then stained with uranyl acetate replacement stain (Electron Microscopy Sciences, USA). After dehydration in ethanol, samples were cleared in propylene oxide and embedded in Araldite (Fluka, Saint Louis, USA). Semithin sections (1 \u0026micro;m thick) were obtained using an ultramicrotome (Ultracut UCT, Leica, Wetzlar, Germany), stained with 1% toluidine blue and 2% borate in distilled water, and then observed under a light microscope to ensure accurate localization of the corpus callosum. For each sample, bright-field images of the medial corpus callosum were acquired at 100x magnification a Nikon Eclipse 80i microscope. Axon density was quantified using ImageJ software. Each image was overlaid with a grid (via the Grid tool), and eight non-overlapping regions were randomly selected. Axons were manually counted within each square, and axon density was calculated as the number of axons \u0026micro;m\u003csup\u003e2\u003c/sup\u003e. For each sample, the mean axon density was obtained by averaging values from the eight 8 squares. The obtained ultrathin sections (70\u0026ndash;100 nm) were examined by TEM and STEM. The G-ratio (inner perimeter/outer perimeter), axon diameter, and myelin thickness were determined by using a JEOL, JEM-1400Flash, (Tokyo, Japan) operated at 80 kV, and equipped with a Mega-View-III digital camera and a Soft-Imaging-System (SIS, M\u0026uuml;nster, Germany). The recorded images were elaborated and measured by using the ImageJ software [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The quantification of G-ratio and axon diameter was performed on at least 50 axons/animal and on 4\u0026ndash;5 mice per genotype.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eProtein analysis\u003c/h2\u003e \u003cp\u003eProtein analysis was performed as previously described [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Mice were anesthetized with isoflurane (Isoflurane-Vet, Merial, Italy) and decapitated. The cerebella were removed, frozen in ice-cold isopentane, and stored at -80\u0026deg;C until use. Cerebella from wild type and \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e mice were resuspended in 20% (w/v) RIPA buffer (25 mM Tris\u0026ndash;HCl pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, 0.5 mM PMSF, 10 \u0026micro;g/ml Aprotinine, 10 \u0026micro;g/ml Leupeptine, 2 mM sodium orthovanadate), and homogenized with a tissue lyser. The lysates were centrifuged at 10,000 g for 20 min at 4\u0026deg;C and the supernatant was collected and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until use. Twenty micrograms of proteins were separated by using a 4\u0026ndash;12% Bis-Tris precast gel (Life Technologies) and transferred onto nitrocellulose membrane (GE‐Healthcare). Membranes were than blocked with 50 g/L (5%) nonfat dry milk (Bio‐Rad) in 50 mM Tris\u0026ndash;HCl pH7.4, containing 200 mM NaCl and 0.5 mM Tween‐20 and then incubated overnight at 4\u0026deg;C with primary antibodies. The following primary antibodies were used: myelin basic protein (MBP) (1:1000, Covance, Cat# SMI‐99P‐500, RRID: AB_10120130), and Gapdh (1:1000, Abcam, Cat# ab181602, RRID: AB_2630358). HRP‐conjugated goat anti‐mouse (1:5000, Bio‐Rad, Cat# 170\u0026ndash;6516, RRID: AB_11125547) and goat anti‐rabbit (1:5000, Bio‐Rad, 170\u0026ndash;6515, RRID: AB_11125142) immunoglobulins, in Tris‐buffered saline Tween containing 20 g/L non‐fat dry milk, were used for detection with Luminata Forte Western substrate (WBLUF0100, Millipore, Darmstadt, Germany). Densitometric values were normalized to gapdh. Images were acquired by Chemidoc (Bio‐Rad) and quantified by ImageLab software (RRID: SCR_014210, Bio‐Rad). The protein extracts were run at least three times to check reproducibility.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePurkinje cell dark cell degeneration assessment\u003c/h3\u003e\n\u003cp\u003e2 to 4 months-old mice were anaesthetized by intraperitoneal injection with zoletil (100 mg/kg body weight) and xylazine (5 mg/kg body weight). They were subsequently fixed by transcardiac perfusion with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3). Sagittal sections of the cerebellum (500 \u0026micro;m thick) were cut using a vibratome and post-fixed by incubation for 2 hours in 1% (wt/vol) OsO4 supplemented with 1.5% (wt/vol) potassium ferrocyanide. The sections were then dehydrated in a graded ethanol series (30% to 100%, 5 minutes per step), followed by two 10-minute passages in propylene oxide and 1 hour in a 1:1 mixture of propylene oxide and Epon resin. Finally, the samples were embedded in resin. Semithin sections (1 \u0026micro;m thick) were prepared using an ultramicrotome (Ultracut UCT, Leica, Wetzlar, Germany) and stained with 1% toluidine blue and 2% borate in distilled water and mounted onto glass cover slips for morphological assessment and quantitation via standard light microscopy [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Dark cell degeneration (DCD) was verified by the appearance of a minimum of two of the following features: cytoplasmic darkening, soma shrinkage, and nuclear darkening/chromatin aggregation [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eElectrophysiology\u003c/h3\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSlices preparation\u003c/h2\u003e \u003cp\u003eCerebellar slices were prepared as previously described [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The animals were anesthetized with isoflurane (Isoflurane-Vet, Merial, Italy) and decapitated. The cerebellar vermis was removed and transferred to an ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM); 125 NaCl, 2.5 KCl, 2 CaCl\u003csub\u003e2\u003c/sub\u003e, 1 MgCl\u003csub\u003e2\u003c/sub\u003e, 1.25 NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 26 NaHCO\u003csub\u003e3\u003c/sub\u003e, 20 glucose, which was bubbled with 95% O\u003csub\u003e2\u003c/sub\u003e/5% CO\u003csub\u003e2\u003c/sub\u003e (pH 7.4). Parasagittal cerebellar slices (200 \u0026micro;m thickness) were obtained using a vibratome (Leica Microsystems GmbH, Wetzlar, Germany) and kept for 1 h at 35\u0026deg;C and then at 31\u0026deg;C. Single slices were placed in the recording chamber, which was perfused at a rate of 2\u0026ndash;3 ml/min with ACSF bubbled with the 95% O\u003csub\u003e2\u003c/sub\u003e/5% CO\u003csub\u003e2\u003c/sub\u003e. All recordings were performed at 31\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C temperature.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eElectrophysiological recordings\u003c/h2\u003e \u003cp\u003eRecordings from PCs were performed by an EPC-10 patch‐clamp amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany). PCs were held at \u0026ndash; 70 mV, and data were filtered at 9.1 kHz and sampled at 20 kHz. For current clamp recordings, patch pipettes were filled with a K-gluconate-based internal solution containing (in mM): 140 K-gluconate, 10 HEPES, 0.5 EGTA, 4 MgCl\u003csub\u003e2\u003c/sub\u003e, 4 Na\u003csub\u003e2\u003c/sub\u003eATP, 0.4 Na\u003csub\u003e3\u003c/sub\u003eGTP and the pH was adjusted to 7.3 with KOH and filtered at 0.2 \u0026micro;m. Hyperpolarizing (form \u0026minus;\u0026thinsp;400pA to -100 pA) and depolarizing (from +\u0026thinsp;100 to +\u0026thinsp;1000 pA) current steps in increments of 100 pA, each lasting 1000 ms and a step interval of 10 s were delivered to PCs. For cell-attached recordings the pipette was filled with NaCl 0.9% solution filtered at 0.2 \u0026micro;m. Gabazine (SR 95531, 20 \u0026micro;M), DAP5 (50 \u0026micro;M) and NBQX (10 \u0026micro;M) were added to the recording chamber to inhibit the GABAA and ionotropic glutamate receptors of PCs, respectively. Data were analyzed using Axograph software (AxoGraph Scientific, Sydney, Australia) and graphs were designed using Igor Pro (Wavemetrics, Lake Oswego, Oregon, USA). Data have been derived from at least three animals per genotype.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDrugs\u003c/h2\u003e \u003cp\u003eAll drugs were purchased from HelloBio (Bristol, UK) and were applied via the chamber perfusion line.