A human Staufen1 BAC transgenic mouse exhibits abnormal autophagy and neurodegeneration across the central nervous system

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Staufen1 (STAU1) is an RBP that regulates mRNA degradation and subcellular localization, and is part of the ATXN2 protein complex. Previously, we showed that STAU1 is overabundant in patient fibroblasts and in mouse models of Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), and spinocerebellar ataxia type 2 (SCA2), where it is associated with impaired autophagic flux due to STAU1-mediated upregulation of mTOR translation. STAU1 overabundance and impaired autophagy cause accumulation of biomolecular condensates and abnormal unfolded protein response (UPR). We generated a mouse model expressing the entire human STAU1 gene (h STAU1 ) in a bacterial artificial chromosome (BAC) construct. hSTAU1 in these mice was expressed in cerebral hemispheres, cerebellum and spinal cord, as well as cultured cortical neurons and cortical and spinal cord astrocytes and microglia. Expression of hSTAU1 caused dysregulated gene expression, abnormal autophagy, glial activation, and changes in neuronal marker proteins. All of these were significantly improved by reducing STAU1 abundance by RNAi, but exacerbated in BAC-STAU1 mice crossed with Prp-TDP-43(Q331K) transgenic mice. Similar results were also obtained in eye phenotypes in ALS- and SCA2-relevant fly models upon changing staufen-1 dosage. Despite the molecular changes, we observed no overt behavioral changes in mice up to 55 weeks of age, suggesting that STAU1 may function as an epistatic modifier of neuronal degeneration. The BAC-hSTAU1 mouse will be useful for developing therapies targeting the human STAU1 gene. Biological sciences/Neuroscience/Diseases of the nervous system/Amyotrophic lateral sclerosis Biological sciences/Neuroscience/Diseases of the nervous system/Dementia/Alzheimer's disease Alzheimer’s disease Amyotrophic lateral sclerosis Spinocerebellar ataxia type 2 Staufen1 STAU1 TDP-43 Autophagy Drosophila Stau Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction STAU1 is a double stranded RNA binding protein (RBD) initially described in the fly for its essential role in the transport and spatiotemporal localization of mRNAs critical for embryonic patterning 1 . It is a multi-functional RBP that also regulates mRNA translation and decay through interactions with double-stranded structures in specific 5′ and 3′ UTRs 2 . STAU1 is an interactor of ATXN2 3 , another RBD that is mutated in spinocerebellar ataxia type 2 (SCA2) 4 and, more rarely, in ALS and PD (reviewed in Scoles and Pulst 5 ). STAU1 is also a stress granule (SG) protein that aggregates with ATXN2 in cytoplasmic condensates within cerebellar Purkinje cells (PCs) of SCA2 patients and mice 3 . STAU1 abundance is increased in multiple neurodegenerative disease (NDD) models and in tissues from patients with SCA2, sporadic and familial ALS, and frontotemporal dementia (FTD) associated with C9ORF72 and TDP-43 pathology 3 , 6 , 7 . In Alzheimer disease and AD related disorders (AD/ADRDs), STAU1 overabundance is seen in APP/PS1 transgenic mice with accumulation of β-amyloid (Aβ) and Tau-neurofibrillary tangles. This was associated with STAU1-mediated stabilization of β-amyloid converting enzyme 1 (BACE1 ) expression 8 . Mechanistically, STAU1 overabundance elevates mTOR translation by directly interacting with the 5’-UTR MTOR mRNA. As mTOR is a negative regulator of autophagy, its increased activity leads to autophagy suppression 7 . Thus, STAU1 overabundance is a post-transcriptional effect associated with decreased autophagic clearance due to impaired autophagy 3 , 7 . We have proposed a model that leads to a maladaptive feed-forward loop that can be initiated by cellular stress, STAU1 overabundance, and reduced autophagy or misfolded proteins 9 . Recently, Zhou et al. independently confirmed the control of MTOR translation by STAU1 and also demonstrated that STAU1 overabundance drives the production of biomolecular condensates rich in MTOR mRNA 10 . Furthermore, STAU1 can act as a modulator of the unfolded protein response (UPR): In fibroblasts from individuals with SCA2 or pathogenic TDP-43 or C9ORF72 mutations, increased CHOP levels were restored to normal by STAU1 knockdown 11 . Other functions of STAU1 include degradation of cell-specific subsets of mRNAS, usually by interaction with the 3’-UTR in a process designated Staufen1-mediated decay (SMD), a UPF1-dependent process linked to cell death 12 , 13 . In neurons, STAU1 traffics mRNAs in coordination with TDP-43, implicating it in synaptic function, cognitive decline, and autophagy-related neuroprotection 14 , 15 , 16 , 17 , 18 . STAU1 also binds Alu and IRAlu elements to control mRNA nuclear export 19 , 20 , 21 . The multi-facetted role of STAU1 in NDDs prompted us to study whether STAU1 was necessary and/or sufficient to mediate changes in autophagy or in UPR in cells with NDD-causing mutations. Indeed, in vitro STAU1 knockdown normalized these cellular processes. Surprisingly, exogenous expression of STAU1 in wildtype cells was sufficient to alter autophagy and UPR 7 , 11 . Here we further extended these studies for understanding the consequences of STAU1 overabundance in vivo. To this end, we generated a new bacterial artificial chromosome (BAC)-STAU1 mouse that includes 133.8 kb of the human STAU1 locus including all exons and introns as well as up- and downstream regions. This mouse line will also be useful for preclinical development of human STAU1 -directed therapies. Materials and Methods Mice The BAC-STAU1 mouse model was developed by the University of Utah Transgenic Mouse Core by pronuclear microinjection of a non-linearized human Staufen1 bacterial artificial chromosome (BAC) construct (BAC-STAU1 clone RP11-120I11, BACPAC Resources) into fertilized oocytes sourced from mice with a B6/D2 mixed hybrid background (B6D2F1J, The Jackson Laboratory stock #100006). Prior to injection, the BAC construct was separated from other nucleotide fragments by pulsed field gel electrophoresis, and gel purified, by the University of Michigan Transgenic Mouse Core. Genotyping of mouse tail DNA was initially done with multiplex PCRs covering all coding exons, 5’ and 3’ UTR and the 5’ upstream promoter region (ENST00000371856.7, STAU1-207, NM_017453.4). Transgenic mouse bone marrow samples were sequenced and analyzed by Cergentis to determine integration sites and vector-vector breakpoints that represent concatemerization. BAC-STAU1 mice were maintained in a B6D2 mixed background by backcrossing to B6D2F1J no less than every 4 generations. The Stau1 tm1Apa(−/−) ( Stau1 −/− ) mouse 22 was a generous gift from Prof. Michael A. Kiebler, Ludwig Maximilian University of Munich, Germany, and maintained in a C57BL/6J background. Mice were maintained in a temperature and humidity-controlled environment on a 12h light/dark cycle with light onset at 6:00 AM. Both males and females were used in animal studies. Genotyping Genotyping was performed by PCR using tail DNAs as previously described 23 . Our approach verified the presence of all STAU1 exons. Genotyping primers are provided in Supplemental Table 1. Primary cell culture Purified astrocytes and microglia were prepared from mixed glial cultures from spinal cord or brain cortex of neonatal mice as previously described 24 , 25 . Mice were sacrificed and brain cortices and spinal cords were isolated for culture and cerebella for genotyping by PCR as described above. To establish primary mixed glial cultures, the corresponding tissue was dissected, digested with trypsin, mechanically dissociated and passed through a 50 µM mesh. The single cell suspension was plated at a density of 2 × 10 4 cells/cm 2 and maintained in DMEM supplemented with 10% fetal bovine serum, penicillin and streptomycin (reagents from ThermoFisher). When the astrocyte monolayers were confluent, the cultures were vigorously tapped to dislodge microglia growing on top. The microglia were collected, seeded into a new culture dish and maintained in the same culture media. Astrocytes were further purified by adding 5 µM cytosine arabinoside (Thermofisher) for 24 hrs. Samples were collected after 2 weeks in culture by lysing cells directly in Laemmli buffer with 5% β-mercaptoethanol (Bio-Rad) for western blot, or in RLT buffer (Qiagen) for qPCR. Primary cortical neuron cultures were prepared from neonatal mouse cortices, and cerebella were used for genotyping by PCR. Cortices were isolated, digested with a papain enzyme kit (Worthington) according to the manufacturer’s instructions, mechanically dissociated and passed through a 50 µM mesh. The single cell suspension was plated at 5 x 10 4 cells/cm 2 in a substrate of poly-l-ornithine and laminin in Neurobasal Plus media supplemented with 2% B27 Plus and 500 mM glutamax (reagents from ThermoFisher). After 72 hrs cultures were incubated with 1 µM cytosine arabinoside for 24 hrs to eliminate glial cells. Samples for western blots were collected after 2 weeks in culture by lysing cells directly in Laemmli buffer with 5% β-mercaptoethanol. Quantitative PCR (qPCR) qPCR assays with mice included cDNA preparations using the Life Sciences cDNA Kit (#4368814). Taqman qPCR kits included ThermoFisher Hs00244999_m1 for human STAU1 , Thermofisher Hs01060665_g1 for human ACTB , ThermoFisher Mm00488465_m1 for mouse Stau1 and Thermofisher Mm01205647_g1 for mouse Actb . Taqman assays were performed using Taqman master mix (ThermoFisher 4440040). Behavioral phenotype testing Forelimb and hindlimb grip strength was determined using a grip strength meter (Ugo Basile cat# 47200). Peak force grip strength values are reported as the mean of three replicates. Rotarod testing was performed using the Rotamex-5 accelerating rotarod (Columbus Instruments). In any week of testing, on day 1 mice are handled in the palm of the hand for at least 5 min each for 3 separate times to acclimate animals to the investigator. On day 2, mice are trained on the rotarod with acceleration of 1 RPM per 15 seconds to a maximum of 10 RPM and total time of 10 min, and this is repeated three times. On days 3–5 mice are placed on the rotarod set to accelerate 1 RPM per 9 sec until mice fall from the rod and the latency to fall (in seconds) is recorded, and 2 trials per day are performed. Each mouse group included 20 or more animals, exceeding the minimum sample size required to achieve 80% power to detect a 50% difference between groups, based on power analysis performed using nQuery Advisor. The analysis assumed a two-sided test with α = 0.05. No mice were subjected to exclusion. The technician was blinded to the genotype when performing motor phenotype tests. Western blot analyses Protein extracts from mouse cerebellar or cerebral hemisphere or spinal cord tissues were prepared by homogenization in an extraction buffer (25 mM Tris-HCl pH 7.6, 300 mM NaCl, 0.5% Nonidet P-40, 2 mM EDTA, 2 mM MgCl 2 , 0.5 M urea and protease inhibitors; Sigma; cat# P-8340). The homogenates were subjected to sonication (3 s for 1 stroke, and level 2; Sonic Dismembrator, Fisher Scientific) followed by centrifugation at 4°C for 20 min at 14,000 rpm. Only the supernatants were used for Western blotting. Protein extracts were resolved by SDS-PAGE and transferred to Hybond P membranes (Amersham Bioscience Inc., USA). After blocking with 3% skim milk in 0.1% Tween 20/PBS, the membranes were incubated with primary antibodies in 3% skim milk in 0.1% Tween 20/PBS for 2 hrs at room temperature or overnight at 4°C. After washing in 0.1% Tween 20/PBS, the membranes were incubated with the corresponding secondary antibodies conjugated with HRP in 3% skim milk in 0.1% Tween 20/PBS for 2 hrs at room temperature and washed again. Signals were detected by using the Immobilon Western Chemiluminescent HRP Substrate (Millipore Inc., USA; cat# WBKLSO100) according to the manufacturer’s protocol, and detected using a ChemiDoc System (Bio-Rad). Band intensities were quantified by ImageJ software analyses after inversion of the images and proteins were quantitated as a ratio to β-actin (ACTB) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We used Precision Plus Protein Dual Color Standards (Bio-Rad Inc., USA, Cat# 1610374) for SDS-PAGE analyses. Antibodies Antibodies included the following: rabbit polyclonal anti-Staufen antibody (Novus Biologicals, NBP1-33202); rabbit polyclonal anti-mTOR antibody (Cell Signaling Technology, 2972); rabbit polyclonal anti-Phospho-mTOR (Ser2448) [(1:3000), Cell Signaling Technology, 2971]; rabbit polyclonal anti-SQSTM1/p62 antibody (Cell Signaling Technology, 5114); rabbit polyclonal anti-LC3B antibody (Novus Biologicals, NB100-2220); mouse monoclonal anti-TDP-43 (human specific) antibody [(1:7000), Proteintech Group, Inc., Cat #60019-2-Ig)]; rabbit polyclonal human/mouse anti-TDP-43 antibody [(1:7000), Proteintech Group, Inc., Cat #10782-2-AP)]; mouse monoclonal anti-Calbindin-D-28K antibody [(1:5000), Sigma-Aldrich, C9848]; rabbit polyclonal anti-RGS8 antibody [(1:5000), Novus Biologicals, NBP2-20153]; mouse monoclonal anti-PCP2 antibody (F-3) [(1:3000), Santa Cruz, sc-137064]; rabbit polyclonal anti-PCP4 antibody [(1:5000), Abcam, ab197377]; Phospho-p70 S6 Kinase (Thr389) antibody [(1:3000), Cell Signaling, Cat# 9205]; GFAP (GA5) mouse mAb [(1:7000), Cell signaling, Cat #3670]; ChAT (E4F9G) Rabbit mAb [(1:5000), Cell Signaling, Cat# 27269]; NeuN (D4G4O) XP® Rabbit mAb [(1:5000), Cell Signaling, Cat# 24307]; GAPDH (14C10) rabbit mAb [(1:7,000), Cell Signaling, Cat# 2118]; Cleaved Caspase-3 (Asp175) (5A1E) rabbit mAb [(1:3000), Cell Signaling, Cat #9664]; Anti-NeuN107B antibody [(1:5,000) Abcam, ab175148); GFP (D5.1) rabbit mAb [(1:6,000), Cell Signaling, Cat #2956]; UNC13A/Munc13-1 polyclonal antibody [(1:3000), Proteintech Group, Inc., Cat #55053-1-AP]; STMN2 polyclonal antibody [(1:3000), Proteintech Group, Inc., Cat #10586-1-AP], and mouse monoclonal anti-β-Actin − peroxidase antibody (clone AC-15) [(1:10,000), Sigma-Aldrich, A3854). Secondary antibodies included anti-mouse IgG, HRP-linked antibody [(1:5000), Cell signaling, Cat #7076], and peroxidase AffiniPure goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, 111-035-144). RNA-seq Total RNA was extracted from tissues using the RNeasy Mini-Kit (Qiagen) according to the manufacturer’s protocol. RNA quality was determined using the Agilent ScreenTape Assay. Library preparation was performed using the Illumina TruSeq Stranded Total RNA library prep Ribo-Zero gold. Paired-end 150 bp reads were generated on a Novaseq 6000 S2 cell sequencing instrument at the High-Throughput Genomics and Bioinformatic Analysis Shared Resource at Huntsman Cancer Institute (University of Utah). The human GRCh38 genome and gene annotation files were downloaded from Ensembl release 100 and a reference database was created using STAR version 2.7.3a with splice junctions optimized for 150 base pair reads 26 . Optical duplicates were removed from the paired end FASTQ files using clumpify v38.34 27 and reads were trimmed of adapters using cutadapt 1.16 28 . The trimmed reads were aligned to the reference database using STAR in two pass mode to output a BAM file sorted by coordinates. Mapped reads were assigned to annotated genes using featureCounts version 1.6.3 29 . The output files from cutadapt, FastQC, FastQ Screen, Picard CollectRnaSeqMetrics, STAR and featureCounts were summarized using MultiQC to check for any sample outliers 30 . Differentially expressed genes were identified for siSTAU1 vs siControl using a 5% false discovery rate with DESeq2 version 1.26.0 31 . Pathways were analyzed using the fast gene set enrichment package with a 10% false discovery rate (FDR) 32 , 33 . Replicate RNA-seq experiments were performed independent of one another four years apart with RNAs prepared by different technicians. Immunofluorescent labeling To confirm the pan-cellular expression of BAC-STAU1, we performed immunofluorescent staining for STAU1. Brains and spinal cords were harvested and fixed in 4% PFA for 24 hours, cryopreserved in sucrose 30% sucrose/PBS, and embedded in OCT. Cryosections were immunostained using a Staufen antibody (Novus Biologicals, NBP1-33202; 1:100 dilution) and a Goat anti-Rabbit Alexa Fluor™ Plus 594 secondary antibody (Thermo Fisher Scientific, A32740, 1:1000 dilution). Nuclei were counterstained with DAPI, and sections were mounted with Fluoromount-G (Southern Biotech, Cat. #0100-01). Fluorescence imaging was performed using a Leica SP8 confocal microscope and an AxioScan 7 at the Cell Imaging Core, University of Utah. AAV production and treatment of mice Artificial microRNAs targeting STAU1 were designed following the method described by Boudreau and Davidson, that begins with siRNA sequences 34 . Two different miRNAs were designed that target human STAU1 that were based on siRNA sequences that were previously determined effective for lowering STAU1 abundance. One of these siRNAs, developed by us (si STAU1 , 5’-GUAACUGCCAUGAUAGCCCGAG-3’), was used to design mi STAU1 -A, and a second was based on a published STAU1 siRNA (5’-CCUAUAACUACAACAUGAG-3’) used to design mi STAU1 -B 35 . PHP.eB serotype AAVs were generated by the Virus Packaging Laboratory of the Drug Discovery Core at University of Utah using pUCmini-iCAP-PHP.eB (Addgene #103005). The viral expression vector that we used (pAAV-U6-sgRNA-CMV-GFP, Addgene #85451) includes the U6 promoter to drive miRNA expression and independently expresses GFP under the control of the CMV promoter. BAC-STAU1 mice and wildtype littermates, 20 wks of age, were treated with AAV (9.6E10 viral genomes (VG) or PBS vehicle as indicated, by single intracerebroventricular (ICV) injections to the right lateral ventricle as previously described 36 . Mice were sacrificed at 23 wks of age when spinal cords were collected for analysis by western blotting. Drosophila studies Fly stocks were maintained on standard cornmeal molasses agar. Transgenic lines included gmr-GAL4 (BDSC_1104); UAS- stau .RNAi (shRNA) (BDSC_82965); UAS-hTDP-43 (BDSC_79587) 37 ; UAS-hATXN2(CAG)64 38 ; UAS- stau (Drosophila stau ) 39 . External eye microscopy, paraffin sectioning and quantification were performed as described 38 , 40 . Stau knockdown in these lines has been verified 41 . The GMR-Gal4 functions as a driver line that expresses the Gal4 transcriptional activator under control of the Glass Multimer Reporter (GMR) element. GMR is active in the developing and mature fly eye allowing targeted eye gene expression. Statistical Analysis Statistical differences between selected groups evaluated by qPCR, western blotting, and the electrophysiological data were evaluated using blocked two-way analysis of variance (ANOVA) tests followed by post-hoc tests of significance (Tukey’s or Bonferroni’s tests for multiple comparison, as indicated). Data shown on western blots are mean and SD of biological replicates (n = 3–4 mice) that were each evaluated on a minimum of three blots (3 technical replicates). Behavioral tests were evaluated with repeated measures ANOVA in GraphPad Prism (grip strength testing) or STATA (rotarod testing). Student’s t -tests were unpaired and two-tailed. All tests were performed using the GraphPad Prism software package. Results Generation of BAC-STAU1 mice We produced a BAC mouse harboring the human STAU1 gene. We utilized a 133.8 kb BAC construct (Fig. 1 A) and generated transgenic mice by pronuclear injection. Sequencing of DNA isolated from BAC-STAU1 mouse bone marrow specimens determined that one of four founders, only BAC-STAU1.6, had a single genomic integration of the transgene, on chromosome 17 (chr17:9,394,829-~9,610,924). The integration occurred simultaneously to a 210 kb genomic deletion within this region. Query of the NCBI Reference Sequence Database (RefSeq) confirmed that there are no annotated genes in this region of the mouse genome and therefore no gene disruption is predicted. No structural variants were identified within the integrated construct. Due to the nature of the sequence at the integration site, BAC copy number via this method could not be estimated. BAC-STAU1.6 mice, now simply referred to as BAC-STAU1, are viable, display no abnormal gross morphology or significant segregation distortion. Further sequencing also demonstrated that the STAU1 isoform expressed in CNS tissues of BAC-STAU1 mice is equivalent to the Ensemble STAU1-203 transcript (ENSMUST00000109236.9), and that it lacks a 6 amino acid insertion in the RNA binding domain 3 (RBD3) known to reduce RNA binding affinity 42 . The mean weights of BAC-STAU1 mice were not significantly different from wildtype littermates up to 80 wks (Fig. 1 B). The presence of the BAC-transgene in brain was demonstrated by quantitative PCR using primers specific for human STAU1 vs mouse Stau1 (Fig. 1 C,D). BAC-STAU1 is expressed in multiple CNS cell types in BAC-STAU1 mice. To confirm widespread hSTAU1 expression in the CNS we performed western blotting and qPCR analyses using cultured astrocytes, microglia, and neurons prepared from neonate spinal cord and cortical tissues. hSTAU1 was highly abundant in all cultures from BAC-STAU1 mice vs wildtype littermates (Supplemental Fig. 1A-E). We verified that neuronal cultures expressed NeuN and lacked IBA1, while microglia cultures expressed IBA1 which was absent in neuronal cultures, and we also verified GFAP expression in astrocyte cultures (not shown). There was also approximately 4-fold more hSTAU1 -mRNA in the cortex vs spinal cord of BAC-STAU1 mice, determined by qPCR, while mouse STAU1 was generally mildly reduced in these tissues in BAC-STAU1 vs wildtype littermates (Supplemental Fig. 1F,G). Immunofluorescent labeling We observed widespread STAU1 expression in BAC-STAU1 mice by immunofluorescent labeling, with a trend of increased signal for STAU1 that mirrored the quantified increases in complimentary western blots. STAU1 was strongly present in tissues of BAC-STAU1 mice including cell bodies and neuropil of ventral horn, dorsal horn of the spinal cord, and various brain tissues including the cerebral cortex, hippocampus, cerebellum and pons, compared to WT mice (Supplemental Fig. 2). Behavioral phenotype testing We investigated BAC-STAU1 mice for behavioral phenotypes using both accelerating rotarod and grip strength tests. No significant behavioral differences in motor strength or coordination were observed in either forelimb or hindlimb grip strength tests or rotarod testing (Supplemental Figs. 3 and 4). Molecular phenotype associated with STAU1 overabundance Transcriptome analysis: We carried out RNA-seq of spinal cords from BAC-STAU1 mice at 25 wks and 57 wks of age. The number of mice included were 2 male and 2 female BAC-STAU1 mice compared to 2 male and 2 female WT littermates for the 25 wk group, and 2 male and 2 female BAC-STAU1 mice compared to 2 male and 1 female WT littermates for the 57 wk group. In both cases the number of differentially expressed genes (DEGs) was low, with an FDR cut-off of 10%. The 25 wk group had 30 DEGs while the 57 wk group had 44 DEGs and 3 were shared including Smg5 , Urm1 and Hdac11 (Fig. 2 A). Volcano plots showed that the most significantly upregulated DEG in both experiments was Smg5 , a regulator of UPF1 in the nonsense mediated decay (NMD) pathway (Fig. B). Hallmark and KEGG pathway analyses revealed progressive transcriptome dysregulation in BAC-STAU1 mice with age, with an increased number of annotated pathways in older mice. Hallmark analysis showed 7 enriched gene sets in the 25 wk group and 22 in the 57 wk group, while KEGG pathway analysis showed 7 in the 25 wk group and 31 in the 57 wk group (Fig. 2 C). Enriched Hallmark gene sets shared between the two groups included Epithelial Mesenchymal Transition, Mtorc1 Signaling, Myogenesis, and Oxidative Phosphorylation. Enriched KEGG gene sets shared between the two groups included Alzheimer’s Disease, Huntington’s Disease, Neuroactive Ligand Receptor Interaction, Oxidative Phosphorylation, Parkinson’s Disease, Ribosome, Taste Transduction. Complete RNA-seq and pathway analyses are provided in Supplemental Table 2. Western immunoblotting for autophagy and pathology markers: Given that analysis of DEG pathways showed gene sets in NDDs and MTORC1 signaling, we wanted to test key proteins for their abundance by western blotting. Overabundant STAU1 in the brain, cerebellum and spinal cord was associated with abnormal abundance of autophagy proteins. These include mTOR, p-mTOR, p62, p-S6K and LC3-II in the brain, cerebellum and spinal cord, of which p-S6K was only evaluated in cerebellum (Fig. 3 ). The profile of these proteins and their phosphorylated forms are consistent with reduced autophagy, previously described in vitro 3 . We next examined protein changes often associated with neurodegeneration such as reduction of key neuronal marker proteins and an increase in astrogliosis. We isolated protein extracts from cerebral hemispheres, cerebella, and spinal cords of WT and transgenic mice at 14 weeks-of-age and analyzed abundance by semi-quantitative western blot analysis (Fig. 3 ). In all three tissues, there was a remarkable increase in GFAP and cleaved caspase-3 consistent with increased gliosis and apoptotic cell death. The pan-neuronal marker protein NEUN was decreased in hemispheres and spinal cord, and PC-specific proteins (CALB1, RGS8, PCP2, PCP4, FAM107B) were decreased in cerebellum. Choline-acetyl transferase (CHAT), the marker protein for cholinergic neurons, was decreased by 50% compared to that in WT tissues, not only in spinal cord but also in hemisphere tissue (Fig. 3 ). Mis-splicing of specific mRNAs is part of TDP-43 proteinopathy. In human spinal cord, UNC13A and STMN2 show cryptic exon splicing in the presence of 43 , 44 . Homologous cryptic exons are not known in mice, yet we observed abnormally reduced UNC13A and STMN2 abundance in BAC-STAU1 mouse brain and spinal cord (Fig. 3 A,B and E,F). Modulation of STAU abundance in flies and mice We had previously shown that reduction of STAU1 by genetic interaction improved the motor phenotype of SCA2 mice 3 . The results described above suggest that STAU1 overabundance by itself induces a cellular pre-degenerative or risk phenotype. We wanted to test an extension of this hypothesis in fly and mouse models by modulating STAU1 levels in the presence of disease-associated mutations, both reducing and increasing levels compared to wildtype levels. Stau dosage modulates retinal degeneration in fly TDP-43 and ATXN2 exp models. We selected fly models expressing human TDP-43 and human ATXN2-CAG 64 and varied fly stau dosage using an upregulation line, and a line that reduced fly stau by RNAi. In the presence of normal stau levels, both hTDP-43 and hATXN2-CAG 64 flies showed reduced retinal depth compared to WT flies, reflecting degeneration. When stau levels were then reduced by co-expressing stau shRNA, the retinal depth in flies was significantly improved (Fig. 4 A-C). Transgenic upregulation of fly stau worsened the eye phenotype in hTDP-43 flies. Modulation of stau in WT flies had no effect on eye phenotype or retinal depth (Fig. 4 D). These data indicate that Stau modulates neurotoxicity of hTDP-43 and hATXN2-CAG64 in Drosophila , supporting the potential for targeting STAU1 for treating neurodegenerative diseases. In vivo genetic interaction of overabundant STAU1 and TDP-43 Previously we demonstrated that STAU1 is elevated in cerebellum and spinal cord of Thy1-TDP-43 transgenic mice and in sporadic ALS patient spinal cords, associated with abnormal abundance or phosphorylation of molecular markers of autophagy 6 , 7 . Because autophagy and other neuronal markers are abnormally expressed in BAC-STAU1 mice (Fig. 3 ), we now sought to demonstrate that adding the expression of human STAU1 to TDP-43 transgenic mice would further worsen the molecular phenotype. To do so we performed a genetic cross of BAC-STAU1 mice with Prp-TDP43(Q331K) mice described in Wils et al. 45 . We then evaluated brain hemisphere tissues from mice of all resultant genotypes for the expression of CHAT, NEUN, cleaved CASP3, mTOR, p62 and GFAP, at 8 and 24 wks of age. Significant changes were observed for each of these proteins in BAC-STAU1 mice and Prp-TDP43(Q331K) mice in both age groups (Fig. 5 ). Additionally, nearly all of the proteins were significantly more elevated (STAU1, GFAP, cCASP3, mTOR, p62) or reduced (CHAT, NEUN) in double-transgenic Prp-TDP43(Q331K);BAC-STAU1 vs single-transgenic BAC-STAU1 mice. This was true in both age groups, with a few exceptions (cCASP3 and p62 in the 8 wk group, and mTOR in the 24 wk group) (Fig. 5 ). In general, the in vivo overexpression of hSTAU1 by crossing BAC-STAU1 mice with Prp-TDP43(Q331K) mice resulted in a significantly more severe molecular phenotype in either age group investigated. Note that in Fig. 5 the TDP-43 antibody used recognized only human TDP-43, and full blots using an antibody recognizing both mouse and human TDP-43 is shown in Fig. S5 . Mouse TDP-43 was significantly elevated in BAC-STAU1 brain vs WT by 20–30% (Fig. S5 C). For all other full blots in the study see Fig. S6 . Artificial microRNAs targeting STAU1 improve the abnormal molecular phenotype in BAC-STAU1 mice. As STAU1 overabundance in BAC-STAU1 is associated with abnormal autophagy and neuronal abundance markers, and lowering Stau in the fly improved the retinal depth neurodegenerative phenotype in TDP-43 and ATX2 flies, we sought to determine if lowering STAU1 abundance in BAC-STAU1 mice could improve these features. To do this we generated two different AAVs that deliver artificial microRNAs targeting human STAU1 , designated AAV-miSTAU1-A and AAV-miSTAU1-B. We treated mice at 20 wks of age which was well after phenotype onset observed in Fig. 3 A-F, and sacrificed mice at 23 wks of age. We then removed spinal cords and evaluated proteins by western blotting and quantification (Fig. 6 A,B). Very little STAU1 was observed in WT mice while a high abundance of STAU1 was observed in BAC-STAU1 mice, as well as significantly reduced abundances of CHAT and NEUN, and normalized abundance of mTOR. BAC-STAU1 mice treated with AAVs were positive for GFP on blots, expressed by an independent promotor, indicating positive uptake of the AAVs. BAC-STAU1 mice that were treated with either of AAV-miSTAU1-A or AAV-miSTAU1-B had significantly reduced STAU1 abundance and normalized CHAT, NEUN, and mTOR compared to the BAC-STAU1 mice treated with vehicle. Levels of CHAT, NEUN and mTOR in the AAV-treated BAC-STAU1 mice were close to those observed in the WT mice (Fig. 6 ). Discussion RNA-binding proteins (RBPs) including STAU1 play important roles in health and disease ranging from viral defense to neurodegeneration 46 . Pathogenic variants in some RBPs such as TDP-43 and FUS have been reported in patients with ALS and FTD, directly linking the respective genes to neurodegenerative diseases (NDDs). Overabundance of TDP-43, its subcellular mislocalization, and cytoplasmic aggregation are also typical in patients without pathogenic variants in ALS genes and TDP-43 aggregates are found in > 90% of spinal cords of individuals with sporadic ALS. Although STAU1 mutations have not yet been identified in NDDs, the protein is overabundant in cells and spinal cord tissue from ALS patients and in ALS model systems as a result of a negative post-transcriptional feedback loop on autophagy 9 . Here we examined whether changes seen in vitro in the presence of STAU1 overabundance are replicated in vivo . We describe a new BAC-STAU1 mouse that expresses the entire human STAU1 gene. The transgene was expressed in cerebral hemispheres, cerebellum, and spinal cord (Fig. 3 ) as well as in cultures of mouse neurons, glia, and microglia (Supplemental Fig. 1). Western blot analysis indicated that the BAC transgene expressed full-length STAU1 (Fig. 3 ). Transcriptomics autophagy neuronal apoptosis Based on our findings in vitro 3 , 6 , 7 , 11 , we analyzed BAC-STAU1 mice for molecular markers of autophagy function and neurodegeneration as well as for transcriptomic changes. Whole-genome RNA-seq provided surprisingly few DEGs in spinal cord of BAC-STAU1 mice at 25 and 57 wks-of-age (Fig. 2 ). In both age groups, the nonsense mediated decay regulator Smg5 appeared as the most significantly elevated transcript, supporting a role for STAU1 in NMD. Pathway analyses revealed a common signature of transcriptomic dysregulation in both the 25 and 57 wk age groups associated with neurodegeneration including MTORC1 signaling, oxidative phosphorylation as well as Alzheimer’s, Parkinson and Huntington disease (Fig. 2 C). Semiquantitative WB analyses showed significant abnormalities in pathways involving key signaling cascades with abnormal expression of mTOR, p-mTOR, p-S6K, p62, LC3-II consistent with reduced autophagic flux (Fig. 3 ). These findings were confirmed in extracts from cerebral hemispheres, cerebellum, and spinal cord pointing to the potential role of STAU1 overabundance in NDDs affecting distinct parts of the CNS. We previously established a biomarker profile of PC-specific proteins (CALB1, RGS8, PCP2, PCP4, FAM107B) that show early and continuous decline in two SCA2 mouse models as proxies for PC health and function 3 , 36 , 47 . These proteins were also dysregulated in the cerebellum of BAC-STAU1 mice consistent with PC dysfunction (Fig. 3 C, D). We applied a similar approach to protein extracts of cerebral hemispheres and spinal cords (Fig. 3 A, B, E, F). Protein analyses were consistent with autophagy dysfunction. Neuronal injury was evidenced by decreased expression of NEUN and CHAT. Cleaved caspase-3 was significantly increased consistent with apoptotic cell death. As typically seen in