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMotor Tests\u003c/h2\u003e \u003cp\u003eWe performed a series of motor tests to evaluate the presence of motor impairment on \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e mice. All the experiments were conducted in the morning and with the same light conditions for all animals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eBalance Beam test\u003c/h2\u003e \u003cp\u003eWe performed balance beam test to evaluate balance and motor coordination on 2, 12, and 24 months old \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e and wild type mice [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. We used a metal beam 1 cm wide, and 100 cm long suspended 12 cm above the bench. For 24-months old animals, a wider beam (1.5 cm) was used. The mice had to cross the beam to reach a cage enriched with toys. The day of the test was preceded by three days of habituation to allow the mice to familiarize with the experimental apparatus. On the day of the test, the animals were placed to acclimate in the behavioral room at least 15 minutes before the experiment. The test consisted of three bar crossings for three consecutive days. We recorded the crossings using a video camera and analyzed offline by an operator blind to the genotype. The performance was assessed by measuring the latency required to cross the beam as well as the number of slips. The data are presented as violin plot (median and 25th to 75th percentiles are shown).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eFootprint Analysis\u003c/h2\u003e \u003cp\u003eThe footprinting test was used to analyze gait parameters. The test was performed as previously described [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. We used a transparent Plexiglass walkway (20 cm high, 67 cm long and 4 cm wide) elevated 70 cm from the floor. A digital camera was placed underneath the clear platform and video recordings of the mice walking were collected. The mice had to walk at least for three consecutive steps per crossing. Still-frames from the recordings were extracted and analyzed offline using the ImageJ software in order to obtain data concerning stride length (the distance of the same paw in consecutive steps), width (the distance between the center of the two hind or fore paws), the distance between ipsilateral fore paw (FP) and hind paw (HP) placements and the fore and hind stance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAccelerated rotarod test\u003c/h2\u003e \u003cp\u003eWe assessed the locomotor function using the accelerated rotarod test as previously described [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. We tested mice for five consecutive days and then again 5 days later (10th day). This protocol was repeated at different ages, at 2, 12, and 24 months, to evaluate long term retention of the motor improvements. In each day, after 1 minute training session at constant speed (4 rpm), mice received three test sessions (with a minute interval between sessions) in which the rod (Mouse Rota-Rod, Ugo Basile Biological Research Apparatus, Comerio, Italy) accelerated continuously from 4 to 65 rpm with an acceleration of 5.5 rpm. The latency to fall off the rod was recorded. The cutoff in this experiment was set to 300 seconds.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. The Shapiro-Wilk test was used to check whether the data followed a normal distribution. For the data sets that passed the normality test, comparisons were done with unpaired two tailed Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test or by one-way or two-way analyses of variance (ANOVA), followed by the appropriate \u003cem\u003epost hoc\u003c/em\u003e correction. Data for which the normality test failed, were compared by the Mann-Whitney u-test or by Kruskal-Wallis test. \u003cem\u003eP\u003c/em\u003e values lesser than 0.05 was accepted as significant. Correlation analysis between proteomic protein expression levels and motor performance was performed using SPSS (IBM SPSS Statistics, version 7). Spearman\u0026rsquo;s rank correlation coefficient was applied to identify proteins significantly associated with motor outcomes. Proteins showing significant correlations were further analyzed using STRING for protein\u0026ndash;protein interaction network construction and functional enrichment analysis. Network outputs were subsequently redrawn and customized using in-house Python scripts.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eAtm\u003c/b\u003e \u003csup\u003e \u003cb\u003eKDF\u003c/b\u003e \u003c/sup\u003e \u003cb\u003emurine model\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOur initial \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKD\u003c/em\u003e\u003c/sup\u003e allele with knocking in D2880A/N2885K (corresponding to D2870A/N2875K in humans) double-mutation into the conserved catalytic loop also contains a single loxP site after the Cre-mediated deletion of the Neo resistance cassette (NeoR) [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Using this KD allele, we successfully demonstrated accelerated oncogenesis using an early and robust Vav-Cre allele, leading to aggressive immature T cell lymphomas [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. But in the slow progressive B cell lymphoma model or in neuronal models as described here, a rare recombination between the residual loxP site in the KD allele and the loxP sites in the Atm conditional/null allele [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] leads to inactivation (null) of both alleles \u003cb\u003e(Supplementary.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e and effectively creates a null model. To avoid this, we have generated a new \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF\u003c/em\u003e\u003c/sup\u003e allele (termed KDF for FRT), in which the LoxP sites flanked NeoR were replaced by a pair of FRT sites (\u003cb\u003eSupplementary Fig.\u0026nbsp;1B\u003c/b\u003e). Therefore, after the neo-deletion, only an FRT is left (not a loxP) that would not recombine with the loxP on the conditional allele (\u003cb\u003eSupplementary Fig.\u0026nbsp;1A-B\u003c/b\u003e). Southern blot analyses and the Sanger sequencing validate the correct targeting and the presence of the double mutations (\u003cb\u003eSupplementary Fig.\u0026nbsp;1C-D\u003c/b\u003e). Using the Vav-Cre that expresses in all hematopoietic lineages, we confirmed that the newly generated \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF\u003c/em\u003e\u003c/sup\u003e allele conferred no ATM kinase activity, evidenced by reduced surface TCRβ-positive mature thymocytes and decreased Immunoglobulin class switch recombination at levels comparable to the \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKD\u003c/em\u003e\u003c/sup\u003e alleles [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] (\u003cb\u003eSupplementary Fig.\u0026nbsp;1E)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDifferentially expressed proteins in the cerebellum of\u003c/b\u003e \u003cb\u003eAtm\u003c/b\u003e\u003csup\u003e\u003cb\u003eKDF/CNS\u0026minus;KO\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo identify molecular alterations induced by Atm kinase deficiency, we performed proteomic analysis of whole-cerebellum lysates from \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e mice and wild type littermates. A total of 8302 master proteins were identified, revealing a profound disruption of cerebellar protein homeostasis in \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e mice. Global differential expression analysis demonstrated a widespread molecular phenotype associated with Atm kinase deficiency, as illustrated by the volcano plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). We identified 299 differentially expressed proteins: 165 proteins were upregulated, and 134 downregulated in \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e mice compared to wild type littermates, with |log2(Fold-change)| \u0026ge;0.5 and statistical significance set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, revealing a coordinated disruption of multiple cellular compartments (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Hierarchical clustering analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) based on the Z-score distribution demonstrates a clear-cut proteomic signature that distinguishes \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e replicates from the wild type littermates, indicating high reproducibility of the molecular phenotype and supporting the existence of a consistent cerebellar proteomic remodeling induced by Atm deficiency. KEGG pathway enrichment analysis revealed a significant enrichment of cellular processes, metabolic, and signaling pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Importantly, KEGG enrichment analyses identified extracellular matrix (ECM)-receptor interaction and focal adhesion pathways, indicating early disruptions in the ECM stability and matrix-cell signaling in the cerebellum of \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). These findings highlight a broad disruption of the cellular homeostasis in the cerebellum of \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e \u003cp\u003eConsistently, Gene Ontology (GO) analysis of the upregulated proteome revealed a strong enrichment of basement membrane and interstitial ECM components together with astrocyte- and immune-related processes (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, \u003cb\u003etop\u003c/b\u003e). In contrast, the downregulated proteome was dominated by pathways related to myelination, oligodendrocyte differentiation, axon ensheathment, and neuronal communication (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, \u003cb\u003ebottom\u003c/b\u003e), suggesting a coordinated impairment of white matter integrity and axon-glia interactions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAtm\u003c/b\u003e \u003csup\u003e \u003cb\u003eKDF/CNS\u0026minus;KO\u003c/b\u003e \u003c/sup\u003e \u003cb\u003emice display myelination defects\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGiven the strong enrichment of ECM, glial and myelin-related pathways, we next asked whether these molecular changes corresponded to structural and ultrastructural alterations. We examined whether the cerebellar white matter was affected in \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e mice. Despite the normal foliation, overall cytoarchitecture, and preserved thickness of the molecular and granular layers (\u003cb\u003eSupplementary Fig.\u0026nbsp;2\u003c/b\u003e), \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e mice showed a significant reduction in the cerebellar slice area compared to wild type controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Unpaired Student\u0026rsquo;s t-test: t\u003csub\u003e(9)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.40) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c). This reduction was primarily driven by with a significant shrinkage of the cerebellar white matter area (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Mann-Whitney test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Moreover, the white matter are/slice area ratio was significantly reduced in \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Mann-Whitney test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). To directly assess myelin integrity, we performed Gallyas staining on cerebellar sections (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). The analysis revealed a marked reduction of myelin staining in \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e mice compared to control animal (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Unpaired Student\u0026rsquo;s t-test: t\u003csub\u003e(12)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.73; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). In parallel with the structural analyses, we assessed the expression level of MBP, the most abundantly expressed myelin protein in the CNS, by performing western blotting of cerebellar extracts. Quantitative analyses revealed a significant reduction in both isoforms of Mbp in \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e mice compared to wild type littermates (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Unpaired Student\u0026rsquo;s t-test; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), supporting the presence of an early hypomyelinating phenotype.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine whether myelin abnormalities were restricted to the cerebellum, we analyzed the corpus callosum (CC), the largest white matter tract in the brain. \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e mice showed a significant reduction in CC thickness compared to wild type littermates (7.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37 vs 6.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32 \u0026micro;m, respectively, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Unpaired Student\u0026rsquo;s t-test: t\u003csub\u003e(7)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.03). Gallyas staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, f\u003cb\u003e)\u003c/b\u003e revealed a significant reduction of the myelin content within the CC (cortex: p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Mann-Whitney test; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg; cortex+corpus callosum: p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Unpaired Student\u0026rsquo;s t-test: t\u003csub\u003e(12)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.17; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh), together with a patchy appearance of the myelin in the overlying cortical regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, g), indicating that white matter pathology in \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e mice is not confined to the cerebellum. To determine whether reduced myelin staining reflected axonal loss or primary myelin defects, we analyzed semithin and ultrastructural sections of the corpus callosum. Quantification of toluidine blue-stained CC sections revealed comparable axonal densities between genotypes, indicating that Atm kinase deficiency does not affect axon number at this stage (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Unpaired Student\u0026rsquo;s t-test; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-c). TEM revealed several abnormalities in myelin ultrastructure in \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, e). We evaluated myelin thickness by calculating the g-ratio (myelin thickness relative to axon diameter), defined as the ratio between the inner axonal diameter and the total diameter of the myelinated fiber. Cumulative frequency analysis revealed a significant rightward shift in the g-ratio distribution in \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e mice, consistent with thinner myelin sheaths (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Mann-Whitney test; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). Two-way ANOVA analysis of g-ratio as a function of axonal diameter showed a significant main effect of genotype (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Two-way ANOVA: genotype effect, F\u003csub\u003e(1, 472)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;21.38; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg), with Sidak\u0026rsquo;s \u003cem\u003epost hoc\u003c/em\u003e multiple comparisons indicating a selective increase in g-ratio in medium-caliber axons in \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e mice compared to wild type littermates (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg\u003cb\u003e)\u003c/b\u003e. Consistent with these findings, direct measurements of myelin thickness confirmed a significant reduction in \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Mann-Whitney test; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). Linear regression analysis of myelin thickness across all axon diameters revealed a significant reduction of the intercept in \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) without a significant change in slope, indicating a global reduction in myelin thickness rather than defective scaling with axon caliber (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei\u003cb\u003e).\u003c/b\u003e To assess whether the reduction in myelin resulted from oligodendrocyte depletion, we quantified cells expressing the transcription factor Sox10, a marker of the oligodendrocyte lineage across all stages of their development. Notably, the density of Sox10-positive cells in both the cerebellum and corpus callosum was comparable between genotypes \u003cb\u003e(Supplementary Fig.\u0026nbsp;3)\u003c/b\u003e, indicating that myelin defects occur in the absence of oligodendrocyte loss and suggesting impaired myelin maintenance rather than reduced cell number.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAtm\u003c/b\u003e \u003csup\u003e \u003cb\u003eKDF/CNS\u0026minus;KO\u003c/b\u003e \u003c/sup\u003e \u003cb\u003emice show cerebellar astroglial reactivity\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGiven the prominent white matter abnormalities, we next investigated whether Atm kinase deficiency was associated with astroglial activation, a hallmark of disrupted neuron\u0026ndash;glia interactions in cerebellar degeneration [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Immunofluorescence of cerebellar slices with GFAP, a canonical indicator of the astrocyte activation, revealed a robust increase in astrogliosis in \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e mice across all cerebellar regions examined (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-d). Specifically, the percentage area covered by GFAP was higher in the molecular layer (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Mann-Whitney test), granular layer (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Mann-Whitney test), and white matter area (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Mann-Whitney test). Similarly, GFAP mean intensity was significantly increased in the molecular layer (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Mann-Whitney test), granular layer (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Unpaired Student\u0026rsquo;s t-test: t\u003csub\u003e(14)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;8.94), and white matter (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Mann-Whitney test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee-g). These findings indicate a widespread astrocytes activation affecting both gray and white matter regions of the cerebellum.