NDDs, there was a significant increase in GFAP expression. Of note, CHAT abundance was not only decreased in spinal cord as a sign of motor neuron degeneration, but also in cerebral hemispheres consistent with degeneration in basal forebrain cholinergic neurons and potential relevance for memory dysfunction in AD and limbic-predominant age-related TDP-43 encephalopathy (LATE). We also examined abundance of UNC13A and STMN2 in spinal cords. Previous studies have shown that TDP-43 mislocalization to the cytoplasm (and loss of nuclear splicing regulation) is associated with the inclusion of cryptic exons in human cells resulting in reduced expression of UNC13A and STMN2 44, 48, 49 . Even though the cryptic exons described in humans have not been observed in mice, the abundances of both proteins were reduced in BAC-STAU1 mice, suggesting that the regulation of these genes, at least in mice, involves some mechanism other than cryptic exons, such as transcription or translation, possibly by way of STAU1-mediated decay. The molecular changes seen in BAC-STAU1 mice were specific and reversible by RNAi. We developed 2 artificial miRNAs to human STAU1 and delivered them by ICV injection. Both constructs worked equally well and reverted marker proteins back to levels seen in WT mice (Fig. 6 ). These findings are in line with our previous observations that genetic reduction of Stau1 improves mouse SCA2 phenotypes 3 and support the development of therapeutics targeting STAU1 for treating neurodegenerative disease. Modulation of Staufen dosage As reducing Stau1 dosage protected cerebellar Purkinje cells and improved motor behavior in an SCA2 mouse model, the question arises whether increasing STAU1 abundance would worsen NDD phenotypes. We therefore examined the effects of staufen dosage in the fly and in a TDP-43 mouse model. Flies expressing human TDP-43 or polyglutamine expanded human ATXN2 (ATXN2-CAG 64 ) were treated with shRNAs targeting the endogenous fly stau gene. The decreased retinal depth seen in flies transgenic for hTDP-43 or hATXN2-CAG 64 was significantly improved by co-expressing stau shRNA (Fig. 4 ). Conversely, upregulation of fly stau further worsened the degenerative phenotype seen in the TDP-43 line, but not in the ATXN2 line. We next investigated the role of Staufen in a mammalian system by crossing BAC-STAU1 mice with Prp-TDP-43(Q331K) transgenic mice, approximately doubling the amount of endogenous Staufen protein. In Prp-TDP-43(Q331K) mice, neuronal marker proteins, glial and apoptotic markers are abnormal already at 8 wks of age, prior to the reported onset of motor abnormalities (Fig. 5 , round symbols). Increasing STAU1 abundance worsened the levels of all marker proteins (CHAT, NEUN, cleaved CASP3, mTOR, p62 and GFAP) in brain with changes increasing from 8 to 24 weeks-of age (Fig. 5 , diamond symbols). Of note, cleaved caspase-3 was 3.5-fold increased in double-transgenic mice compared to a 2.5-fold increase in single transgenic mice. These studies suggest that variability in STAU1 levels in model systems and possibly also in humans can modify neurodegeneration. Genetic and environmental factors affecting STAU1 abundance deserve further study. Implications for human disease Despite the pronounced molecular phenotype in BAC-STAU1 mice including activation of apoptosis, we did not see a progressive motor phenotype, at least for the observation period of up to 1 year and the motor tests used in our studies (grip strength, rotarod) (Supplemental Figs. 3 and 4). It is quite likely that longer observation periods are needed and that more nuanced behavioral tests need to be applied. The sensitivity of NDD phenotypes to levels of STAU1 protein may have implications for mouse models of human NDDs as well as for the human disease. Based on our studies it is reasonable to predict that genetic or environmental variation affecting STAU1 levels will affect NDD phenotypes. Mouse background is known to affect NDD phenotypes 50 , but little is known about CNS STAU1 levels in different mouse strains. Large-scale sequencing projects comparing individuals with ALS to controls so far have not identified STAU1 Mendelian pathogenic variants or suggested risk variants 51 although analyses searching for protective variants have received less attention 52 . It may be that STAU1 variation is not a modifier of disease risk itself, but age-of-onset or rate of progression. It is also possible that the role of STAU1 in early development may limit the extent of genetic variation and that genetic or epigenetic variation of genes acting on STAU1 play a role. Finally, it is possible that environmental factors such as brain trauma affect STAU1 levels, as has been shown for stress granule formation in the fly 53 . Conclusions Our findings in the BAC-STAU1 mouse model demonstrate that STAU1 overabundance alone at levels found in neurodegenerative mouse models is sufficient to impair autophagy and to promote apoptotic cell death in the brain. Moreover, altering STAU1 levels in the presence of disease-associated mutations influences the severity of the resulting phenotypes. These results strengthen the case for STAU1 as a therapeutic target in human neurodegenerative disorders. Abbreviations alzheimer’s disease AD; ataxin-2 ATXN2; antisense oligonucleotide ASO; bacterial artificial chromosome BAC; beta actin ACTB; choline O-acetyltransferase CHAT; central nervous system CNS; differentially expressed gene DEG; glial fibrillary acidic protein GFAP; gram g; human STAU1 hSTAU1; kilogram kg; Kyoto Encyclopedia of Genes and Genomes KEGG; litre l; microtubule-associated protein 1 light chain 3 LC3; mechanistic target of rapamycin mTOR; mechanistic target of rapamycin complex 1 mTORC1; milligram mg; neuronal nuclei (RNA binding Fox-1 homolog 3 (RBFOX3) gene product) NEUN; spinocerebellar ataxia type 2 SCA2; standard deviation SD; standard error of the mean SE; staufen-1 STAU1; stathmin-2 STMN2; trans-activation response DNA-binding protein 43 TDP-43; transgenic TG; Unc-13 homolog A (C. elegans analog) UNC13A; wild-type WT; Declarations Competing Interests: The authors declare no competing interests Competing interests The authors declair no competing financial interests. Ethics All mouse procedures were performed under protocol 2040 approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Utah, SLC, UT. Availability of data The data that support the findings of this study are available on request from the corresponding authors (S.M.P. and D.R.S.). Acknowledgments We thank Scott Redlin and Gentrie Maag for contributing to work in this study. We thank Leeanne McGurk for sharing flies. Viral vectors were provided by Viviana Gradinaru and Hetian Lei. We also thank the University of Utah Cell Imaging Core for assistance. The graphical abstract was created with BioRender.com. This work was supported by a Harrington Discovery Institute Rare Disease Scholarship to D.R.S., and National Institutes of Health (NIH) / National Institute of Neurological Disorders and Stroke (NINDS) grants, R21NS127028, R01NS137233 and R01NS097903 to D.R.S., R21NS128630 to D.R.S. and M.G., R56NS033123, R37NS033123, R35NS127253 to S.M.P., R61/R33NS124965 to D.R.S. and S.M.P., and R35NS097275 to N.M.B. Author contributions S.M.P., D.R.S. conceived the study and jointly supervised research. N.M.B. conceived and supervised drosophila studies. S.P., H.N., M.G., W.D, K.P.F. designed and performed experiments. W.D. and K.P.F. generated transgenic mice. M.G. and W.D. performed RNA-seq studies. S.P. performed western blotting of mouse tissues. H.N. performed drosophila studies. M.G. produced primary cell cultures and conducted their analyses. M.G. performed immunohistological stains. K.P.F. and D.R.S. supervised motor phenotype testing. S.P., H.N., M.G., and D.R.S. performed statistical tests. S.M.P., S.P., M.G., K.P.F., N.M.B. and D.R.S. contributed to writing the manuscript. References St Johnston D, Beuchle D, Nusslein-Volhard C. Staufen, a gene required to localize maternal RNAs in the Drosophila egg. Cell 1991, 66(1): 51–63. Tosar LJ, Thomas MG, Baez MV, Ibanez I, Chernomoretz A, Boccaccio GL. Staufen: from embryo polarity to cellular stress and neurodegeneration. Front Biosci (Schol Ed) 2012, 4: 432–452. Paul S, Dansithong W, Figueroa KP, Scoles DR, Pulst SM. Staufen1 links RNA stress granules and autophagy in a model of neurodegeneration. Nat Commun 2018, 9(1): 3648. Pulst SM, Nechiporuk A, Nechiporuk T, Gispert S, Chen XN, Lopes-Cendes I, et al. Moderate expansion of a normally biallelic trinucleotide repeat in spinocerebellar ataxia type 2. Nat Genet 1996, 14(3): 269–276. Scoles DR, Pulst SM. Spinocerebellar Ataxia Type 2. Adv Exp Med Biol 2018, 1049: 175–195. Paul S, Dansithong W, Figueroa KP, Gandelman M, Scoles DR, Pulst SM. Staufen1 in Human Neurodegeneration. Ann Neurol 2021, 89(6): 1114–1128. Paul S, Dansithong W, Gandelman M, Figueroa KP, Zu T, Ranum LPW, et al. Staufen impairs autophagy in neurodegeneration. Ann Neurol 2023, 93(2): 398–416. Li CL, Zhou GF, Xie XY, Wang L, Chen X, Pan QL, et al. STAU1 exhibits a dual function by promoting amyloidogenesis and tau phosphorylation in cultured cells. Exp Neurol 2024, 377: 114805. Pulst SM, Scoles DR, Paul S. Effects of STAU1/staufen1 on autophagy in neurodegenerative diseases. Autophagy 2023, 19(9): 2607–2608. Zhao R, Huang S, Li J, Gu A, Fu M, Hua W, et al. Excessive STAU1 condensate drives mTOR translation and autophagy dysfunction in neurodegeneration. The Journal of cell biology 2024, 223(8). Gandelman M, Dansithong W, Figueroa KP, Paul S, Scoles DR, Pulst SM. Staufen 1 amplifies proapoptotic activation of the unfolded protein response. Cell Death Differ 2020, 27(10): 2942–2951. Sakurai M, Shiromoto Y, Ota H, Song C, Kossenkov AV, Wickramasinghe J, et al. ADAR1 controls apoptosis of stressed cells by inhibiting Staufen1-mediated mRNA decay. Nat Struct Mol Biol 2017, 24(6): 534–543. Park E, Maquat LE. Staufen-mediated mRNA decay. Wiley interdisciplinary reviews RNA 2013, 4(4): 423–435. Lebeau G, Maher-Laporte M, Topolnik L, Laurent CE, Sossin W, Desgroseillers L, et al. Staufen1 regulation of protein synthesis-dependent long-term potentiation and synaptic function in hippocampal pyramidal cells. Mol Cell Biol 2008, 28(9): 2896–2907. Kohrmann M, Luo M, Kaether C, DesGroseillers L, Dotti CG, Kiebler MA. Microtubule-dependent recruitment of Staufen-green fluorescent protein into large RNA-containing granules and subsequent dendritic transport in living hippocampal neurons. Mol Biol Cell 1999, 10(9): 2945–2953. Yu Z, Fan D, Gui B, Shi L, Xuan C, Shan L, et al. Neurodegeneration-associated TDP-43 interacts with fragile X mental retardation protein (FMRP)/Staufen (STAU1) and regulates SIRT1 expression in neuronal cells. J Biol Chem 2012, 287(27): 22560–22572. Chu JF, Majumder P, Chatterjee B, Huang SL, Shen CJ. TDP-43 Regulates Coupled Dendritic mRNA Transport-Translation Processes in Co-operation with FMRP and Staufen1. Cell reports 2019, 29(10): 3118–3133 e3116. Iguchi Y, Katsuno M, Niwa JI, Yamada SI, Sone J, Waza M, et al. TDP-43 depletion induces neuronal cell damage through dysregulation of Rho family GTPases. J Biol Chem 2009, 284(33): 22059–22066. Lucas BA, Lavi E, Shiue L, Cho H, Katzman S, Miyoshi K, et al. Evidence for convergent evolution of SINE-directed Staufen-mediated mRNA decay. Proc Natl Acad Sci U S A 2018, 115(5): 968–973. Elbarbary RA, Li W, Tian B, Maquat LE. STAU1 binding 3' UTR IRAlus complements nuclear retention to protect cells from PKR-mediated translational shutdown. Genes Dev 2013, 27(13): 1495–1510. Gong C, Maquat LE. lncRNAs transactivate STAU1-mediated mRNA decay by duplexing with 3' UTRs via Alu elements. Nature 2011, 470(7333): 284–288. Vessey JP, Macchi P, Stein JM, Mikl M, Hawker KN, Vogelsang P, et al. A loss of function allele for murine Staufen1 leads to impairment of dendritic Staufen1-RNP delivery and dendritic spine morphogenesis. Proc Natl Acad Sci U S A 2008, 105(42): 16374–16379. Dansithong W, Paul S, Figueroa KP, Rinehart MD, Wiest S, Pflieger LT, et al. Ataxin-2 regulates RGS8 translation in a new BAC-SCA2 transgenic mouse model. PLoS Genet 2015, 11(4): e1005182. Gandelman M, Peluffo H, Beckman JS, Cassina P, Barbeito L. Extracellular ATP and the P2X7 receptor in astrocyte-mediated motor neuron death: implications for amyotrophic lateral sclerosis. J Neuroinflammation 2010, 7: 33. Gandelman M, Levy M, Cassina P, Barbeito L, Beckman JS. P2X7 receptor-induced death of motor neurons by a peroxynitrite/FAS-dependent pathway. J Neurochem 2013, 126(3): 382–388. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 2013, 29(1): 15–21. Bushnell B, Rood J, Singer E. BBMerge - Accurate paired shotgun read merging via overlap. PLoS ONE 2017, 12(10): e0185056. Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet j 2011, 17(1): 3. Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 2014, 30(7): 923–930. Ewels P, Magnusson M, Lundin S, Kaller M. MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics 2016, 32(19): 3047–3048. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 2014, 15(12): 550. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A 2005, 102(43): 15545–15550. Castanza AS, Recla JM, Eby D, Thorvaldsdottir H, Bult CJ, Mesirov JP. Extending support for mouse data in the Molecular Signatures Database (MSigDB). Nat Methods 2023, 20(11): 1619–1620. Boudreau RL, Davidson BL. Generation of hairpin-based RNAi vectors for biological and therapeutic application. Methods Enzymol 2012, 507: 275–296. Kim YK, Furic L, Desgroseillers L, Maquat LE. Mammalian Staufen1 recruits Upf1 to specific mRNA 3'UTRs so as to elicit mRNA decay. Cell 2005, 120(2): 195–208. Scoles DR, Meera P, Schneider MD, Paul S, Dansithong W, Figueroa KP, et al. Antisense oligonucleotide therapy for spinocerebellar ataxia type 2. Nature 2017, 544(7650): 362–366. Elden AC, Kim HJ, Hart MP, Chen-Plotkin AS, Johnson BS, Fang X, et al. Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS. Nature 2010, 466(7310): 1069–1075. McGurk L, Rifai OM, Shcherbakova O, Perlegos AE, Byrns CN, Carranza FR, et al. Toxicity of pathogenic ataxin-2 in Drosophila shows dependence on a pure CAG repeat sequence. Hum Mol Genet 2021, 30(19): 1797–1810. Jia M, Shan Z, Yang Y, Liu C, Li J, Luo ZG, et al. The structural basis of Miranda-mediated Staufen localization during Drosophila neuroblast asymmetric division. Nat Commun 2015, 6: 8381. McGurk L, Mojsilovic-Petrovic J, Van Deerlin VM, Shorter J, Kalb RG, Lee VM, et al. Nuclear poly(ADP-ribose) activity is a therapeutic target in amyotrophic lateral sclerosis. Acta neuropathologica communications 2018, 6(1): 84. Song C, Leahy SN, Rushton EM, Broadie K. RNA-binding FMRP and Staufen sequentially regulate the Coracle scaffold to control synaptic glutamate receptor and bouton development. Development 2022, 149(9). Monshausen M, Putz U, Rehbein M, Schweizer M, DesGroseillers L, Kuhl D, et al. Two rat brain staufen isoforms differentially bind RNA. J Neurochem 2001, 76(1): 155–165. Brown AL, Wilkins OG, Keuss MJ, Hill SE, Zanovello M, Lee WC, et al. TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A. Nature 2022, 603(7899): 131–137. Prudencio M, Humphrey J, Pickles S, Brown AL, Hill SE, Kachergus JM, et al. Truncated stathmin-2 is a marker of TDP-43 pathology in frontotemporal dementia. J Clin Invest 2020, 130(11): 6080–6092. Wils H, Kleinberger G, Janssens J, Pereson S, Joris G, Cuijt I, et al. TDP-43 transgenic mice develop spastic paralysis and neuronal inclusions characteristic of ALS and frontotemporal lobar degeneration. Proc Natl Acad Sci U S A 2010, 107(8): 3858–3863. Cottrell KA, Andrews RJ, Bass BL. The competitive landscape of the dsRNA world. Mol Cell 2024, 84(1): 107–119. Hansen ST, Meera P, Otis TS, Pulst SM. Changes in Purkinje cell firing and gene expression precede behavioral pathology in a mouse model of SCA2. Hum Mol Genet 2013, 22(2): 271–283. Melamed Z, Lopez-Erauskin J, Baughn MW, Zhang O, Drenner K, Sun Y, et al. Premature polyadenylation-mediated loss of stathmin-2 is a hallmark of TDP-43-dependent neurodegeneration. Nat Neurosci 2019, 22(2): 180–190. Ma XR, Prudencio M, Koike Y, Vatsavayai SC, Kim G, Harbinski F, et al. TDP-43 represses cryptic exon inclusion in the FTD-ALS gene UNC13A. Nature 2022, 603(7899): 124–130. Zhong MZ, Peng T, Duarte ML, Wang M, Cai D. Updates on mouse models of Alzheimer's disease. Mol Neurodegener 2024, 19(1): 23. van Rheenen W, van der Spek RAA, Bakker MK, van Vugt J, Hop PJ, Zwamborn RAJ, et al. Common and rare variant association analyses in amyotrophic lateral sclerosis identify 15 risk loci with distinct genetic architectures and neuron-specific biology. Nat Genet 2021, 53(12): 1636–1648. Pottinger TD, Motelow JE, Povysil G, Moreno CAM, Ren Z, Phatnani H, et al. Rare variant analyses validate known ALS genes in a multi-ethnic population and identifies ANTXR2 as a candidate in PLS. BMC Genomics 2024, 25(1): 651. Anderson EN, Gochenaur L, Singh A, Grant R, Patel K, Watkins S, et al. Traumatic injury induces stress granule formation and enhances motor dysfunctions in ALS/FTD models. Hum Mol Genet 2018, 27(8): 1366–1381. Additional Declarations (Not answered) Supplementary Files FigS1primarycellcultures.tif Supplemental Fig. 1. Expression of STAU1 in cultured cells of the brain cortex and spinal cord of BAC-STAU1 mice. Spinal cord cultures were prepared from neonate BAC-STAU1 mice (TG) or wildtype littermates (WT), cultured for 3 wks with conditions enriching for microglia and astrocytes, or for production of neurons. A-E) Western blotting: STAU1 was highly abundant in microglia of the cortex and SC (A&B), in astrocytes of cortex and SC (C&D), and cortical neurons (E). In A-E each lane represents one culture from different mice. F-G) qPCR: Human STAU1 (F) and mouse Stau1 (G) detected in cultured SC astrocytes and cultured cortical astrocytes. RQ, relative quantity to Actb . ***, p<0.001, ANOVA with Bonferroni post-hoc correction. FigS2histologyCMYK.tif Supplemental Fig. 2. Immunofluorescent labeling of BAC-STAU1 compared to WT mouse tissues. STAU1 labeling trends stronger in BAC-STAU1 mice vs WT mice in A) spinal cord tissues including in the cell bodies and neuropil in ventral horn and dorsal horn, and B) brain tissues including the cerebral cortex, hippocampus, cerebellum and pons. Bars: 100 µm (A, ventral and dorsal horn), 50 µm (A, white matter), 20 µm (B). FigS3grip.tif Supplemental Fig. 3. Grip strength testing. BAC-STAU1 mice were tested for forelimb and hindlimb strength at ages 16 wks (A,B) and 20 wks (C,D). Significant difference was observed for hindlimb strength at 16 wks (B), however statistical difference was not observed at 20 wks (D). N=22 WT & 23 TG (A,B) and 21 WT & 21 TG (C,D). Significance was determined using repeated measures ANOVA. Values shown are means of 3 replicates per mouse, and bars represent group means. ns, not significant; *, p<0.05. FigS4rotarodtestingCMYK.tif Supplemental Fig. 4. Rotarod behavioral testing. BAC-STAU1 mice and wildtype littermates were tested on the rotarod in 5 timepoints as indicated. Testing was performed in duplicate over three days in a week of testing. Values shown are mean ±SEM. No significant differences were observed (repeated measures ANOVA). N= 28 WT & 30 TG (10 wks), 23 WT & 27 TG (20 wks), 22 WT & 23 TG (40, 50, 55 wks). FigS5mTDP43CMYK.tif Supplemental Fig. 5. STAU1 genetic interaction with TDP-43. Western blots corresponding to those in Fig. 5 but using an antibody that recognizes both mouse TDP-43 (mTDP-43) and human TDP-43 (hTDP-43), presenting full blots. As in Fig. 5, BAC-STAU1 mice were crossed with Prp-TDP43(Q331K) mice and brain hemisphere tissues from mice of each resultant genotype were evaluated by western blotting. Western blotting and quantification of the indicated proteins from brain of 8 wk old mice (A) and 24 wk old mice (B), and quantifications of mTDP-43 (C). Each lane represents an individual mouse, N=3-4 mice per group. Means ± SD of biological replicates are plotted; the SD of technical replicates ranged from 0.01-1.0 (8 wks), 0.01 to 0.06 (24 wks).. ns, non-significant; **, p<0.01, two-way ANOVA with Bonferroni post-hoc correction. FigS6allfullblots.pdf Supplemental Fig. 6. Full blots. Presentation of full-blots for all western blot experiments, except for Supplemental Fig. 5 that is shown separately. TableS1genotypingprimers.xlsx Supplemental Table 1: BAC-STAU1 genotyping primers. TableS2RNAseqdata.xlsx Supplemental Table 2: RNA-seq and pathway analyses. 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1","display":"","copyAsset":false,"role":"figure","size":1531571,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeneration and characterization of a BAC-STAU1 transgenic mouse model.\u003c/strong\u003e A) Schematic of the BAC construct showing the relative position of the \u003cem\u003eSTAU1\u003c/em\u003e gene and flanking sequences. B) Weights of female and male WT and BAC-STAU1 (TG) mice for 7 age groups over 80 wks. N = 11-14 mice (WT-F and WT-M), 10-16 mice (TG-F), 13-14 mice (TG-M). Two outlier mice remain in the chart that were 5.7 (WT-F) and 7.7 (WT-M) greater than the SD in the 80\u003csup\u003eth\u003c/sup\u003e wk. C,D) qPCR using brain extracts for human \u003cem\u003eSTAU1\u003c/em\u003e (C) and mouse \u003cem\u003eStau1\u003c/em\u003e (D) for 8 and 24 wk old BAC-STAU1 mice compared to wildtype mice. ns, non-significant; **, p\u0026lt;0.01; ****, p\u0026lt;0.0001, two-way ANOVA with Bonferroni post-hoc correction.\u003c/p\u003e","description":"","filename":"Fig1weightsandqPCR.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-7411941/v1/3579b4e1cf8f25f816b842d9.png"},{"id":91931587,"identity":"c4971d18-f6c1-4112-b26d-8223f22d9ebb","added_by":"auto","created_at":"2025-09-23 02:31:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3687120,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBAC-STAU1 mouse spinal cord transcriptomes.\u003c/strong\u003e Two independent RNA-seq experiments were performed using 25 wk old and 57 wk old BAC-STAU1 mice. A) 30 and 44 DEGs were identified (FDR=10), including 22 up- and 8 downregulated DEGs in the 25 wk group, and 31 up- and 13 downregulated DEGs in the 57 wk group. Three DEGs upregulated in both are marked by an asterisk. B) Volcano plots showing distribution of up- and downregulated DEGs showing \u003cem\u003eSmg5\u003c/em\u003e as the most upregulated DEG in both experiments. C) Hallmark and KEGG pathway analyses showing an increased number of significant annotated pathways in the 57 wk group, and VENN diagrams indicating the numbers of shared pathways between the 25 and 57 wk groups.\u003c/p\u003e","description":"","filename":"Fig2RNAseqCMYK.png","url":"https://assets-eu.researchsquare.com/files/rs-7411941/v1/a5c6692bfa818a089f7e51ea.png"},{"id":91931581,"identity":"b404fe71-c085-46b8-bdd4-34bf94330d62","added_by":"auto","created_at":"2025-09-23 02:31:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":11600744,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular characterization of BAC-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSTAU1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emice.\u003c/strong\u003e Western blotting showing autophagy and other abnormal molecular marker proteins as indicated in the cerebral hemispheres (A \u0026amp; B), cerebellum (C \u0026amp; D) and spinal cord (E \u0026amp; F). Mice were 14 weeks of age. Each lane represents an individual mouse, N=3-4 mice per group. Means ± SD of biological replicates are plotted; the SD of technical replicates ranged from 0.03-0.54 (B), 0.02-0.48 (D), 0.05 to 0.37 (F). ****, p\u0026lt;0.0001, two-way ANOVA with Bonferroni post-hoc correction.\u003c/p\u003e","description":"","filename":"Fig3autophagyphenotype.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-7411941/v1/9239c6ea69e1daaa6a0ffa2e.png"},{"id":91931579,"identity":"74a55dc8-9070-4814-8a6b-f62b6984cf3d","added_by":"auto","created_at":"2025-09-23 02:31:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":9922609,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModulation of fly STAU levels.\u003c/strong\u003e Upregulation of fly STAU enhances the retinal degeneration phenotype in flies expressing hTDP-43 (a) or hATXN2-CAG\u003csub\u003e64\u003c/sub\u003e (b), whereas reduction of Stau by shRNA improves retinal depth. (c) Quantification of retinal depth. (d) Modulation of Stau levels in wildtype (WT) flies has no effect on the external eye or retinal depth eye. Control indicates lines crossed to \u003cem\u003ew\u003c/em\u003e\u003csup\u003e\u003cem\u003e1118\u003c/em\u003e\u003c/sup\u003e, a line with normal WT Stau levels. ns, non-significant; **, p\u0026lt;0.01, two-way ANOVA with Bonferroni post-hoc correction.\u003c/p\u003e","description":"","filename":"Fig4DrosophilaCMYK.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-7411941/v1/17c78194317d57867cb61698.png"},{"id":91931597,"identity":"0526b6d2-2f41-4a9b-bac7-ddc376e422b5","added_by":"auto","created_at":"2025-09-23 02:31:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":11687535,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSTAU1 genetic interaction with TDP-43.\u003c/strong\u003e BAC-STAU1 mice were crossed with Prp-TDP43(Q331K) mice and brain hemisphere tissues from mice of each resultant genotype were evaluated by western blotting. Western blotting and quantification of the indicated proteins from brain of 8 wk old mice (A,B) and 24 wk old mice (C,D). Each lane represents an individual mouse, N=3-4 mice per group. Means ± SD of biological replicates are plotted; the SD of technical replicates ranged from 0.13-1.2 (B), 0.15 to 1.2 (D). ns, non-significant; *, p\u0026lt;0.05; **, p\u0026lt;0.01; ***, p\u0026lt;0.001; ****, p\u0026lt;0.0001, two-way ANOVA with Bonferroni post-hoc correction.\u003c/p\u003e","description":"","filename":"Fig5geneticinteractionREVISED.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-7411941/v1/d0600e5c3083c18ca75e573a.png"},{"id":91934180,"identity":"a3e729be-005b-40f4-bc34-7d00bb6ecdf3","added_by":"auto","created_at":"2025-09-23 02:39:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2550701,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSTAU1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in BAC-STAU1 mice with an artificial microRNA normalizes CHAT, NEUN and mTOR abundance in spinal cord.\u003c/strong\u003e BAC-STAU1 mice 20 wks of age were treated with 9.6E10 VG of AAV-\u003cem\u003emiSTAU1-A\u003c/em\u003e or AAV-\u003cem\u003emiSTAU1-B\u003c/em\u003e by single ICV injections. Control mice were treated with PBS vehicle. Mice were sacrificed at 23 wks of age and spinal cord proteins were evaluated by western blotting (A). Quantifications of three replicate blots demonstrated 50% reduction of STAU1 in SC, and that mice treated with either of the miRNAs had normalized levels of CHAT, NEUN and mTOR compared to levels in WT mice (B). The lower STAU1 band is non-specific to the antibody. Each lane represents a different mouse, N=3-4 mice. Means ± SD of biological replicates are plotted; the SD of the technical replicates ranged from 0.03-0.37. \u0026nbsp;****, p\u0026lt;0.0001, two-way ANOVA with Bonferroni post-hoc correction.\u003c/p\u003e","description":"","filename":"Fig6AAVmiRNAmTORadded.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-7411941/v1/05258eeb970fbe6c13e8ec0d.png"},{"id":91937942,"identity":"c30c7505-2b95-4050-b8ce-2f201c8e8a1b","added_by":"auto","created_at":"2025-09-23 03:04:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":42873003,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7411941/v1/2056c678-e075-420f-9f89-3960cff58a99.pdf"},{"id":91931586,"identity":"aafa1602-65e2-4b4f-a096-566ed0a56873","added_by":"auto","created_at":"2025-09-23 02:31:36","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":398064,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Fig. 1.\u003c/strong\u003e \u003cstrong\u003eExpression of STAU1 in cultured cells of the brain cortex and spinal cord of BAC-STAU1 mice.\u003c/strong\u003e Spinal cord cultures were prepared from neonate BAC-STAU1 mice (TG) or wildtype littermates (WT), cultured for 3 wks with conditions enriching for microglia and astrocytes, or for production of neurons. A-E) Western blotting: STAU1 was highly abundant in microglia of the cortex and SC (A\u0026amp;B), in astrocytes of cortex and SC (C\u0026amp;D), and cortical neurons (E). In A-E each lane represents one culture from different mice. F-G) qPCR: Human \u003cem\u003eSTAU1\u003c/em\u003e (F) and mouse \u003cem\u003eStau1\u003c/em\u003e(G) detected in cultured SC astrocytes and cultured cortical astrocytes. RQ, relative quantity to \u003cem\u003eActb\u003c/em\u003e. ***, p\u0026lt;0.001, ANOVA with Bonferroni post-hoc correction.\u003c/p\u003e","description":"","filename":"FigS1primarycellcultures.tif","url":"https://assets-eu.researchsquare.com/files/rs-7411941/v1/62cefb236ae5e6854c54d03a.tif"},{"id":91937450,"identity":"5ebb3b85-425f-46f4-97a0-91799fceb9a0","added_by":"auto","created_at":"2025-09-23 02:55:36","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12493240,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Fig. 2. Immunofluorescent labeling of BAC-STAU1 compared to WT mouse tissues. \u003c/strong\u003eSTAU1 labeling trends stronger in BAC-STAU1 mice vs WT mice in A) spinal cord tissues including in the cell bodies and neuropil in ventral horn and dorsal horn, and B) brain tissues including the cerebral cortex, hippocampus, cerebellum and pons. Bars: 100 µm (A, ventral and dorsal horn), 50 µm (A, white matter), 20 µm (B).\u003c/p\u003e","description":"","filename":"FigS2histologyCMYK.tif","url":"https://assets-eu.researchsquare.com/files/rs-7411941/v1/36aa0e8fa4b46cf910653565.tif"},{"id":91934177,"identity":"6b64989a-927d-4b78-a28f-bf18abfad7d9","added_by":"auto","created_at":"2025-09-23 02:39:36","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":275096,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Fig. 3. Grip strength testing.\u003c/strong\u003e BAC-STAU1 mice were tested for forelimb and hindlimb strength at ages 16 wks (A,B) and 20 wks (C,D). Significant difference was observed for hindlimb strength at 16 wks (B), however statistical difference was not observed at 20 wks (D). N=22 WT \u0026amp; 23 TG (A,B) and 21 WT \u0026amp; 21 TG (C,D). Significance was determined using repeated measures ANOVA. Values shown are means of 3 replicates per mouse, and bars represent group means. ns, not significant; *, p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"FigS3grip.tif","url":"https://assets-eu.researchsquare.com/files/rs-7411941/v1/9d93b53beb7e1047e4517028.tif"},{"id":91934179,"identity":"351e7ab1-8b0d-40ef-a2a6-e604475eea1d","added_by":"auto","created_at":"2025-09-23 02:39:36","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":468940,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Fig. 4. Rotarod behavioral testing.\u003c/strong\u003e BAC-STAU1 mice and wildtype littermates were tested on the rotarod in 5 timepoints as indicated. Testing was performed in duplicate over three days in a week of testing. Values shown are mean ±SEM. No significant differences were observed (repeated measures ANOVA). N= 28 WT \u0026amp; 30 TG (10 wks), 23 WT \u0026amp; 27 TG (20 wks), 22 WT \u0026amp; 23 TG (40, 50, 55 wks).\u003c/p\u003e","description":"","filename":"FigS4rotarodtestingCMYK.tif","url":"https://assets-eu.researchsquare.com/files/rs-7411941/v1/c9b3b9781e1b32e1aff71f32.tif"},{"id":91936371,"identity":"b74bcc74-ff64-4df8-89bc-3097f639a880","added_by":"auto","created_at":"2025-09-23 02:47:36","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":2513024,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Fig. 5. STAU1 genetic interaction with TDP-43.\u003c/strong\u003e Western blots corresponding to those in Fig. 5 but using an antibody that recognizes both mouse TDP-43 (mTDP-43) and human TDP-43 (hTDP-43), presenting full blots. As in Fig. 5, BAC-STAU1 mice were crossed with Prp-TDP43(Q331K) mice and brain hemisphere tissues from mice of each resultant genotype were evaluated by western blotting. Western blotting and quantification of the indicated proteins from brain of 8 wk old mice (A) and 24 wk old mice (B), and quantifications of mTDP-43 (C). Each lane represents an individual mouse, N=3-4 mice per group. Means ± SD of biological replicates are plotted; the SD of technical replicates ranged from 0.01-1.0 (8 wks), 0.01 to 0.06 (24 wks).. ns, non-significant; **, p\u0026lt;0.01, two-way ANOVA with Bonferroni post-hoc correction.\u003c/p\u003e","description":"","filename":"FigS5mTDP43CMYK.tif","url":"https://assets-eu.researchsquare.com/files/rs-7411941/v1/e0f17e7d105a627b9a032033.tif"},{"id":91936370,"identity":"85f80ff4-7d49-41bc-8cc5-36a53d7aae83","added_by":"auto","created_at":"2025-09-23 02:47:36","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":2387801,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Fig. 6. Full blots. \u003c/strong\u003ePresentation of full-blots for all western blot experiments, except for Supplemental Fig. 5 that is shown separately.\u003c/p\u003e","description":"","filename":"FigS6allfullblots.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7411941/v1/4c87b40f810d39a35effe4ec.pdf"},{"id":91931594,"identity":"a652fb73-e2ee-41f6-bf03-c0be90e17541","added_by":"auto","created_at":"2025-09-23 02:31:36","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":12846,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Table 1: BAC-STAU1 genotyping primers.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"TableS1genotypingprimers.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7411941/v1/246f3f8563f672687bb8bf8a.xlsx"},{"id":91931613,"identity":"50ebc6d1-5d1b-474b-a944-8f10dbebb278","added_by":"auto","created_at":"2025-09-23 02:31:37","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":6219215,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Table 2: RNA-seq and pathway analyses.