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAtm\u003c/b\u003e \u003csup\u003e \u003cb\u003eKDF/CNS\u0026minus;KO\u003c/b\u003e \u003c/sup\u003e \u003cb\u003emice show early signs of Purkinje cell degeneration\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWhite matter disruption and astroglial reactivity are known to alter ionic homeostasis and axonal conduction, potentially increasing the vulnerability of neuronal cells [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. To assess whether PCs were affected at early stages of the disease, we first quantified PC density on calbindin-stained cerebellar sections. At 2 months of age, PC density was comparable between genotypes, indicating the absence of neuronal cell loss at this stage (wild type: 28.69\u0026thinsp;\u0026plusmn;\u0026thinsp;1.35 cells/mm vs \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e: 28.26\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14 cells/mm, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Unpaired Student\u0026rsquo;s t-test: t\u003csub\u003e(16)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.24). We next investigated whether PCs exhibited early degenerative changes by assessing DCD [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. DCD is a type of neuronal death characterized by a shrunken, electron-dense appearance of the cell body and nucleus [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. While no DCD PCs were detected in wild type mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-b\u003cb\u003e)\u003c/b\u003e, \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e mice displayed multiple PCs with DCD-like morphology \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec-f\u003cb\u003e).\u003c/b\u003e The presence of DCD PCs in the absence of any exogenous insult suggests an increased intrinsic vulnerability of \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e PCs to degeneration compared to wild type ones.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePurkinje cells exhibit early hyperexcitable phenotype in\u003c/b\u003e \u003cb\u003eAtm\u003c/b\u003e\u003csup\u003e\u003cb\u003eKDF/CNS\u0026minus;KO\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine whether increased vulnerability of PCs was associated with altered intrinsic excitability, we performed electrophysiological recordings from PCs in acute cerebellar slices. Analysis of spontaneous firing recorded in cell-attached mode revealed comparable interspike interval and coefficient of variation between genotypes \u003cb\u003e(Supplementary Fig.\u0026nbsp;4)\u003c/b\u003e. In contrast, analysis of evoked firing, recorded in whole-cell current clamp configuration, revealed marked differences between \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e and wild type PCs. We performed the analysis of evoked action potential discharge by applying a fixed depolarizing current step (600 pA) and found a significant shift to the left in the distribution of interspike intervals (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, K-S test) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea-c\u003cb\u003e)\u003c/b\u003e, an increase of the instantaneous frequency (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Unpaired Student\u0026rsquo;s t-test) without significant changes, between genotypes, in the coefficient of variation (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Unpaired Student\u0026rsquo;s t-test) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e. When PCs were challenged with progressively increasing depolarizing current steps, \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e PCs displayed a significant higher number of action potentials compared to wild type mice across stimulus intensities (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Two-way ANOVA: genotype effect, F\u003csub\u003e(1, 21)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;10.34) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea, b). Consistent with a hyperexcitable phenotype, the latency to the first action potential was significantly shorter for \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e PCs compared to wild type littermates (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Unpaired Student\u0026rsquo;s t-test: t\u003csub\u003e(17)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.22) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed). The analysis of the features of the first action potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec) revealed a significant reduction of the threshold (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Unpaired Student\u0026rsquo;s t-test: t\u003csub\u003e(19)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.44) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed) and of the after hyperpolarization (AHP) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Unpaired Student\u0026rsquo;s t-test: t\u003csub\u003e(19)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.04) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ef). Overall, our analysis demonstrates that \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e PCs present an early increase in intrinsic excitability compared to wild type mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEarly motor coordination impairment and gait disturbance in\u003c/b\u003e \u003cb\u003eAtm\u003c/b\u003e\u003csup\u003e\u003cb\u003eKDF/CNS\u0026minus;KO\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine whether the structural and functional cerebellar alterations at early stages translated into motor impairment, we assessed motor coordination and gait across disease progression, starting at 2 months of age, an early phase of the disease, and repeating the analysis at 12 and 24 months to monitoring the progressive worsening of the phenotype over time. The balance beam test is very effective in detecting very slight deficits in both motor coordination and balance \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. At 2 months of age, \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e mice already displayed impaired motor performance in the balance beam task, characterized by increased crossing latency (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Two-way ANOVA: genotype effect, F\u003csub\u003e(1, 114)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;61.49); for 2 months old mice: (wild type: 4.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27; \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e: 7.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55), for 12 months old: (wild type: 5.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39; \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e: 6.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60), and for 24 months old: (wild type: 8.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77; \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e: 15.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.37) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. The number of slips was higher compared to controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Two-way ANOVA: genotype effect, F\u003csub\u003e(1, 185)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;134.4); for 2 months old mice (wild type: 0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05; \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e: 1.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29), for 12 months old: (wild type: 0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08; \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e: 1.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32) and for 24 months old: (wild type: 1.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35; \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e: 5.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e. These deficits progressively worsened with age, indicating a gradual deterioration of motor coordination (genotype per age interaction effect: for latency, F\u003csub\u003e(2, 72)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;9.97; for number of slips, F\u003csub\u003e(2, 185)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;20.53).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe performed the footprinting test to analyze the gait characteristics in mice of both genotypes at 2 months of age. Our analysis indicated a significant reduction of the FP-HP placement (wild type: 1.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 cm and \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e: 0.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 cm; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Unpaired Student\u0026rsquo;s t-test) as well as an increased coefficient of variation of FP-HP placement (wild type: 0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 and \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e: 0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Unpaired Student\u0026rsquo;s t-test) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ed-f\u003cb\u003e).\u003c/b\u003e No significant changes between genotypes were observed for the other gait parameters and their coefficient of variation (for stride length: wild type: 5.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 cm and \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e: 6.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09; for fore limb width: wild type: 3.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 cm and \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e: 3.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05; for hind limb width: wild type: 3.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 cm and \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e : 3.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 cm; for each gait parameter: p\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Unpaired Student\u0026rsquo;s t-test).\u003c/p\u003e \u003cp\u003eBy means of accelerated rotarod test we evaluated the motor learning and memory in \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e and wild type mice. Two-way ANOVA repeated measures revealed a significant time x genotype interaction effect (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; F(5, 335)\u0026thinsp;=\u0026thinsp;2.72) suggesting a different response of genotypes over time. Indeed, the learning over days of \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e mice was delayed compared to wild type mice (2nd day \u003cem\u003evs\u003c/em\u003e 1st day; one way ANOVA repeated measures, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05 Tukey\u0026rsquo;s \u003cem\u003ePost hoc\u003c/em\u003e). Moreover, the 10th day \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e mice did not have the retention of the memory (10th day \u003cem\u003evs\u003c/em\u003e 1st day; one-way ANOVA repeated measures, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05 Tukey\u0026rsquo;s \u003cem\u003ePost hoc\u003c/em\u003e) compared to wild type littermates \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eg, h\u003cb\u003e)\u003c/b\u003e. At 12 months of age \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e mice could not learn the task over days compared to wild type littermates and they reached a plateau from day 2 to 10 (one way ANOVA repeated measures, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eh\u003cb\u003e)\u003c/b\u003e. This indicates a progressive deficit of motor learning in \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e mice. The absolute time on the rotating rod is influenced by the body mouse weight while the motor learning is not dependent on it. We found a significant reduction in body weight of \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e compared to wild type mice at each age point under analysis: for 2 months old mice: wild type (23.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40 g) and \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e (20.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46 g); p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Mann-Whitney test), for 12 months old: wild type (37.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26 g) and \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e (31.01\u0026thinsp;\u0026plusmn;\u0026thinsp;1.40 g); p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Unpaired Student\u0026rsquo;s t-test), and for 24 months old: wild type (34.34\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33 g) and \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e (29.23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.73 g); p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Unpaired Student\u0026rsquo;s t-test) which might explain the slightly higher time on the rotating rod.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, we show that loss of Atm kinase activity in the CNS induces an early and coordinated remodeling of the cerebellar microenvironment without overt neuronal loss. By combining proteomic, structural, ultrastructural, electrophysiological and behavioral analysis, we demonstrate that Atm kinase deficiency is associated with ECM alterations, astroglial reactivity, impaired white matter homeostasis, increased PC vulnerability, and early circuit dysfunction. Together, these data indicate that cerebellar pathology in \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e mice emerges from a destabilization of the tissue environment that compromises circuit integrity at early stages of the disease.\u003c/p\u003e \u003cp\u003eOur proteomic profiling revealed broad alteration across four major pathways in \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e mice: \u003cem\u003ei)\u003c/em\u003e disorganization of the ECM, \u003cem\u003eii)\u003c/em\u003e astrocytosis, iii) downregulation of myelin/oligodendroglial lineage proteins and \u003cem\u003eiv)\u003c/em\u003e alteration in neuronal excitability and synaptic organization. These findings indicate a global destabilization of cerebellar homeostasis rather than isolated molecular changes.\u003c/p\u003e \u003cp\u003eA central finding of our study is an early and extensive remodeling of the ECM in the cerebellum of \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e mice. The ECM remodeling is a well-recognized consequence of oxidative stress and DNA damage, hallmark features of A-T, through their effects on ECM synthesis, turnover, and stabilization [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The upregulation of the components of the basement membrane and the interstitial matrix suggests that ECM could be biochemically remodeled toward greater stiffness and rigidity. Such changes are known to aggravate oxidative stress and DNA damage [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], stressors particularly detrimental in the absence of ATM. A properly regulated ECM is required throughout development and remains crucial in adulthood. It preserves network function by regulating cell survival, proliferation, migration and differentiation. Furthermore, it modulates synaptic plasticity and provides structural anchorage for both neuronal and glial processes [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough ECM is secreted by both neurons and glial cells, astrocytes and microglia are the primary regulators of ECM remodeling [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Accordingly, we observed increased expression of astrocytic and astrocyte endfoot markers, indicative of reactive astrogliosis and vascular endfoot remodeling, consistent with the profound ECM reorganization. The diffused astrocytosis in the cerebellar cortex of \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e mice likely contributes to ECM remodeling, although ECM alterations can themselves activate astrocytes [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This reciprocal influence suggests a self-reinforcing cycle in which mechanical and biochemical changes of the ECM and astrocytic reactivity perpetuate each other, progressively destabilizing the cerebellar microenvironment.