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"TableS2RNAseqdata.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7411941/v1/5f39a62b49f3bd588dd91e36.xlsx"}],"financialInterests":"(Not answered)","formattedTitle":"A human Staufen1 BAC transgenic mouse exhibits abnormal autophagy and neurodegeneration across the central nervous system","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSTAU1 is a double stranded RNA binding protein (RBD) initially described in the fly for its essential role in the transport and spatiotemporal localization of mRNAs critical for embryonic patterning\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. It is a multi-functional RBP that also regulates mRNA translation and decay through interactions with double-stranded structures in specific 5\u0026prime; and 3\u0026prime; UTRs\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSTAU1 is an interactor of ATXN2\u003csup\u003e3\u003c/sup\u003e, another RBD that is mutated in spinocerebellar ataxia type 2 (SCA2)\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e and, more rarely, in ALS and PD (reviewed in Scoles and Pulst\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e). STAU1 is also a stress granule (SG) protein that aggregates with ATXN2 in cytoplasmic condensates within cerebellar Purkinje cells (PCs) of SCA2 patients and mice\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. STAU1 abundance is increased in multiple neurodegenerative disease (NDD) models and in tissues from patients with SCA2, sporadic and familial ALS, and frontotemporal dementia (FTD) associated with C9ORF72 and TDP-43 pathology\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In Alzheimer disease and AD related disorders (AD/ADRDs), STAU1 overabundance is seen in APP/PS1 transgenic mice with accumulation of β-amyloid (Aβ) and Tau-neurofibrillary tangles. This was associated with STAU1-mediated stabilization of β-amyloid converting enzyme 1 (BACE1\u003cem\u003e)\u003c/em\u003e expression\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eMechanistically, STAU1 overabundance elevates mTOR translation by directly interacting with the 5\u0026rsquo;-UTR \u003cem\u003eMTOR\u003c/em\u003e mRNA. As mTOR is a negative regulator of autophagy, its increased activity leads to autophagy suppression\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Thus, STAU1 overabundance is a post-transcriptional effect associated with decreased autophagic clearance due to impaired autophagy\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. We have proposed a model that leads to a maladaptive feed-forward loop that can be initiated by cellular stress, STAU1 overabundance, and reduced autophagy or misfolded proteins\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Recently, Zhou \u003cem\u003eet al.\u003c/em\u003e independently confirmed the control of \u003cem\u003eMTOR\u003c/em\u003e translation by STAU1 and also demonstrated that STAU1 overabundance drives the production of biomolecular condensates rich in \u003cem\u003eMTOR\u003c/em\u003e mRNA\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Furthermore, STAU1 can act as a modulator of the unfolded protein response (UPR): In fibroblasts from individuals with SCA2 or pathogenic TDP-43 or C9ORF72 mutations, increased CHOP levels were restored to normal by STAU1 knockdown\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eOther functions of STAU1 include degradation of cell-specific subsets of mRNAS, usually by interaction with the 3\u0026rsquo;-UTR in a process designated Staufen1-mediated decay (SMD), a UPF1-dependent process linked to cell death\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. In neurons, STAU1 traffics mRNAs in coordination with TDP-43, implicating it in synaptic function, cognitive decline, and autophagy-related neuroprotection\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. STAU1 also binds Alu and IRAlu elements to control mRNA nuclear export\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe multi-facetted role of STAU1 in NDDs prompted us to study whether STAU1 was necessary and/or sufficient to mediate changes in autophagy or in UPR in cells with NDD-causing mutations. Indeed, \u003cem\u003ein vitro\u003c/em\u003e STAU1 knockdown normalized these cellular processes. Surprisingly, exogenous expression of STAU1 in wildtype cells was sufficient to alter autophagy and UPR\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eHere we further extended these studies for understanding the consequences of STAU1 overabundance \u003cem\u003ein vivo.\u003c/em\u003e To this end, we generated a new bacterial artificial chromosome (BAC)-STAU1 mouse that includes 133.8 kb of the human STAU1 locus including all exons and introns as well as up- and downstream regions. This mouse line will also be useful for preclinical development of human \u003cem\u003eSTAU1\u003c/em\u003e-directed therapies.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eMice\u003c/p\u003e\u003cp\u003eThe BAC-STAU1 mouse model was developed by the University of Utah Transgenic Mouse Core by pronuclear microinjection of a non-linearized human Staufen1 bacterial artificial chromosome (BAC) construct (BAC-STAU1 clone RP11-120I11, BACPAC Resources) into fertilized oocytes sourced from mice with a B6/D2 mixed hybrid background (B6D2F1J, The Jackson Laboratory stock #100006). Prior to injection, the BAC construct was separated from other nucleotide fragments by pulsed field gel electrophoresis, and gel purified, by the University of Michigan Transgenic Mouse Core. Genotyping of mouse tail DNA was initially done with multiplex PCRs covering all coding exons, 5\u0026rsquo; and 3\u0026rsquo; UTR and the 5\u0026rsquo; upstream promoter region (ENST00000371856.7, STAU1-207, NM_017453.4). Transgenic mouse bone marrow samples were sequenced and analyzed by Cergentis to determine integration sites and vector-vector breakpoints that represent concatemerization. BAC-STAU1 mice were maintained in a B6D2 mixed background by backcrossing to B6D2F1J no less than every 4 generations. The \u003cem\u003eStau1\u003c/em\u003e\u003csup\u003e\u003cem\u003etm1Apa(\u0026minus;/\u0026minus;)\u003c/em\u003e\u003c/sup\u003e (\u003cem\u003eStau1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) mouse\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e was a generous gift from Prof. Michael A. Kiebler, Ludwig Maximilian University of Munich, Germany, and maintained in a C57BL/6J background. Mice were maintained in a temperature and humidity-controlled environment on a 12h light/dark cycle with light onset at 6:00 AM. Both males and females were used in animal studies.\u003c/p\u003e\u003cp\u003eGenotyping\u003c/p\u003e\u003cp\u003eGenotyping was performed by PCR using tail DNAs as previously described\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Our approach verified the presence of all \u003cem\u003eSTAU1\u003c/em\u003e exons. Genotyping primers are provided in Supplemental Table\u0026nbsp;1.\u003c/p\u003e\u003cp\u003ePrimary cell culture\u003c/p\u003e\u003cp\u003ePurified astrocytes and microglia were prepared from mixed glial cultures from spinal cord or brain cortex of neonatal mice as previously described\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Mice were sacrificed and brain cortices and spinal cords were isolated for culture and cerebella for genotyping by PCR as described above. To establish primary mixed glial cultures, the corresponding tissue was dissected, digested with trypsin, mechanically dissociated and passed through a 50 \u0026micro;M mesh. The single cell suspension was plated at a density of 2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e and maintained in DMEM supplemented with 10% fetal bovine serum, penicillin and streptomycin (reagents from ThermoFisher). When the astrocyte monolayers were confluent, the cultures were vigorously tapped to dislodge microglia growing on top. The microglia were collected, seeded into a new culture dish and maintained in the same culture media. Astrocytes were further purified by adding 5 \u0026micro;M cytosine arabinoside (Thermofisher) for 24 hrs. Samples were collected after 2 weeks in culture by lysing cells directly in Laemmli buffer with 5% β-mercaptoethanol (Bio-Rad) for western blot, or in RLT buffer (Qiagen) for qPCR.\u003c/p\u003e\u003cp\u003ePrimary cortical neuron cultures were prepared from neonatal mouse cortices, and cerebella were used for genotyping by PCR. Cortices were isolated, digested with a papain enzyme kit (Worthington) according to the manufacturer\u0026rsquo;s instructions, mechanically dissociated and passed through a 50 \u0026micro;M mesh. The single cell suspension was plated at 5 x 10\u003csup\u003e4\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e in a substrate of poly-l-ornithine and laminin in Neurobasal Plus media supplemented with 2% B27 Plus and 500 mM glutamax (reagents from ThermoFisher). After 72 hrs cultures were incubated with 1 \u0026micro;M cytosine arabinoside for 24 hrs to eliminate glial cells. Samples for western blots were collected after 2 weeks in culture by lysing cells directly in Laemmli buffer with 5% β-mercaptoethanol.\u003c/p\u003e\u003cp\u003eQuantitative PCR (qPCR)\u003c/p\u003e\u003cp\u003eqPCR assays with mice included cDNA preparations using the Life Sciences cDNA Kit (#4368814). Taqman qPCR kits included ThermoFisher Hs00244999_m1 for human \u003cem\u003eSTAU1\u003c/em\u003e, Thermofisher Hs01060665_g1 for human \u003cem\u003eACTB\u003c/em\u003e, ThermoFisher Mm00488465_m1 for mouse \u003cem\u003eStau1\u003c/em\u003e and Thermofisher Mm01205647_g1 for mouse \u003cem\u003eActb\u003c/em\u003e. Taqman assays were performed using Taqman master mix (ThermoFisher 4440040).\u003c/p\u003e\u003cp\u003eBehavioral phenotype testing\u003c/p\u003e\u003cp\u003eForelimb and hindlimb grip strength was determined using a grip strength meter (Ugo Basile cat# 47200). Peak force grip strength values are reported as the mean of three replicates. Rotarod testing was performed using the Rotamex-5 accelerating rotarod (Columbus Instruments). In any week of testing, on day 1 mice are handled in the palm of the hand for at least 5 min each for 3 separate times to acclimate animals to the investigator. On day 2, mice are trained on the rotarod with acceleration of 1 RPM per 15 seconds to a maximum of 10 RPM and total time of 10 min, and this is repeated three times. On days 3\u0026ndash;5 mice are placed on the rotarod set to accelerate 1 RPM per 9 sec until mice fall from the rod and the latency to fall (in seconds) is recorded, and 2 trials per day are performed. Each mouse group included 20 or more animals, exceeding the minimum sample size required to achieve 80% power to detect a 50% difference between groups, based on power analysis performed using nQuery Advisor. The analysis assumed a two-sided test with α\u0026thinsp;=\u0026thinsp;0.05. No mice were subjected to exclusion. The technician was blinded to the genotype when performing motor phenotype tests.\u003c/p\u003e\u003cp\u003eWestern blot analyses\u003c/p\u003e\u003cp\u003eProtein extracts from mouse cerebellar or cerebral hemisphere or spinal cord tissues were prepared by homogenization in an extraction buffer (25 mM Tris-HCl pH 7.6, 300 mM NaCl, 0.5% Nonidet P-40, 2 mM EDTA, 2 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.5 M urea and protease inhibitors; Sigma; cat# P-8340). The homogenates were subjected to sonication (3 s for 1 stroke, and level 2; Sonic Dismembrator, Fisher Scientific) followed by centrifugation at 4\u0026deg;C for 20 min at 14,000 rpm. Only the supernatants were used for Western blotting. Protein extracts were resolved by SDS-PAGE and transferred to Hybond P membranes (Amersham Bioscience Inc., USA). After blocking with 3% skim milk in 0.1% Tween 20/PBS, the membranes were incubated with primary antibodies in 3% skim milk in 0.1% Tween 20/PBS for 2 hrs at room temperature or overnight at 4\u0026deg;C. After washing in 0.1% Tween 20/PBS, the membranes were incubated with the corresponding secondary antibodies conjugated with HRP in 3% skim milk in 0.1% Tween 20/PBS for 2 hrs at room temperature and washed again. Signals were detected by using the Immobilon Western Chemiluminescent HRP Substrate (Millipore Inc., USA; cat# WBKLSO100) according to the manufacturer\u0026rsquo;s protocol, and detected using a ChemiDoc System (Bio-Rad). Band intensities were quantified by ImageJ software analyses after inversion of the images and proteins were quantitated as a ratio to β-actin (ACTB) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We used Precision Plus Protein Dual Color Standards (Bio-Rad Inc., USA, Cat# 1610374) for SDS-PAGE analyses.\u003c/p\u003e\u003cp\u003eAntibodies\u003c/p\u003e\u003cp\u003eAntibodies included the following: rabbit polyclonal anti-Staufen antibody (Novus Biologicals, NBP1-33202); rabbit polyclonal anti-mTOR antibody (Cell Signaling Technology, 2972); rabbit polyclonal anti-Phospho-mTOR (Ser2448) [(1:3000), Cell Signaling Technology, 2971]; rabbit polyclonal anti-SQSTM1/p62 antibody (Cell Signaling Technology, 5114); rabbit polyclonal anti-LC3B antibody (Novus Biologicals, NB100-2220); mouse monoclonal anti-TDP-43 (human specific) antibody [(1:7000), Proteintech Group, Inc., Cat #60019-2-Ig)]; rabbit polyclonal human/mouse anti-TDP-43 antibody [(1:7000), Proteintech Group, Inc., Cat #10782-2-AP)]; mouse monoclonal anti-Calbindin-D-28K antibody [(1:5000), Sigma-Aldrich, C9848]; rabbit polyclonal anti-RGS8 antibody [(1:5000), Novus Biologicals, NBP2-20153]; mouse monoclonal anti-PCP2 antibody (F-3) [(1:3000), Santa Cruz, sc-137064]; rabbit polyclonal anti-PCP4 antibody [(1:5000), Abcam, ab197377]; Phospho-p70 S6 Kinase (Thr389) antibody [(1:3000), Cell Signaling, Cat# 9205]; GFAP (GA5) mouse mAb [(1:7000), Cell signaling, Cat #3670]; ChAT (E4F9G) Rabbit mAb [(1:5000), Cell Signaling, Cat# 27269]; NeuN (D4G4O) XP\u0026reg; Rabbit mAb [(1:5000), Cell Signaling, Cat# 24307]; GAPDH (14C10) rabbit mAb [(1:7,000), Cell Signaling, Cat# 2118]; Cleaved Caspase-3 (Asp175) (5A1E) rabbit mAb [(1:3000), Cell Signaling, Cat #9664]; Anti-NeuN107B antibody [(1:5,000) Abcam, ab175148); GFP (D5.1) rabbit mAb [(1:6,000), Cell Signaling, Cat #2956]; UNC13A/Munc13-1 polyclonal antibody [(1:3000), Proteintech Group, Inc., Cat #55053-1-AP]; STMN2 polyclonal antibody [(1:3000), Proteintech Group, Inc., Cat #10586-1-AP], and mouse monoclonal anti-β-Actin\u0026thinsp;\u0026minus;\u0026thinsp;peroxidase antibody (clone AC-15) [(1:10,000), Sigma-Aldrich, A3854). Secondary antibodies included anti-mouse IgG, HRP-linked antibody [(1:5000), Cell signaling, Cat #7076], and peroxidase AffiniPure goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, 111-035-144).\u003c/p\u003e\u003cp\u003eRNA-seq\u003c/p\u003e\u003cp\u003eTotal RNA was extracted from tissues using the RNeasy Mini-Kit (Qiagen) according to the manufacturer\u0026rsquo;s protocol. RNA quality was determined using the Agilent ScreenTape Assay. Library preparation was performed using the Illumina TruSeq Stranded Total RNA library prep Ribo-Zero gold. Paired-end 150 bp reads were generated on a Novaseq 6000 S2 cell sequencing instrument at the High-Throughput Genomics and Bioinformatic Analysis Shared Resource at Huntsman Cancer Institute (University of Utah). The human GRCh38 genome and gene annotation files were downloaded from Ensembl release 100 and a reference database was created using STAR version 2.7.3a with splice junctions optimized for 150 base pair reads\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Optical duplicates were removed from the paired end FASTQ files using clumpify v38.34\u003csup\u003e27\u003c/sup\u003e and reads were trimmed of adapters using cutadapt 1.16\u003csup\u003e28\u003c/sup\u003e. The trimmed reads were aligned to the reference database using STAR in two pass mode to output a BAM file sorted by coordinates. Mapped reads were assigned to annotated genes using featureCounts version 1.6.3 \u003csup\u003e29\u003c/sup\u003e. The output files from cutadapt, FastQC, FastQ Screen, Picard CollectRnaSeqMetrics, STAR and featureCounts were summarized using MultiQC to check for any sample outliers\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Differentially expressed genes were identified for siSTAU1 vs siControl using a 5% false discovery rate with DESeq2 version 1.26.0\u003csup\u003e31\u003c/sup\u003e. Pathways were analyzed using the fast gene set enrichment package with a 10% false discovery rate (FDR)\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Replicate RNA-seq experiments were performed independent of one another four years apart with RNAs prepared by different technicians.\u003c/p\u003e\u003cp\u003eImmunofluorescent labeling\u003c/p\u003e\u003cp\u003eTo confirm the pan-cellular expression of BAC-STAU1, we performed immunofluorescent staining for STAU1. Brains and spinal cords were harvested and fixed in 4% PFA for 24 hours, cryopreserved in sucrose 30% sucrose/PBS, and embedded in OCT. Cryosections were immunostained using a Staufen antibody (Novus Biologicals, NBP1-33202; 1:100 dilution) and a Goat anti-Rabbit Alexa Fluor\u0026trade; Plus 594 secondary antibody (Thermo Fisher Scientific, A32740, 1:1000 dilution). Nuclei were counterstained with DAPI, and sections were mounted with Fluoromount-G (Southern Biotech, Cat. #0100-01). Fluorescence imaging was performed using a Leica SP8 confocal microscope and an AxioScan 7 at the Cell Imaging Core, University of Utah.\u003c/p\u003e\u003cp\u003eAAV production and treatment of mice\u003c/p\u003e\u003cp\u003eArtificial microRNAs targeting \u003cem\u003eSTAU1\u003c/em\u003e were designed following the method described by Boudreau and Davidson, that begins with siRNA sequences\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Two different miRNAs were designed that target human \u003cem\u003eSTAU1\u003c/em\u003e that were based on siRNA sequences that were previously determined effective for lowering STAU1 abundance. One of these siRNAs, developed by us (si\u003cem\u003eSTAU1\u003c/em\u003e, 5\u0026rsquo;-GUAACUGCCAUGAUAGCCCGAG-3\u0026rsquo;), was used to design mi\u003cem\u003eSTAU1\u003c/em\u003e-A, and a second was based on a published \u003cem\u003eSTAU1\u003c/em\u003e siRNA (5\u0026rsquo;-CCUAUAACUACAACAUGAG-3\u0026rsquo;) used to design mi\u003cem\u003eSTAU1\u003c/em\u003e-B\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. PHP.eB serotype AAVs were generated by the Virus Packaging Laboratory of the Drug Discovery Core at University of Utah using pUCmini-iCAP-PHP.eB (Addgene #103005). The viral expression vector that we used (pAAV-U6-sgRNA-CMV-GFP, Addgene #85451) includes the U6 promoter to drive miRNA expression and independently expresses GFP under the control of the CMV promoter. BAC-STAU1 mice and wildtype littermates, 20 wks of age, were treated with AAV (9.6E10 viral genomes (VG) or PBS vehicle as indicated, by single intracerebroventricular (ICV) injections to the right lateral ventricle as previously described\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Mice were sacrificed at 23 wks of age when spinal cords were collected for analysis by western blotting.\u003c/p\u003e\u003cp\u003e\u003cem\u003eDrosophila\u003c/em\u003e studies\u003c/p\u003e\u003cp\u003eFly stocks were maintained on standard cornmeal molasses agar. Transgenic lines included gmr-GAL4 (BDSC_1104); UAS-\u003cem\u003estau\u003c/em\u003e.RNAi (shRNA) (BDSC_82965); UAS-hTDP-43 (BDSC_79587)\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e; UAS-hATXN2(CAG)64\u003csup\u003e38\u003c/sup\u003e; UAS-\u003cem\u003estau\u003c/em\u003e (Drosophila \u003cem\u003estau\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. External eye microscopy, paraffin sectioning and quantification were performed as described\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Stau knockdown in these lines has been verified\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. The GMR-Gal4 functions as a driver line that expresses the Gal4 transcriptional activator under control of the Glass Multimer Reporter (GMR) element. GMR is active in the developing and mature fly eye allowing targeted eye gene expression.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eStatistical differences between selected groups evaluated by qPCR, western blotting, and the electrophysiological data were evaluated using blocked two-way analysis of variance (ANOVA) tests followed by post-hoc tests of significance (Tukey\u0026rsquo;s or Bonferroni\u0026rsquo;s tests for multiple comparison, as indicated). Data shown on western blots are mean and SD of biological replicates (n\u0026thinsp;=\u0026thinsp;3\u0026ndash;4 mice) that were each evaluated on a minimum of three blots (3 technical replicates). Behavioral tests were evaluated with repeated measures ANOVA in GraphPad Prism (grip strength testing) or STATA (rotarod testing). Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-tests were unpaired and two-tailed. All tests were performed using the GraphPad Prism software package.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003eGeneration of BAC-STAU1 mice\u003c/h2\u003e\u003cp\u003eWe produced a BAC mouse harboring the human \u003cem\u003eSTAU1\u003c/em\u003e gene. We utilized a 133.8 kb BAC construct (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and generated transgenic mice by pronuclear injection. Sequencing of DNA isolated from BAC-STAU1 mouse bone marrow specimens determined that one of four founders, only BAC-STAU1.6, had a single genomic integration of the transgene, on chromosome 17 (chr17:9,394,829-~9,610,924). The integration occurred simultaneously to a 210 kb genomic deletion within this region. Query of the NCBI Reference Sequence Database (RefSeq) confirmed that there are no annotated genes in this region of the mouse genome and therefore no gene disruption is predicted. No structural variants were identified within the integrated construct. Due to the nature of the sequence at the integration site, BAC copy number via this method could not be estimated. BAC-STAU1.6 mice, now simply referred to as BAC-STAU1, are viable, display no abnormal gross morphology or significant segregation distortion. Further sequencing also demonstrated that the \u003cem\u003eSTAU1\u003c/em\u003e isoform expressed in CNS tissues of BAC-STAU1 mice is equivalent to the Ensemble STAU1-203 transcript (ENSMUST00000109236.9), and that it lacks a 6 amino acid insertion in the RNA binding domain 3 (RBD3) known to reduce RNA binding affinity\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe mean weights of BAC-STAU1 mice were not significantly different from wildtype littermates up to 80 wks (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The presence of the BAC-transgene in brain was demonstrated by quantitative PCR using primers specific for human \u003cem\u003eSTAU1\u003c/em\u003e vs mouse \u003cem\u003eStau1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC,D).\u003c/p\u003e\u003cp\u003e\u003cb\u003eBAC-STAU1 is expressed in multiple CNS cell types in BAC-STAU1 mice.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo confirm widespread hSTAU1 expression in the CNS we performed western blotting and qPCR analyses using cultured astrocytes, microglia, and neurons prepared from neonate spinal cord and cortical tissues. hSTAU1 was highly abundant in all cultures from BAC-STAU1 mice vs wildtype littermates (Supplemental Fig.\u0026nbsp;1A-E). We verified that neuronal cultures expressed NeuN and lacked IBA1, while microglia cultures expressed IBA1 which was absent in neuronal cultures, and we also verified GFAP expression in astrocyte cultures (not shown). There was also approximately 4-fold more \u003cem\u003ehSTAU1\u003c/em\u003e-mRNA in the cortex vs spinal cord of BAC-STAU1 mice, determined by qPCR, while mouse STAU1 was generally mildly reduced in these tissues in BAC-STAU1 vs wildtype littermates (Supplemental Fig.\u0026nbsp;1F,G).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eImmunofluorescent labeling\u003c/h3\u003e\n\u003cp\u003eWe observed widespread STAU1 expression in BAC-STAU1 mice by immunofluorescent labeling, with a trend of increased signal for STAU1 that mirrored the quantified increases in complimentary western blots. STAU1 was strongly present in tissues of BAC-STAU1 mice including cell bodies and neuropil of ventral horn, dorsal horn of the spinal cord, and various brain tissues including the cerebral cortex, hippocampus, cerebellum and pons, compared to WT mice (Supplemental Fig.\u0026nbsp;2).\u003c/p\u003e\n\u003ch3\u003eBehavioral phenotype testing\u003c/h3\u003e\n\u003cp\u003eWe investigated BAC-STAU1 mice for behavioral phenotypes using both accelerating rotarod and grip strength tests. No significant behavioral differences in motor strength or coordination were observed in either forelimb or hindlimb grip strength tests or rotarod testing (Supplemental Figs.\u0026nbsp;3 and 4).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eMolecular phenotype associated with STAU1 overabundance\u003c/h2\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003eTranscriptome analysis:\u003c/h2\u003e\u003cp\u003eWe carried out RNA-seq of spinal cords from BAC-STAU1 mice at 25 wks and 57 wks of age. The number of mice included were 2 male and 2 female BAC-STAU1 mice compared to 2 male and 2 female WT littermates for the 25 wk group, and 2 male and 2 female BAC-STAU1 mice compared to 2 male and 1 female WT littermates for the 57 wk group. In both cases the number of differentially expressed genes (DEGs) was low, with an FDR cut-off of 10%. The 25 wk group had 30 DEGs while the 57 wk group had 44 DEGs and 3 were shared including \u003cem\u003eSmg5\u003c/em\u003e, \u003cem\u003eUrm1\u003c/em\u003e and \u003cem\u003eHdac11\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Volcano plots showed that the most significantly upregulated DEG in both experiments was \u003cem\u003eSmg5\u003c/em\u003e, a regulator of UPF1 in the nonsense mediated decay (NMD) pathway (Fig. B). Hallmark and KEGG pathway analyses revealed progressive transcriptome dysregulation in BAC-STAU1 mice with age, with an increased number of annotated pathways in older mice. Hallmark analysis showed 7 enriched gene sets in the 25 wk group and 22 in the 57 wk group, while KEGG pathway analysis showed 7 in the 25 wk group and 31 in the 57 wk group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Enriched Hallmark gene sets shared between the two groups included Epithelial Mesenchymal Transition, Mtorc1 Signaling, Myogenesis, and Oxidative Phosphorylation. Enriched KEGG gene sets shared between the two groups included Alzheimer\u0026rsquo;s Disease, Huntington\u0026rsquo;s Disease, Neuroactive Ligand Receptor Interaction, Oxidative Phosphorylation, Parkinson\u0026rsquo;s Disease, Ribosome, Taste Transduction. Complete RNA-seq and pathway analyses are provided in Supplemental Table\u0026nbsp;2.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\n\u003ch3\u003eWestern immunoblotting for autophagy and pathology markers:\u003c/h3\u003e\n\u003cp\u003eGiven that analysis of DEG pathways showed gene sets in NDDs and MTORC1 signaling, we wanted to test key proteins for their abundance by western blotting. Overabundant STAU1 in the brain, cerebellum and spinal cord was associated with abnormal abundance of autophagy proteins. These include mTOR, p-mTOR, p62, p-S6K and LC3-II in the brain, cerebellum and spinal cord, of which p-S6K was only evaluated in cerebellum (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The profile of these proteins and their phosphorylated forms are consistent with reduced autophagy, previously described \u003cem\u003ein vitro\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe next examined protein changes often associated with neurodegeneration such as reduction of key neuronal marker proteins and an increase in astrogliosis. We isolated protein extracts from cerebral hemispheres, cerebella, and spinal cords of WT and transgenic mice at 14 weeks-of-age and analyzed abundance by semi-quantitative western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In all three tissues, there was a remarkable increase in GFAP and cleaved caspase-3 consistent with increased gliosis and apoptotic cell death. The pan-neuronal marker protein NEUN was decreased in hemispheres and spinal cord, and PC-specific proteins (CALB1, RGS8, PCP2, PCP4, FAM107B) were decreased in cerebellum. Choline-acetyl transferase (CHAT), the marker protein for cholinergic neurons, was decreased by 50% compared to that in WT tissues, not only in spinal cord but also in hemisphere tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMis-splicing of specific mRNAs is part of TDP-43 proteinopathy. In human spinal cord, \u003cem\u003eUNC13A\u003c/em\u003e and \u003cem\u003eSTMN2\u003c/em\u003e show cryptic exon splicing in the presence of\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Homologous cryptic exons are not known in mice, yet we observed abnormally reduced UNC13A and STMN2 abundance in BAC-STAU1 mouse brain and spinal cord (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA,B and E,F).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eModulation of STAU abundance in flies and mice\u003c/h2\u003e\u003cp\u003eWe had previously shown that reduction of STAU1 by genetic interaction improved the motor phenotype of SCA2 mice\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The results described above suggest that STAU1 overabundance by itself induces a cellular pre-degenerative or risk phenotype. We wanted to test an extension of this hypothesis in fly and mouse models by modulating STAU1 levels in the presence of disease-associated mutations, both reducing and increasing levels compared to wildtype levels.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStau dosage modulates retinal degeneration in fly TDP-43 and ATXN2\u003c/b\u003e\u003csup\u003e\u003cb\u003eexp\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emodels.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe selected fly models expressing human TDP-43 and human ATXN2-CAG\u003csub\u003e64\u003c/sub\u003e and varied fly \u003cem\u003estau\u003c/em\u003e dosage using an upregulation line, and a line that reduced fly \u003cem\u003estau\u003c/em\u003e by RNAi. In the presence of normal \u003cem\u003estau\u003c/em\u003e levels, both hTDP-43 and hATXN2-CAG\u003csub\u003e64\u003c/sub\u003e flies showed reduced retinal depth compared to WT flies, reflecting degeneration. When \u003cem\u003estau\u003c/em\u003e levels were then reduced by co-expressing \u003cem\u003estau\u003c/em\u003e shRNA, the retinal depth in flies was significantly improved (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). Transgenic upregulation of fly \u003cem\u003estau\u003c/em\u003e worsened the eye phenotype in hTDP-43 flies. Modulation of \u003cem\u003estau\u003c/em\u003e in WT flies had no effect on eye phenotype or retinal depth (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These data indicate that Stau modulates neurotoxicity of hTDP-43 and hATXN2-CAG64 in \u003cem\u003eDrosophila\u003c/em\u003e, supporting the potential for targeting STAU1 for treating neurodegenerative diseases.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003egenetic interaction of overabundant STAU1 and TDP-43\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePreviously we demonstrated that STAU1 is elevated in cerebellum and spinal cord of Thy1-TDP-43 transgenic mice and in sporadic ALS patient spinal cords, associated with abnormal abundance or phosphorylation of molecular markers of autophagy\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Because autophagy and other neuronal markers are abnormally expressed in BAC-STAU1 mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), we now sought to demonstrate that adding the expression of human STAU1 to TDP-43 transgenic mice would further worsen the molecular phenotype. To do so we performed a genetic cross of BAC-STAU1 mice with Prp-TDP43(Q331K) mice described in Wils \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. We then evaluated brain hemisphere tissues from mice of all resultant genotypes for the expression of CHAT, NEUN, cleaved CASP3, mTOR, p62 and GFAP, at 8 and 24 wks of age. Significant changes were observed for each of these proteins in BAC-STAU1 mice and Prp-TDP43(Q331K) mice in both age groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Additionally, nearly all of the proteins were significantly more elevated (STAU1, GFAP, cCASP3, mTOR, p62) or reduced (CHAT, NEUN) in double-transgenic Prp-TDP43(Q331K);BAC-STAU1 vs single-transgenic BAC-STAU1 mice. This was true in both age groups, with a few exceptions (cCASP3 and p62 in the 8 wk group, and mTOR in the 24 wk group) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In general, the \u003cem\u003ein vivo\u003c/em\u003e overexpression of hSTAU1 by crossing BAC-STAU1 mice with Prp-TDP43(Q331K) mice resulted in a significantly more severe molecular phenotype in either age group investigated. Note that in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e the TDP-43 antibody used recognized only human TDP-43, and full blots using an antibody recognizing both mouse and human TDP-43 is shown in Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e. Mouse TDP-43 was significantly elevated in BAC-STAU1 brain vs WT by 20\u0026ndash;30% (Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eC). For all other full blots in the study see Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eArtificial microRNAs targeting STAU1 improve the abnormal molecular phenotype in BAC-STAU1 mice.