\u003c/p\u003e \u003cp\u003eThe proteomic profile also revealed a coordinated downregulation of multiple myelin and oligodendrocyte lineage proteins in the cerebellum of \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e mice. This pattern is consistent with a failure of the oligodendrocyte lineage to mature and sustain myelination. Structural and ultrastructural analyses confirmed a marked reduction of myelination in cerebellum and corpus callosum despite preserved axon and oligodendrocyte numbers, supporting a defect in myelin maintenance rather than cell loss. Oxidative stress and DNA damage are known to impair the oligodendrocyte lineage and Atm deficiency may exacerbate this vulnerability [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. However, the myelin deficits occur in the context of a profoundly remodeled and stiffened ECM and reactive astrocytosis, both conditions that are known to create a non-permissive environment for the correct oligodendrocyte lineage development and myelination [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Thus, the myelin phenotype likely reflects both cell-autonomous vulnerability and non-cell-autonomous constraints imposed by the remodeled microenvironment.\u003c/p\u003e \u003cp\u003eThe combination of stiffened ECM, astrogliosis and defective myelination in \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e mice is associated with PCs exhibiting DCD and increased intrinsic excitability, indicating that these neurons are highly vulnerable in early stages of the disease. The influence of astrocytes, ECM and oligodendrocytes on neuronal excitability and synaptic function is well established [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Moreover, the relationship between myelin and excitability is bidirectional: neuronal activity regulates myelination and myelin integrity modulates neuronal firing properties [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Thus, the altered excitability of PCs likely reflects the cumulative impact of microenvironment instability. Although most ataxias are characterized by reduced PC excitability [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], the increased activity observed in \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS-KO\u003c/em\u003e\u003c/sup\u003e mice likely represents an early and potentially transient compensatory response aimed at maintaining cerebellar output in the face of widespread microenvironmental disruption. Such compensation is likely unsustainable, ultimately contributing to circuit failure and motor impairment even in the absence of PC loss.\u003c/p\u003e \u003cp\u003eIn conclusion, although our data provide a comprehensive view of the molecular, structural, and functional alterations in the cerebellum of \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e mice, they do not allow us to infer a temporal sequence or to identify a single primary causal event. Instead, our findings support a coordinated disruption across multiple cellular compartments, both intra- and extracellular, suggesting that Atm kinase deficiency alters cerebellar homeostasis through a network level mechanism rather than a single pathway. The simultaneous upregulation of basement-membrane and interstitial ECM proteins, the downregulation of myelin and oligodendrocyte maturation markers, the activation of astrocytic pathways and the increased excitability of PCs point to a multifaceted microenvironmental imbalance in which these processes likely interact bidirectionally. In this context, ECM remodeling may impair oligodendrocyte maturation; dysfunctional oligodendrocytes may trigger astrocytic activation; reactive astrocytes may further reshape the ECM; or early neuronal alterations may secondarily affect myelination and glial reactivity. Thus, while causality cannot be assigned to any single component, the convergence of these alterations strongly supports a model in which ATM is a master regulator of cerebellar microenvironment stability, and identify ECM-glia-neuron interaction as early therapeutic target in A-T.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures have been carried out in accordance with the European Communities Parliament and Council Directives of 24 November 1986 (86/609/EEC) and 22 September 2010 (2010/63/EU), approved by the Ethical Committee of the University of Torino and authorized by the Italian Ministry of Health (authorization number: 466/2021-PR).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are available either within the paper or from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll other authors declare they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was funded by Associazione Nazionale Atassia Telangiectasia (ANAT), Fondazione Cassa di Risparmio di Torino (CRT Foundation), Banca d’Italia, a local grant of the University of Torino (2021), and the Department of Excellence funding from the Ministry of University and Research (MUR) for 2023–2027, awarded to the Department of Neuroscience “Rita Levi Montalcini” (University of Turin).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contribution:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFM and EH performed and supervised the experiments and data analysis and wrote the manuscript. FM, LB, AMR, ML, IB, AR, GPS, GM, RP, KY, and EH performed the experiments and data analysis. FM, MM, FT, RV, AB, SZ and EH contributed to the conception of the work and to the discussion of the results. All authors approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBakkenist CJ, Kastan MB (2003) DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 421:499\u0026ndash;506. doi: 10.1038/nature01368\u003c/li\u003e\n \u003cli\u003eBoder E, Sedgwick RP (1958) Ataxia-telangiectasia; a familial syndrome of progressive cerebellar ataxia, oculocutaneous telangiectasia and frequent pulmonary infection. Pediatrics 21:526\u0026ndash;554\u003c/li\u003e\n \u003cli\u003eBorghesani PR, Alt FW, Bottaro A, Davidson L, Aksoy S, Rathbun GA, Roberts TM, Swat W, Segal RA, Gu Y (2000) Abnormal development of Purkinje cells and lymphocytes in \u003cem\u003eAtm\u003c/em\u003e mutant mice. Proc Natl Acad Sci USA 97:3336\u0026ndash;3341. doi: 10.1073/pnas.97.7.3336\u003c/li\u003e\n \u003cli\u003eCanman CE, Lim D-S, Cimprich KA, Taya Y, Tamai K, Sakaguchi K, Appella E, Kastan MB, Siliciano JD (1998) Activation of the ATM Kinase by Ionizing Radiation and Phosphorylation of p53. Science 281:1677\u0026ndash;1679. doi: 10.1126/science.281.5383.1677\u003c/li\u003e\n \u003cli\u003eCerrato V (2020) Cerebellar Astrocytes: Much More Than Passive Bystanders In Ataxia Pathophysiology. JCM 9:757. doi: 10.3390/jcm9030757\u003c/li\u003e\n \u003cli\u003eCho S, Vashisth M, Abbas A, Majkut S, Vogel K, Xia Y, Ivanovska IL, Irianto J, Tewari M, Zhu K, Tichy ED, Mourkioti F, Tang H-Y, Greenberg RA, Prosser BL, Discher DE (2019) Mechanosensing by the Lamina Protects against Nuclear Rupture, DNA Damage, and Cell-Cycle Arrest. Developmental Cell 49:920-935.e5. doi: 10.1016/j.devcel.2019.04.020\u003c/li\u003e\n \u003cli\u003eChow H-M, Cheng A, Song X, Swerdel MR, Hart RP, Herrup K (2019) ATM is activated by ATP depletion and modulates mitochondrial function through NRF1. Journal of Cell Biology 218:909\u0026ndash;928. doi: 10.1083/jcb.201806197\u003c/li\u003e\n \u003cli\u003eChung EO, Bodensteiner JB, Noorani PA, Schochet SS (1994) Cerebral White-Matter Changes Suggesting Leukodystrophy in Ataxia Telangiectasia. J Child Neurol 9:31\u0026ndash;35. doi: 10.1177/088307389400900106\u003c/li\u003e\n \u003cli\u003eFarley FW, Soriano P, Steffen LS, Dymecki SM (2000) Widespread recombinase expression using FLPeR (flipper) mice. Genesis 28:106\u0026ndash;110\u003c/li\u003e\n \u003cli\u003eFırat AK, Karakaş HM, Fırat Y, Yakıncı C (2005) Quantitative evaluation of brain involvement in ataxia telangiectasia by diffusion weighted MR imaging. European Journal of Radiology 56:192\u0026ndash;196. doi: 10.1016/j.ejrad.2005.04.009\u003c/li\u003e\n \u003cli\u003eGallyas F (1979) Silver Staining of Myelin by Means of Physical Development. Neurological Research 1:203\u0026ndash;209. doi: 10.1080/01616412.1979.11739553\u003c/li\u003e\n \u003cli\u003eGatti RA, Berkel I, Boder E, Braedt G, Charmley P, Concannon P, Ersoy F, Foroud T, Jaspers NGJ, Lange K, Lathrop GM, Leppert M, Nakamura Y, O\u0026rsquo;Connell P, Paterson M, Salser W, Sanal O, Silver J, Sparkes RS, Susi E, Weeks DE, Wei S, White R, Yoder F (1988) Localization of an ataxia-telangiectasia gene to chromosome 11q22\u0026ndash;23. Nature 336:577\u0026ndash;580. doi: 10.1038/336577a0\u003c/li\u003e\n \u003cli\u003eGatti RA, Vinters HV (1985) Cerebellar pathology in ataxia-telangiectasia: the significance of basket cells. Kroc Found Ser 19:225\u0026ndash;232\u003c/li\u003e\n \u003cli\u003eGuo Z, Deshpande R, Paull TT (2010) ATM activation in the presence of oxidative stress. Cell Cycle 9:4805\u0026ndash;4811. doi: 10.4161/cc.9.24.14323\u003c/li\u003e\n \u003cli\u003eHoxha E, Balbo I, Miniaci MC, Tempia F (2018) Purkinje Cell Signaling Deficits in Animal Models of Ataxia. Front Synaptic Neurosci 10:6. doi: 10.3389/fnsyn.2018.00006\u003c/li\u003e\n \u003cli\u003eHoxha E, Balbo I, Parolisi R, Audano M, Montarolo F, Ravera F, Guglielmotto M, Muratori L, Raimondo S, DiGregorio E, Buffo A, Brusco A, Borroni B, Mitro N, Caruso D, Tempia F (2021) Elovl5 is required for proper action potential conduction along peripheral myelinated fibers. Glia 69:2419\u0026ndash;2428. doi: 10.1002/glia.24048\u003c/li\u003e\n \u003cli\u003eHoxha E, Gabriele RMC, Balbo I, Ravera F, Masante L, Zambelli V, Albergo C, Mitro N, Caruso D, Di Gregorio E, Brusco A, Borroni B, Tempia F (2017) Motor Deficits and Cerebellar Atrophy in Elovl5 Knock Out Mice. Front Cell Neurosci 11:343. doi: 10.3389/fncel.2017.00343\u003c/li\u003e\n \u003cli\u003eHoxha E, Marcinn\u0026ograve; A, Montarolo F, Masante L, Balbo I, Ravera F, Laezza F, Tempia F (2019) Emerging roles of Fgf14 in behavioral control. Behavioural Brain Research 356:257\u0026ndash;265. doi: 10.1016/j.bbr.2018.08.034\u003c/li\u003e\n \u003cli\u003eHoxha E, Tonini R, Montarolo F, Croci L, Consalez GG, Tempia F (2013) Motor dysfunction and cerebellar Purkinje cell firing impairment in Ebf2 null mice. Molecular and Cellular Neuroscience 52:51\u0026ndash;61. doi: 10.1016/j.mcn.2012.09.002\u003c/li\u003e\n \u003cli\u003eKanner S, Goldin M, Galron R, Ben Jacob E, Bonifazi P, Barzilai A (2018) Astrocytes restore connectivity and synchronization in dysfunctional cerebellar networks. Proc Natl Acad Sci USA 115:8025\u0026ndash;8030. doi: 10.1073/pnas.1718582115\u003c/li\u003e\n \u003cli\u003eKrishnaswamy VR, Benbenishty A, Blinder P, Sagi I (2019) Demystifying the extracellular matrix and its proteolytic remodeling in the brain: structural and functional insights. Cell Mol Life Sci 76:3229\u0026ndash;3248. doi: 10.1007/s00018-019-03182-6\u003c/li\u003e\n \u003cli\u003eLai J, Demirbas D, Kim J, Jeffries AM, Tolles A, Park J, Chittenden TW, Buckley PG, Yu TW, Lodato MA, Lee EA (2024) ATM-deficiency-induced microglial activation promotes neurodegeneration in ataxia-telangiectasia. Cell Reports 43:113622. doi: 10.1016/j.celrep.2023.113622\u003c/li\u003e\n \u003cli\u003eLau LW, Cua R, Keough MB, Haylock-Jacobs S, Yong VW (2013) Pathophysiology of the brain extracellular matrix: a new target for remyelination. Nat Rev Neurosci 14:722\u0026ndash;729. doi: 10.1038/nrn3550\u003c/li\u003e\n \u003cli\u003eLawrence JM, Schardien K, Wigdahl B, Nonnemacher MR (2023) Roles of neuropathology-associated reactive astrocytes: a systematic review. acta neuropathol commun 11:42. doi: 10.1186/s40478-023-01526-9\u003c/li\u003e\n \u003cli\u003eLi J, Han YR, Plummer MR, Herrup K (2009) Cytoplasmic ATM in Neurons Modulates Synaptic Function. Current Biology 19:2091\u0026ndash;2096. doi: 10.1016/j.cub.2009.10.039\u003c/li\u003e\n \u003cli\u003eLumsden JM, McCarty T, Petiniot LK, Shen R, Barlow C, Wynn TA, Morse HC, Gearhart PJ, Wynshaw-Boris A, Max EE, Hodes RJ (2004) Immunoglobulin Class Switch Recombination Is Impaired in \u003cem\u003eAtm\u003c/em\u003e -deficient Mice. The Journal of Experimental Medicine 200:1111\u0026ndash;1121. doi: 10.1084/jem.20041074\u003c/li\u003e\n \u003cli\u003eMancini C, Hoxha E, Iommarini L, Brussino A, Richter U, Montarolo F, Cagnoli C, Parolisi R, Gondor Morosini DI, Nicol\u0026ograve; V, Maltecca F, Muratori L, Ronchi G, Geuna S, Arnaboldi F, Donetti E, Giorgio E, Cavalieri S, Di Gregorio E, Pozzi E, Ferrero M, Riberi E, Casari G, Altruda F, Turco E, Gasparre G, Battersby BJ, Porcelli AM, Ferrero E, Brusco A, Tempia F (2019) Mice harbouring a SCA28 patient mutation in AFG3L2 develop late-onset ataxia associated with enhanced mitochondrial proteotoxicity. Neurobiology of Disease 124:14\u0026ndash;28. doi: 10.1016/j.nbd.2018.10.018\u003c/li\u003e\n \u003cli\u003eMansouri B, Henne WM, Oomman SK, Bliss R, Attridge J, Finckbone V, Zeitouni T, Hoffman T, Bahr BA, Strahlendorf HK, Strahlendorf JC (2007) Involvement of calpain in AMPA-induced toxicity to rat cerebellar Purkinje neurons. European Journal of Pharmacology 557:106\u0026ndash;114. doi: 10.1016/j.ejphar.2006.11.032\u003c/li\u003e\n \u003cli\u003eMarangon D, Boccazzi M, Lecca D, Fumagalli M (2020) Regulation of Oligodendrocyte Functions: Targeting Lipid Metabolism and Extracellular Matrix for Myelin Repair. JCM 9:470. doi: 10.3390/jcm9020470\u003c/li\u003e\n \u003cli\u003eMartins SG, Zilh\u0026atilde;o R, Thorsteinsd\u0026oacute;ttir S, Carlos AR (2021) Linking Oxidative Stress and DNA Damage to Changes in the Expression of Extracellular Matrix Components. Front Genet 12:673002. doi: 10.3389/fgene.2021.673002\u003c/li\u003e\n \u003cli\u003eMenolfi D, Lee BJ, Zhang H, Jiang W, Bowen NE, Wang Y, Zhao J, Holmes A, Gershik S, Rabadan R, Kim B, Zha S (2023) ATR kinase supports normal proliferation in the early S phase by preventing replication resource exhaustion. Nat Commun 14:3618. doi: 10.1038/s41467-023-39332-5\u003c/li\u003e\n \u003cli\u003eMitiagin Y, Barzilai A (2023) Ataxia-telangiectasia mutated plays an important role in cerebellar integrity and functionality. Neural Regen Res 18:497. doi: 10.4103/1673-5374.350194\u003c/li\u003e\n \u003cli\u003eMitiagin Y, Barzilai A (2023) Ataxia-telangiectasia mutated plays an important role in cerebellar integrity and functionality. Neural Regen Res 18:497. doi: 10.4103/1673-5374.350194\u003c/li\u003e\n \u003cli\u003eMontarolo F, Gao F, Loddo M, Milek J, Zawadzka M, Miniaci MC, Brusco A, Balbo I, Dziembowska M, Tempia F, Skowronska-Krawczyk D, Hoxha E (2025) Impact of Elovl5 deficiency on cerebellar excitatory synaptic transmission in mice. J Neurosci e1529242025. doi: 10.1523/JNEUROSCI.1529-24.2025\u003c/li\u003e\n \u003cli\u003eMontarolo F, Thielens S, Bove M, Bertolotto A, Tempia F, Hoxha E (2025) NURR1 Deficiency Is Associated to Altered Microglial Phenotype in Male Mice. Mol Neurobiol 62:8887\u0026ndash;8899. doi: 10.1007/s12035-025-04787-8\u003c/li\u003e\n \u003cli\u003ePease-Raissi SE, Chan JR (2021) Building a (w)rapport between neurons and oligodendroglia: Reciprocal interactions underlying adaptive myelination. Neuron 109:1258\u0026ndash;1273. doi: 10.1016/j.neuron.2021.02.003\u003c/li\u003e\n \u003cli\u003ePepper RE, Pitman KA, Cullen CL, Young KM (2018) How Do Cells of the Oligodendrocyte Lineage Affect Neuronal Circuits to Influence Motor Function, Memory and Mood? Front Cell Neurosci 12:399. doi: 10.3389/fncel.2018.00399\u003c/li\u003e\n \u003cli\u003ePetersen AJ, Rimkus SA, Wassarman DA (2012) ATM kinase inhibition in glial cells activates the innate immune response and causes neurodegeneration in \u003cem\u003eDrosophila\u003c/em\u003e. Proc Natl Acad Sci USA 109. doi: 10.1073/pnas.1110470109\u003c/li\u003e\n \u003cli\u003ePistorio AL, Hendry SH, Wang X (2006) A modified technique for high-resolution staining of myelin. Journal of Neuroscience Methods 153:135\u0026ndash;146. doi: 10.1016/j.jneumeth.2005.10.014\u003c/li\u003e\n \u003cli\u003ePizzamiglio L, Focchi E, Murru L, Tamborini M, Passafaro M, Menna E, Matteoli M, Antonucci F (2016) New Role of ATM in Controlling GABAergic Tone During Development. Cereb Cortex 26:3879\u0026ndash;3888. doi: 10.1093/cercor/bhw125\u003c/li\u003e\n \u003cli\u003eReina-San-Martin B, Chen HT, Nussenzweig A, Nussenzweig MC (2004) ATM is required for efficient recombination between immunoglobulin switch regions. J Exp Med 200:1103\u0026ndash;1110. doi: 10.1084/jem.20041162\u003c/li\u003e\n \u003cli\u003eRothblum-Oviatt C, Wright J, Lefton-Greif MA, McGrath-Morrow SA, Crawford TO, Lederman HM (2016) Ataxia telangiectasia: a review. Orphanet J Rare Dis 11:159. doi: 10.1186/s13023-016-0543-7\u003c/li\u003e\n \u003cli\u003eSahama I, Sinclair K, Fiori S, Doecke J, Pannek K, Reid L, Lavin M, Rose S (2015) Motor pathway degeneration in young ataxia telangiectasia patients: A diffusion tractography study. NeuroImage: Clinical 9:206\u0026ndash;215. doi: 10.1016/j.nicl.2015.08.007\u003c/li\u003e\n \u003cli\u003eSahama I, Sinclair K, Pannek K, Lavin M, Rose S (2014) Radiological Imaging in Ataxia Telangiectasia: a Review. Cerebellum 13:521\u0026ndash;530. doi: 10.1007/s12311-014-0557-4\u003c/li\u003e\n \u003cli\u003eSchindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez J-Y, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676\u0026ndash;682. doi: 10.1038/nmeth.2019\u003c/li\u003e\n \u003cli\u003eSong I, Dityatev A (2018) Crosstalk between glia, extracellular matrix and neurons. Brain Research Bulletin 136:101\u0026ndash;108. doi: 10.1016/j.brainresbull.2017.03.003\u003c/li\u003e\n \u003cli\u003eStrahlendorf J, Box C, Attridge J, Diertien J, Finckbone V, Henne WM, Medina MS, Miles R, Oomman S, Schneider M, Singh H, Veliyaparambil M, Strahlendorf H (2003) AMPA-induced dark cell degeneration of cerebellar