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs STAU1 overabundance in BAC-STAU1 is associated with abnormal autophagy and neuronal abundance markers, and lowering Stau in the fly improved the retinal depth neurodegenerative phenotype in TDP-43 and ATX2 flies, we sought to determine if lowering STAU1 abundance in BAC-STAU1 mice could improve these features. To do this we generated two different AAVs that deliver artificial microRNAs targeting human \u003cem\u003eSTAU1\u003c/em\u003e, designated AAV-miSTAU1-A and AAV-miSTAU1-B. We treated mice at 20 wks of age which was well after phenotype onset observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-F, and sacrificed mice at 23 wks of age. We then removed spinal cords and evaluated proteins by western blotting and quantification (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA,B). Very little STAU1 was observed in WT mice while a high abundance of STAU1 was observed in BAC-STAU1 mice, as well as significantly reduced abundances of CHAT and NEUN, and normalized abundance of mTOR. BAC-STAU1 mice treated with AAVs were positive for GFP on blots, expressed by an independent promotor, indicating positive uptake of the AAVs. BAC-STAU1 mice that were treated with either of AAV-miSTAU1-A or AAV-miSTAU1-B had significantly reduced STAU1 abundance and normalized CHAT, NEUN, and mTOR compared to the BAC-STAU1 mice treated with vehicle. Levels of CHAT, NEUN and mTOR in the AAV-treated BAC-STAU1 mice were close to those observed in the WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eRNA-binding proteins (RBPs) including STAU1 play important roles in health and disease ranging from viral defense to neurodegeneration\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Pathogenic variants in some RBPs such as TDP-43 and FUS have been reported in patients with ALS and FTD, directly linking the respective genes to neurodegenerative diseases (NDDs). Overabundance of TDP-43, its subcellular mislocalization, and cytoplasmic aggregation are also typical in patients without pathogenic variants in ALS genes and TDP-43 aggregates are found in \u0026gt;\u0026thinsp;90% of spinal cords of individuals with sporadic ALS. Although STAU1 mutations have not yet been identified in NDDs, the protein is overabundant in cells and spinal cord tissue from ALS patients and in ALS model systems as a result of a negative post-transcriptional feedback loop on autophagy\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eHere we examined whether changes seen \u003cem\u003ein vitro\u003c/em\u003e in the presence of STAU1 overabundance are replicated \u003cem\u003ein vivo\u003c/em\u003e. We describe a new BAC-STAU1 mouse that expresses the entire human \u003cem\u003eSTAU1\u003c/em\u003e gene. The transgene was expressed in cerebral hemispheres, cerebellum, and spinal cord (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) as well as in cultures of mouse neurons, glia, and microglia (Supplemental Fig.\u0026nbsp;1). Western blot analysis indicated that the BAC transgene expressed full-length STAU1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eTranscriptomics autophagy neuronal apoptosis\u003c/h2\u003e\u003cp\u003eBased on our findings \u003cem\u003ein vitro\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, we analyzed BAC-STAU1 mice for molecular markers of autophagy function and neurodegeneration as well as for transcriptomic changes. Whole-genome RNA-seq provided surprisingly few DEGs in spinal cord of BAC-STAU1 mice at 25 and 57 wks-of-age (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In both age groups, the nonsense mediated decay regulator \u003cem\u003eSmg5\u003c/em\u003e appeared as the most significantly elevated transcript, supporting a role for STAU1 in NMD. Pathway analyses revealed a common signature of transcriptomic dysregulation in both the 25 and 57 wk age groups associated with neurodegeneration including MTORC1 signaling, oxidative phosphorylation as well as Alzheimer\u0026rsquo;s, Parkinson and Huntington disease (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eSemiquantitative WB analyses showed significant abnormalities in pathways involving key signaling cascades with abnormal expression of mTOR, p-mTOR, p-S6K, p62, LC3-II consistent with reduced autophagic flux (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These findings were confirmed in extracts from cerebral hemispheres, cerebellum, and spinal cord pointing to the potential role of STAU1 overabundance in NDDs affecting distinct parts of the CNS.\u003c/p\u003e\u003cp\u003eWe previously established a biomarker profile of PC-specific proteins (CALB1, RGS8, PCP2, PCP4, FAM107B) that show early and continuous decline in two SCA2 mouse models as proxies for PC health and function \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. These proteins were also dysregulated in the cerebellum of BAC-STAU1 mice consistent with PC dysfunction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D).\u003c/p\u003e\u003cp\u003eWe applied a similar approach to protein extracts of cerebral hemispheres and spinal cords (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B, E, F). Protein analyses were consistent with autophagy dysfunction. Neuronal injury was evidenced by decreased expression of NEUN and CHAT. Cleaved caspase-3 was significantly increased consistent with apoptotic cell death. As typically seen in NDDs, there was a significant increase in GFAP expression. Of note, CHAT abundance was not only decreased in spinal cord as a sign of motor neuron degeneration, but also in cerebral hemispheres consistent with degeneration in basal forebrain cholinergic neurons and potential relevance for memory dysfunction in AD and limbic-predominant age-related TDP-43 encephalopathy (LATE).\u003c/p\u003e\u003cp\u003eWe also examined abundance of UNC13A and STMN2 in spinal cords. Previous studies have shown that TDP-43 mislocalization to the cytoplasm (and loss of nuclear splicing regulation) is associated with the inclusion of cryptic exons in human cells resulting in reduced expression of UNC13A and STMN2\u003csup\u003e44, 48, 49\u003c/sup\u003e. Even though the cryptic exons described in humans have not been observed in mice, the abundances of both proteins were reduced in BAC-STAU1 mice, suggesting that the regulation of these genes, at least in mice, involves some mechanism other than cryptic exons, such as transcription or translation, possibly by way of STAU1-mediated decay.\u003c/p\u003e\u003cp\u003eThe molecular changes seen in BAC-STAU1 mice were specific and reversible by RNAi. We developed 2 artificial miRNAs to human \u003cem\u003eSTAU1\u003c/em\u003e and delivered them by ICV injection. Both constructs worked equally well and reverted marker proteins back to levels seen in WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These findings are in line with our previous observations that genetic reduction of \u003cem\u003eStau1\u003c/em\u003e improves mouse SCA2 phenotypes\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e and support the development of therapeutics targeting STAU1 for treating neurodegenerative disease.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eModulation of Staufen dosage\u003c/h2\u003e\u003cp\u003eAs reducing \u003cem\u003eStau1\u003c/em\u003e dosage protected cerebellar Purkinje cells and improved motor behavior in an SCA2 mouse model, the question arises whether increasing STAU1 abundance would worsen NDD phenotypes. We therefore examined the effects of staufen dosage in the fly and in a TDP-43 mouse model. Flies expressing human TDP-43 or polyglutamine expanded human ATXN2 (ATXN2-CAG\u003csub\u003e64\u003c/sub\u003e) were treated with shRNAs targeting the endogenous fly \u003cem\u003estau\u003c/em\u003e gene. The decreased retinal depth seen in flies transgenic for hTDP-43 or hATXN2-CAG\u003csub\u003e64\u003c/sub\u003e was significantly improved by co-expressing \u003cem\u003estau\u003c/em\u003e shRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Conversely, upregulation of fly \u003cem\u003estau\u003c/em\u003e further worsened the degenerative phenotype seen in the TDP-43 line, but not in the ATXN2 line.\u003c/p\u003e\u003cp\u003eWe next investigated the role of Staufen in a mammalian system by crossing BAC-STAU1 mice with Prp-TDP-43(Q331K) transgenic mice, approximately doubling the amount of endogenous Staufen protein. In Prp-TDP-43(Q331K) mice, neuronal marker proteins, glial and apoptotic markers are abnormal already at 8 wks of age, prior to the reported onset of motor abnormalities (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, round symbols). Increasing STAU1 abundance worsened the levels of all marker proteins (CHAT, NEUN, cleaved CASP3, mTOR, p62 and GFAP) in brain with changes increasing from 8 to 24 weeks-of age (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, diamond symbols). Of note, cleaved caspase-3 was 3.5-fold increased in double-transgenic mice compared to a 2.5-fold increase in single transgenic mice. These studies suggest that variability in STAU1 levels in model systems and possibly also in humans can modify neurodegeneration. Genetic and environmental factors affecting STAU1 abundance deserve further study.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eImplications for human disease\u003c/h2\u003e\u003cp\u003eDespite the pronounced molecular phenotype in BAC-STAU1 mice including activation of apoptosis, we did not see a progressive motor phenotype, at least for the observation period of up to 1 year and the motor tests used in our studies (grip strength, rotarod) (Supplemental Figs.\u0026nbsp;3 and 4). It is quite likely that longer observation periods are needed and that more nuanced behavioral tests need to be applied.\u003c/p\u003e\u003cp\u003eThe sensitivity of NDD phenotypes to levels of STAU1 protein may have implications for mouse models of human NDDs as well as for the human disease. Based on our studies it is reasonable to predict that genetic or environmental variation affecting STAU1 levels will affect NDD phenotypes. Mouse background is known to affect NDD phenotypes\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, but little is known about CNS STAU1 levels in different mouse strains. Large-scale sequencing projects comparing individuals with ALS to controls so far have not identified \u003cem\u003eSTAU1\u003c/em\u003e Mendelian pathogenic variants or suggested risk variants\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e although analyses searching for protective variants have received less attention\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. It may be that STAU1 variation is not a modifier of disease risk itself, but age-of-onset or rate of progression. It is also possible that the role of STAU1 in early development may limit the extent of genetic variation and that genetic or epigenetic variation of genes acting on STAU1 play a role. Finally, it is possible that environmental factors such as brain trauma affect STAU1 levels, as has been shown for stress granule formation in the fly\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur findings in the BAC-STAU1 mouse model demonstrate that STAU1 overabundance alone at levels found in neurodegenerative mouse models is sufficient to impair autophagy and to promote apoptotic cell death in the brain. Moreover, altering STAU1 levels in the presence of disease-associated mutations influences the severity of the resulting phenotypes. These results strengthen the case for STAU1 as a therapeutic target in human neurodegenerative disorders.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ealzheimer’s disease AD;\u003c/p\u003e\n\u003cp\u003eataxin-2 ATXN2;\u003c/p\u003e\n\u003cp\u003eantisense oligonucleotide ASO;\u003c/p\u003e\n\u003cp\u003ebacterial artificial chromosome BAC;\u003c/p\u003e\n\u003cp\u003ebeta actin ACTB;\u003c/p\u003e\n\u003cp\u003echoline O-acetyltransferase CHAT;\u003c/p\u003e\n\u003cp\u003ecentral nervous system CNS;\u003c/p\u003e\n\u003cp\u003edifferentially expressed gene DEG;\u003c/p\u003e\n\u003cp\u003eglial fibrillary acidic protein GFAP;\u003c/p\u003e\n\u003cp\u003egram g;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ehuman STAU1 hSTAU1;\u003c/p\u003e\n\u003cp\u003ekilogram kg; Kyoto Encyclopedia of Genes and Genomes KEGG;\u003c/p\u003e\n\u003cp\u003elitre l;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003emicrotubule-associated protein 1 light chain 3 LC3;\u003c/p\u003e\n\u003cp\u003emechanistic target of rapamycin mTOR;\u003c/p\u003e\n\u003cp\u003emechanistic target of rapamycin complex 1 mTORC1;\u003c/p\u003e\n\u003cp\u003emilligram mg;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eneuronal nuclei (RNA binding Fox-1 homolog 3 (RBFOX3) gene product) NEUN;\u003c/p\u003e\n\u003cp\u003espinocerebellar ataxia type 2 SCA2;\u003c/p\u003e\n\u003cp\u003estandard deviation SD;\u003c/p\u003e\n\u003cp\u003estandard error of the mean SE;\u003c/p\u003e\n\u003cp\u003estaufen-1 STAU1;\u003c/p\u003e\n\u003cp\u003estathmin-2 STMN2;\u003c/p\u003e\n\u003cp\u003etrans-activation response DNA-binding protein 43 TDP-43;\u003c/p\u003e\n\u003cp\u003etransgenic TG;\u003c/p\u003e\n\u003cp\u003eUnc-13 homolog A (C. elegans analog) UNC13A;\u003c/p\u003e\n\u003cp\u003ewild-type WT;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u0026nbsp;\u003c/strong\u003e The authors declare no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declair no competing financial interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll mouse procedures were performed under protocol 2040 approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Utah, SLC, UT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available on request from the corresponding authors (S.M.P. and D.R.S.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Scott Redlin and Gentrie Maag for contributing to work in this study. We thank Leeanne McGurk for sharing flies. Viral vectors were provided by Viviana Gradinaru and Hetian Lei. We also thank the University of Utah Cell Imaging Core for assistance. The graphical abstract was created with BioRender.com. This work was supported by a Harrington Discovery Institute Rare Disease Scholarship to D.R.S., and National Institutes of Health (NIH) / National Institute of Neurological Disorders and Stroke (NINDS) grants, R21NS127028, R01NS137233 and R01NS097903 to D.R.S., R21NS128630 to D.R.S. and M.G., R56NS033123, R37NS033123, R35NS127253 to S.M.P., R61/R33NS124965 to D.R.S. and S.M.P., and R35NS097275 to N.M.B.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.M.P., D.R.S. conceived the study and jointly supervised research. N.M.B. conceived and supervised drosophila studies. S.P., H.N., M.G., W.D, K.P.F. designed and performed experiments. W.D. and K.P.F. generated transgenic mice. M.G. and W.D. performed RNA-seq studies. S.P. performed western blotting of mouse tissues. H.N. performed drosophila studies. M.G. produced primary cell cultures and conducted their analyses. M.G. performed immunohistological stains. K.P.F. and D.R.S. supervised motor phenotype testing. S.P., H.N., M.G., and D.R.S. performed statistical tests. S.M.P., S.P., M.G., K.P.F., N.M.B. and D.R.S. contributed to writing the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSt Johnston D, Beuchle D, Nusslein-Volhard C. Staufen, a gene required to localize maternal RNAs in the Drosophila egg. \u003cem\u003eCell\u003c/em\u003e 1991, 66(1): 51\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTosar LJ, Thomas MG, Baez MV, Ibanez I, Chernomoretz A, Boccaccio GL. Staufen: from embryo polarity to cellular stress and neurodegeneration. \u003cem\u003eFront Biosci (Schol Ed)\u003c/em\u003e 2012, 4: 432\u0026ndash;452.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePaul S, Dansithong W, Figueroa KP, Scoles DR, Pulst SM. Staufen1 links RNA stress granules and autophagy in a model of neurodegeneration. \u003cem\u003eNat Commun\u003c/em\u003e 2018, 9(1): 3648.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePulst SM, Nechiporuk A, Nechiporuk T, Gispert S, Chen XN, Lopes-Cendes I, \u003cem\u003eet al.\u003c/em\u003e Moderate expansion of a normally biallelic trinucleotide repeat in spinocerebellar ataxia type 2. \u003cem\u003eNat Genet\u003c/em\u003e 1996, 14(3): 269\u0026ndash;276.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eScoles DR, Pulst SM. Spinocerebellar Ataxia Type 2. \u003cem\u003eAdv Exp Med Biol\u003c/em\u003e 2018, 1049: 175\u0026ndash;195.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePaul S, Dansithong W, Figueroa KP, Gandelman M, Scoles DR, Pulst SM. Staufen1 in Human Neurodegeneration. \u003cem\u003eAnn Neurol\u003c/em\u003e 2021, 89(6): 1114\u0026ndash;1128.