Purkinje neurons involves activation of caspases and apparent mitochondrial dysfunction. Brain Research 994:146\u0026ndash;159. doi: 10.1016/j.brainres.2003.09.048\u003c/li\u003e\n \u003cli\u003eTal E, Alfo M, Zha S, Barzilai A, De Zeeuw CI, Ziv Y, Shiloh Y (2018) Inactive Atm abrogates DSB repair in mouse cerebellum more than does Atm loss, without causing a neurological phenotype. DNA Repair 72:10\u0026ndash;17. doi: 10.1016/j.dnarep.2018.10.001\u003c/li\u003e\n \u003cli\u003eTewari BP, Chaunsali L, Prim CE, Sontheimer H (2022) A glial perspective on the extracellular matrix and perineuronal net remodeling in the central nervous system. Front Cell Neurosci 16:1022754. doi: 10.3389/fncel.2022.1022754\u003c/li\u003e\n \u003cli\u003eTse K-H, Cheng A, Yeung SH-S, Ng J-N, Cheng GW-Y, Wang Q, Zhu B, Cui Y, Jiang L, Kofler J, Herrup K (2021) Myelin pathology in ataxia-telangiectasia is the cell autonomous effect of ATM deficiency in oligodendrocytes\u003c/li\u003e\n \u003cli\u003eTse K-H, Herrup K (2017) DNA damage in the oligodendrocyte lineage and its role in brain aging. Mechanisms of Ageing and Development 161:37\u0026ndash;50. doi: 10.1016/j.mad.2016.05.006\u003c/li\u003e\n \u003cli\u003eUrbanski MM, Kingsbury L, Moussouros D, Kassim I, Mehjabeen S, Paknejad N, Melendez-Vasquez CV (2016) Myelinating glia differentiation is regulated by extracellular matrix elasticity. Sci Rep 6:33751. doi: 10.1038/srep33751\u003c/li\u003e\n \u003cli\u003eVail G, Cheng A, Han YR, Zhao T, Du S, Loy MMT, Herrup K, Plummer MR (2016) ATM protein is located on presynaptic vesicles and its deficit leads to failures in synaptic plasticity. Journal of Neurophysiology 116:201\u0026ndash;209. doi: 10.1152/jn.00006.2016\u003c/li\u003e\n \u003cli\u003eVerhoog QP, Holtman L, Aronica E, van Vliet EA (2020) Astrocytes as Guardians of Neuronal Excitability: Mechanisms Underlying Epileptogenesis. Front Neurol 11:591690. doi: 10.3389/fneur.2020.591690\u003c/li\u003e\n \u003cli\u003eXu K, Zhang Y, Chen Y, Zhu X, Li Y, Lv L, He X, Hu Z, Li Y, Ye M, Jiang D, He Z, Jin W, Li Y, Yu X, Zhang D-F, Herrup K, Zheng P, Yao Y-G, Wu D-D, Li J (2025) ATM deficiency drives phenotypic diversity and Purkinje cell degeneration in a macaque model of ataxia-telangiectasia. Cell Reports Medicine 6:102355. doi: 10.1016/j.xcrm.2025.102355\u003c/li\u003e\n \u003cli\u003eYamamoto K, Wang J, Sprinzen L, Xu J, Haddock CJ, Li C, Lee BJ, Loredan DG, Jiang W, Vindigni A, Wang D, Rabadan R, Zha S (2016) Kinase-dead ATM protein is highly oncogenic and can be preferentially targeted by Topo-isomerase I inhibitors. eLife 5:e14709. doi: 10.7554/eLife.14709\u003c/li\u003e\n \u003cli\u003eYamamoto K, Wang Y, Jiang W, Liu X, Dubois RL, Lin C-S, Ludwig T, Bakkenist CJ, Zha S (2012) Kinase-dead ATM protein causes genomic instability and early embryonic lethality in mice. Journal of Cell Biology 198:305\u0026ndash;313. doi: 10.1083/jcb.201204098\u003c/li\u003e\n \u003cli\u003eYang K, Dunn M, Torres-Ramirez G, Dobbs N, Shakkottai VG, Yan N (2025) Autonomous STING signaling in Purkinje cells drives neurodegeneration independent of type I interferon. Cell Reports 44:116480. doi: 10.1016/j.celrep.2025.116480\u003c/li\u003e\n \u003cli\u003eZha S, Sekiguchi J, Brush JW, Bassing CH, Alt FW (2008) Complementary functions of ATM and H2AX in development and suppression of genomic instability. Proc Natl Acad Sci USA 105:9302\u0026ndash;9306. doi: 10.1073/pnas.0803520105\u003c/li\u003e\n \u003cli\u003eZhang J, Tripathi DN, Jing J, Alexander A, Kim J, Powell RT, Dere R, Tait-Mulder J, Lee J-H, Paull TT, Pandita RK, Charaka VK, Pandita TK, Kastan MB, Walker CL (2015) ATM functions at the peroxisome to induce pexophagy in response to ROS. Nat Cell Biol 17:1259\u0026ndash;1269. doi: 10.1038/ncb3\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"acta-neuropathologica-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"anec","sideBox":"Learn more about [Acta Neuropathologica Communications](https://actaneurocomms.biomedcentral.com/)","snPcode":"40478","submissionUrl":"https://submission.springernature.com/new-submission/40478/3","title":"Acta Neuropathologica Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Ataxia Telangiectasia (A-T), cerebellar atrophy, motor deficit, Purkinje cell, degeneration, excitability","lastPublishedDoi":"10.21203/rs.3.rs-8832557/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8832557/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAtaxia Telangiectasia (A-T) is a neurodegenerative disorder characterized by early onset, cerebellar ataxia and progressive motor decline. The causative gene, \u003cem\u003eATM\u003c/em\u003e (A-T Mutated), encodes a Ser/Thr kinase, that belongs to the phosphoinositide 3-kinase-related protein kinase family and is crucial for the response to DNA double-strand breaks. While ATM is classically known for its role in the DNA damage response, increasing evidence points to its critical function in maintaining cellular homeostasis, particularly in the central nervous system (CNS). Yet the mechanisms linking ATM-kinase deficiency to cerebellar circuit dysfunction remain poorly defined.\u003c/p\u003e \u003cp\u003eUsing a CNS Nestin-Cre-restricted mouse model carrying a kinase-dead \u003cem\u003eAtm\u003c/em\u003e allele combined with a null allele (\u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e)\u003c/em\u003e, we integrated proteomics, structural and ultrastructural analyses, electrophysiology, and behavioral testing to understand how the loss of Atm kinase activity influence the cerebellar microenvironment from the earliest stages of the disease.\u003c/p\u003e \u003cp\u003eProteomic profiling revealed alterations across four major pathways in \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e mice, such as disorganization of the extracellular matrix, astrocytosis, downregulation of myelin and oligodendrocyte lineage proteins and changes in neuronal excitability. Histological and electron microscopy analyses confirmed astrocytosis and reduction of myelin content without axonal or oligodendrocyte loss, consistent with impaired myelination at early stages. These microenvironmental changes were associated to Purkinje cell dark cell degeneration and increased intrinsic excitability, demonstrating early circuit dysfunction in the absence of overt neuronal loss. Consistently, the compromised cerebellar output is confirmed by the progressive motor impairment developed by \u003cem\u003eAtm\u003c/em\u003e\u003csup\u003e\u003cem\u003eKDF/CNS\u0026minus;KO\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e \u003cp\u003eOur findings indicate that Atm kinase deficiency disrupts cerebellar homeostasis through interconnected and bidirectional mechanisms involving ECM remodeling, astrocytic activation, impaired oligodendrocyte maturation and altered intrinsic excitability, reflecting a network level destabilization of the cerebellar microenvironment.\u003c/p\u003e","manuscriptTitle":"ATM-kinase deficiency triggers early multi-compartment remodeling of the cerebellar microenvironment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-17 14:00:37","doi":"10.21203/rs.3.rs-8832557/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-01T11:21:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-01T09:59:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-25T16:53:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"199137675431825255793772549177843213017","date":"2026-02-19T20:08:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"174657507249733448516345508523676167887","date":"2026-02-19T02:48:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-17T19:30:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-14T00:08:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-11T14:53:29+00:00","index":"","fulltext":""},{"type":"submitted","content":"Acta Neuropathologica Communications","date":"2026-02-09T15:44:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"acta-neuropathologica-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"anec","sideBox":"Learn more about [Acta Neuropathologica Communications](https://actaneurocomms.biomedcentral.com/)","snPcode":"40478","submissionUrl":"https://submission.springernature.com/new-submission/40478/3","title":"Acta Neuropathologica Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ca380318-659e-468b-80f2-7b6f751a831e","owner":[],"postedDate":"February 17th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-15T00:53:58+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-17 14:00:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8832557","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8832557","identity":"rs-8832557","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}