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePaul S, Dansithong W, Gandelman M, Figueroa KP, Zu T, Ranum LPW, \u003cem\u003eet al.\u003c/em\u003e Staufen impairs autophagy in neurodegeneration. \u003cem\u003eAnn Neurol\u003c/em\u003e 2023, 93(2): 398\u0026ndash;416.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi CL, Zhou GF, Xie XY, Wang L, Chen X, Pan QL, \u003cem\u003eet al.\u003c/em\u003e STAU1 exhibits a dual function by promoting amyloidogenesis and tau phosphorylation in cultured cells. \u003cem\u003eExp Neurol\u003c/em\u003e 2024, 377: 114805.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePulst SM, Scoles DR, Paul S. Effects of STAU1/staufen1 on autophagy in neurodegenerative diseases. \u003cem\u003eAutophagy\u003c/em\u003e 2023, 19(9): 2607\u0026ndash;2608.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhao R, Huang S, Li J, Gu A, Fu M, Hua W, \u003cem\u003eet al.\u003c/em\u003e Excessive STAU1 condensate drives mTOR translation and autophagy dysfunction in neurodegeneration. \u003cem\u003eThe Journal of cell biology\u003c/em\u003e 2024, 223(8).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGandelman M, Dansithong W, Figueroa KP, Paul S, Scoles DR, Pulst SM. Staufen 1 amplifies proapoptotic activation of the unfolded protein response. \u003cem\u003eCell Death Differ\u003c/em\u003e 2020, 27(10): 2942\u0026ndash;2951.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSakurai M, Shiromoto Y, Ota H, Song C, Kossenkov AV, Wickramasinghe J, \u003cem\u003eet al.\u003c/em\u003e ADAR1 controls apoptosis of stressed cells by inhibiting Staufen1-mediated mRNA decay. \u003cem\u003eNat Struct Mol Biol\u003c/em\u003e 2017, 24(6): 534\u0026ndash;543.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePark E, Maquat LE. Staufen-mediated mRNA decay. \u003cem\u003eWiley interdisciplinary reviews RNA\u003c/em\u003e 2013, 4(4): 423\u0026ndash;435.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLebeau G, Maher-Laporte M, Topolnik L, Laurent CE, Sossin W, Desgroseillers L, \u003cem\u003eet al.\u003c/em\u003e Staufen1 regulation of protein synthesis-dependent long-term potentiation and synaptic function in hippocampal pyramidal cells. \u003cem\u003eMol Cell Biol\u003c/em\u003e 2008, 28(9): 2896\u0026ndash;2907.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKohrmann M, Luo M, Kaether C, DesGroseillers L, Dotti CG, Kiebler MA. Microtubule-dependent recruitment of Staufen-green fluorescent protein into large RNA-containing granules and subsequent dendritic transport in living hippocampal neurons. \u003cem\u003eMol Biol Cell\u003c/em\u003e 1999, 10(9): 2945\u0026ndash;2953.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYu Z, Fan D, Gui B, Shi L, Xuan C, Shan L, \u003cem\u003eet al.\u003c/em\u003e Neurodegeneration-associated TDP-43 interacts with fragile X mental retardation protein (FMRP)/Staufen (STAU1) and regulates SIRT1 expression in neuronal cells. \u003cem\u003eJ Biol Chem\u003c/em\u003e 2012, 287(27): 22560\u0026ndash;22572.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChu JF, Majumder P, Chatterjee B, Huang SL, Shen CJ. TDP-43 Regulates Coupled Dendritic mRNA Transport-Translation Processes in Co-operation with FMRP and Staufen1. \u003cem\u003eCell reports\u003c/em\u003e 2019, 29(10): 3118\u0026ndash;3133 e3116.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIguchi Y, Katsuno M, Niwa JI, Yamada SI, Sone J, Waza M, \u003cem\u003eet al.\u003c/em\u003e TDP-43 depletion induces neuronal cell damage through dysregulation of Rho family GTPases. \u003cem\u003eJ Biol Chem\u003c/em\u003e 2009, 284(33): 22059\u0026ndash;22066.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLucas BA, Lavi E, Shiue L, Cho H, Katzman S, Miyoshi K, \u003cem\u003eet al.\u003c/em\u003e Evidence for convergent evolution of SINE-directed Staufen-mediated mRNA decay. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e 2018, 115(5): 968\u0026ndash;973.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eElbarbary RA, Li W, Tian B, Maquat LE. STAU1 binding 3' UTR IRAlus complements nuclear retention to protect cells from PKR-mediated translational shutdown. \u003cem\u003eGenes Dev\u003c/em\u003e 2013, 27(13): 1495\u0026ndash;1510.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGong C, Maquat LE. lncRNAs transactivate STAU1-mediated mRNA decay by duplexing with 3' UTRs via Alu elements. \u003cem\u003eNature\u003c/em\u003e 2011, 470(7333): 284\u0026ndash;288.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVessey JP, Macchi P, Stein JM, Mikl M, Hawker KN, Vogelsang P, \u003cem\u003eet al.\u003c/em\u003e A loss of function allele for murine Staufen1 leads to impairment of dendritic Staufen1-RNP delivery and dendritic spine morphogenesis. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e 2008, 105(42): 16374\u0026ndash;16379.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDansithong W, Paul S, Figueroa KP, Rinehart MD, Wiest S, Pflieger LT, \u003cem\u003eet al.\u003c/em\u003e Ataxin-2 regulates RGS8 translation in a new BAC-SCA2 transgenic mouse model. \u003cem\u003ePLoS Genet\u003c/em\u003e 2015, 11(4): e1005182.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGandelman M, Peluffo H, Beckman JS, Cassina P, Barbeito L. Extracellular ATP and the P2X7 receptor in astrocyte-mediated motor neuron death: implications for amyotrophic lateral sclerosis. \u003cem\u003eJ Neuroinflammation\u003c/em\u003e 2010, 7: 33.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGandelman M, Levy M, Cassina P, Barbeito L, Beckman JS. P2X7 receptor-induced death of motor neurons by a peroxynitrite/FAS-dependent pathway. \u003cem\u003eJ Neurochem\u003c/em\u003e 2013, 126(3): 382\u0026ndash;388.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, \u003cem\u003eet al.\u003c/em\u003e STAR: ultrafast universal RNA-seq aligner. \u003cem\u003eBioinformatics\u003c/em\u003e 2013, 29(1): 15\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBushnell B, Rood J, Singer E. BBMerge - Accurate paired shotgun read merging via overlap. \u003cem\u003ePLoS ONE\u003c/em\u003e 2017, 12(10): e0185056.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMartin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. \u003cem\u003eEMBnet j\u003c/em\u003e 2011, 17(1): 3.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. \u003cem\u003eBioinformatics\u003c/em\u003e 2014, 30(7): 923\u0026ndash;930.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEwels P, Magnusson M, Lundin S, Kaller M. MultiQC: summarize analysis results for multiple tools and samples in a single report. \u003cem\u003eBioinformatics\u003c/em\u003e 2016, 32(19): 3047\u0026ndash;3048.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLove MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. \u003cem\u003eGenome Biol\u003c/em\u003e 2014, 15(12): 550.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSubramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, \u003cem\u003eet al.\u003c/em\u003e Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e 2005, 102(43): 15545\u0026ndash;15550.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCastanza AS, Recla JM, Eby D, Thorvaldsdottir H, Bult CJ, Mesirov JP. Extending support for mouse data in the Molecular Signatures Database (MSigDB). \u003cem\u003eNat Methods\u003c/em\u003e 2023, 20(11): 1619\u0026ndash;1620.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBoudreau RL, Davidson BL. Generation of hairpin-based RNAi vectors for biological and therapeutic application. \u003cem\u003eMethods Enzymol\u003c/em\u003e 2012, 507: 275\u0026ndash;296.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKim YK, Furic L, Desgroseillers L, Maquat LE. Mammalian Staufen1 recruits Upf1 to specific mRNA 3'UTRs so as to elicit mRNA decay. \u003cem\u003eCell\u003c/em\u003e 2005, 120(2): 195\u0026ndash;208.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eScoles DR, Meera P, Schneider MD, Paul S, Dansithong W, Figueroa KP, \u003cem\u003eet al.\u003c/em\u003e Antisense oligonucleotide therapy for spinocerebellar ataxia type 2. \u003cem\u003eNature\u003c/em\u003e 2017, 544(7650): 362\u0026ndash;366.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eElden AC, Kim HJ, Hart MP, Chen-Plotkin AS, Johnson BS, Fang X, \u003cem\u003eet al.\u003c/em\u003e Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS. \u003cem\u003eNature\u003c/em\u003e 2010, 466(7310): 1069\u0026ndash;1075.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMcGurk L, Rifai OM, Shcherbakova O, Perlegos AE, Byrns CN, Carranza FR, \u003cem\u003eet al.\u003c/em\u003e Toxicity of pathogenic ataxin-2 in Drosophila shows dependence on a pure CAG repeat sequence. \u003cem\u003eHum Mol Genet\u003c/em\u003e 2021, 30(19): 1797\u0026ndash;1810.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJia M, Shan Z, Yang Y, Liu C, Li J, Luo ZG, \u003cem\u003eet al.\u003c/em\u003e The structural basis of Miranda-mediated Staufen localization during Drosophila neuroblast asymmetric division. \u003cem\u003eNat Commun\u003c/em\u003e 2015, 6: 8381.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMcGurk L, Mojsilovic-Petrovic J, Van Deerlin VM, Shorter J, Kalb RG, Lee VM, \u003cem\u003eet al.\u003c/em\u003e Nuclear poly(ADP-ribose) activity is a therapeutic target in amyotrophic lateral sclerosis. \u003cem\u003eActa neuropathologica communications\u003c/em\u003e 2018, 6(1): 84.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSong C, Leahy SN, Rushton EM, Broadie K. RNA-binding FMRP and Staufen sequentially regulate the Coracle scaffold to control synaptic glutamate receptor and bouton development. \u003cem\u003eDevelopment\u003c/em\u003e 2022, 149(9).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMonshausen M, Putz U, Rehbein M, Schweizer M, DesGroseillers L, Kuhl D, \u003cem\u003eet al.\u003c/em\u003e Two rat brain staufen isoforms differentially bind RNA. \u003cem\u003eJ Neurochem\u003c/em\u003e 2001, 76(1): 155\u0026ndash;165.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBrown AL, Wilkins OG, Keuss MJ, Hill SE, Zanovello M, Lee WC, \u003cem\u003eet al.\u003c/em\u003e TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A. \u003cem\u003eNature\u003c/em\u003e 2022, 603(7899): 131\u0026ndash;137.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePrudencio M, Humphrey J, Pickles S, Brown AL, Hill SE, Kachergus JM, \u003cem\u003eet al.\u003c/em\u003e Truncated stathmin-2 is a marker of TDP-43 pathology in frontotemporal dementia. \u003cem\u003eJ Clin Invest\u003c/em\u003e 2020, 130(11): 6080\u0026ndash;6092.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWils H, Kleinberger G, Janssens J, Pereson S, Joris G, Cuijt I, \u003cem\u003eet al.\u003c/em\u003e TDP-43 transgenic mice develop spastic paralysis and neuronal inclusions characteristic of ALS and frontotemporal lobar degeneration. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e 2010, 107(8): 3858\u0026ndash;3863.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCottrell KA, Andrews RJ, Bass BL. The competitive landscape of the dsRNA world. \u003cem\u003eMol Cell\u003c/em\u003e 2024, 84(1): 107\u0026ndash;119.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHansen ST, Meera P, Otis TS, Pulst SM. Changes in Purkinje cell firing and gene expression precede behavioral pathology in a mouse model of SCA2. \u003cem\u003eHum Mol Genet\u003c/em\u003e 2013, 22(2): 271\u0026ndash;283.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMelamed Z, Lopez-Erauskin J, Baughn MW, Zhang O, Drenner K, Sun Y, \u003cem\u003eet al.\u003c/em\u003e Premature polyadenylation-mediated loss of stathmin-2 is a hallmark of TDP-43-dependent neurodegeneration. \u003cem\u003eNat Neurosci\u003c/em\u003e 2019, 22(2): 180\u0026ndash;190.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMa XR, Prudencio M, Koike Y, Vatsavayai SC, Kim G, Harbinski F, \u003cem\u003eet al.\u003c/em\u003e TDP-43 represses cryptic exon inclusion in the FTD-ALS gene UNC13A. \u003cem\u003eNature\u003c/em\u003e 2022, 603(7899): 124\u0026ndash;130.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhong MZ, Peng T, Duarte ML, Wang M, Cai D. Updates on mouse models of Alzheimer's disease. \u003cem\u003eMol Neurodegener\u003c/em\u003e 2024, 19(1): 23.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003evan Rheenen W, van der Spek RAA, Bakker MK, van Vugt J, Hop PJ, Zwamborn RAJ, \u003cem\u003eet al.\u003c/em\u003e Common and rare variant association analyses in amyotrophic lateral sclerosis identify 15 risk loci with distinct genetic architectures and neuron-specific biology. \u003cem\u003eNat Genet\u003c/em\u003e 2021, 53(12): 1636\u0026ndash;1648.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePottinger TD, Motelow JE, Povysil G, Moreno CAM, Ren Z, Phatnani H, \u003cem\u003eet al.\u003c/em\u003e Rare variant analyses validate known ALS genes in a multi-ethnic population and identifies ANTXR2 as a candidate in PLS. \u003cem\u003eBMC Genomics\u003c/em\u003e 2024, 25(1): 651.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAnderson EN, Gochenaur L, Singh A, Grant R, Patel K, Watkins S, \u003cem\u003eet al.\u003c/em\u003e Traumatic injury induces stress granule formation and enhances motor dysfunctions in ALS/FTD models. \u003cem\u003eHum Mol Genet\u003c/em\u003e 2018, 27(8): 1366\u0026ndash;1381.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Alzheimer’s disease, Amyotrophic lateral sclerosis, Spinocerebellar ataxia type 2, Staufen1, STAU1, TDP-43, Autophagy, Drosophila Stau","lastPublishedDoi":"10.21203/rs.3.rs-7411941/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7411941/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRNA-binding proteins (RBPs) play an essential role in development, normal functioning and human disease. Staufen1 (STAU1) is an RBP that regulates mRNA degradation and subcellular localization, and is part of the ATXN2 protein complex. Previously, we showed that STAU1 is overabundant in patient fibroblasts and in mouse models of Alzheimer\u0026rsquo;s disease (AD), amyotrophic lateral sclerosis (ALS), and spinocerebellar ataxia type 2 (SCA2), where it is associated with impaired autophagic flux due to STAU1-mediated upregulation of mTOR translation. STAU1 overabundance and impaired autophagy cause accumulation of biomolecular condensates and abnormal unfolded protein response (UPR). We generated a mouse model expressing the entire human \u003cem\u003eSTAU1\u003c/em\u003e gene (h\u003cem\u003eSTAU1\u003c/em\u003e) in a bacterial artificial chromosome (BAC) construct. hSTAU1 in these mice was expressed in cerebral hemispheres, cerebellum and spinal cord, as well as cultured cortical neurons and cortical and spinal cord astrocytes and microglia. Expression of hSTAU1 caused dysregulated gene expression, abnormal autophagy, glial activation, and changes in neuronal marker proteins. All of these were significantly improved by reducing STAU1 abundance by RNAi, but exacerbated in BAC-STAU1 mice crossed with Prp-TDP-43(Q331K) transgenic mice. Similar results were also obtained in eye phenotypes in ALS- and SCA2-relevant fly models upon changing staufen-1 dosage. Despite the molecular changes, we observed no overt behavioral changes in mice up to 55 weeks of age, suggesting that STAU1 may function as an epistatic modifier of neuronal degeneration. The BAC-hSTAU1 mouse will be useful for developing therapies targeting the human STAU1 gene.\u003c/p\u003e","manuscriptTitle":"A human Staufen1 BAC transgenic mouse exhibits abnormal autophagy and neurodegeneration across the central nervous system","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-23 02:31:31","doi":"10.21203/rs.3.rs-7411941/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-09-30T13:32:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-09-29T09:56:23+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-09-22T08:42:19+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-09-16T09:24:21+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-09-15T00:08:16+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-09-12T12:32:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-20T11:04:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2025-08-19T22:29:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-19T22:29:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"59a1b4d3-f918-46cd-9d2a-fff1ae4f24d9","owner":[],"postedDate":"September 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":54630306,"name":"Biological sciences/Neuroscience/Diseases of the nervous system/Amyotrophic lateral sclerosis"},{"id":54630307,"name":"Biological sciences/Neuroscience/Diseases of the nervous system/Dementia/Alzheimer's disease"}],"tags":[],"updatedAt":"2026-04-27T08:58:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-23 02:31:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7411941","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7411941","identity":"rs-7411941","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}