Imbalance in Redox Homeostasis is Associated with Neurodegeneration in the Murine Model of Tay-Sachs Disease

Imbalance in Redox Homeostasis is Associated with Neurodegeneration in the Murine Model of Tay-Sachs Disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Imbalance in Redox Homeostasis is Associated with Neurodegeneration in the Murine Model of Tay-Sachs Disease Hande Basırlı, Nurselin Ateş, Volkan Seyrantepe This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5293300/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Mar, 2025 Read the published version in Molecular Biology Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Background Tay-Sachs disease is a type of neurodegenerative disorder with a build-up of GM2 ganglioside in the brain, which results in progressive central nervous system dysfunction. Our group recently generated Hexa-/-Neu3-/- mice, a murine model with neuropathological abnormalities similar to the infantile form of Tay-Sachs disease. Previously, we reported progressive neurodegeneration with neuronal loss in the brain sections of Hexa-/-Neu3-/- mice. However, the relationship of the severity of neurodegeneration to imbalance in redox homeostasis has not been clarified in Hexa-/-Neu3-/- mice. Here, we evaluated whether neurodegeneration is associated with oxidative stress in the tissues and cells of Hexa-/-Neu3-/- mice and neuroglia cells from Tay-Sachs patients. Methods and Results In four brain regions and fibroblasts of 5-month-old WT , Hexa-/- , Neu3-/- , and Hexa-/-Neu3-/- mice and human neuroglia cells, apoptosis and oxidative stress-related markers were evaluated using Western blot, RT-PCR, and immunohistochemistry analyses. We further analyzed oxidative stress levels using flow cytometry analyses. We discovered neuronal death, alterations in intracellular ROS levels, and damaging effects of oxidative stress, especially in the cerebellum and fibroblasts of Hexa-/-Neu3-/- mice. Conclusions Our results showed that alteration in redox homeostasis might be related to neurodegeneration in the murine model of Tay-Sachs Disease. These findings suggest that targeting the altered redox balance and increased oxidative stress might be a rational therapeutic approach for alleviating neurodegeneration and treating Tay-Sachs disease. Tay-Sachs Disease Neurodegeneration Apoptosis Oxidative stress Reactive Oxygen Species Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Tay-Sachs disease (TSD) is categorized as a rare neurodegenerative disorder with the dysfunctionality of β-hexosaminidase A (HexA). Progressively accumulating GM2 ganglioside in the brain of TS patients results in a deteriorated central nervous system (CNS) and disrupted mental and motor functions. TS patients eventually die at 2–4 years of age. Currently, no effective treatment has been provided for TS patients. Previously, the Hexa-/- mice were presented as a model of TSD. On the other hand, it did not show any neuropathological symptoms noticed in patients. In Hexa-/- mice different from humans, degradation of GA2 to GM2 facilitated by neuraminidase was proposed [ 1 , 2 ]. The role of neuraminidase 3 (Neu3) in the degradation of glycolipids and TSD in mice was discovered, and Hexa-/-Neu3-/- mice were introduced as the murine model of TSD. At birth, Hexa-/-Neu3-/- mice were healthy, but death occurs at five months of age because of progressive loss of neurons and Purkinje cells and increased active astrocytes. Additionally, Hexa-/-Neu3-/- mice prominently showed notable neurological deteriorations, including movement problems, ataxia, and tremor [ 3 , 4 ]. Predominant pathology in lysosomal storage disorders (LSDs) is larger sizes and numbers of lysosomes due to the progressive accumulation of substrates, which may cause an imbalance in redox homeostasis and neurodegeneration [ 5 – 7 ]. A bunch of studies reported increased reactive oxygen species (ROS) and altered redox homeostasis, which is one of the mediators of neurodegeneration in many LSDs [ 8 , 9 ]. The basal level of endogenous ROS is essential for redox homeostasis. However, the excess amount of ROS is removed by antioxidant defense systems comprising non-enzymatic antioxidants or oxidoreductases like superoxide dismutase, catalase, and Ttase1. When the net endogenous ROS levels are more significant than the capacity of antioxidant defense mechanisms to remove the excessive ROS, an imbalance in cellular redox homeostasis is generated in the cell, provoking the oxidation of biological macromolecules [ 10 ]. Notably, proteins are oxidized on their side chains, and thus, they become carbonylated. The detection of carbonylated protein content is generally performed to measure the levels of protein oxidations in cells of several neurodegenerative diseases [ 11 , 12 ]. Besides protein damage, ROS also alters DNA structure and causes oxidative DNA damage. It has been shown that the APE1 protein has a crucial activity in the DNA Base Excision Repair (BER) pathway against oxidative DNA damage. Several studies have reported that APE1 has a protective effect through the DNA BER pathway against imbalance in redox homeostasis, mediating neurodegeneration, oxidative signaling, and cellular injury [ 13 , 14 ]. Thus, the APE1 protein has a significant role in preventing ROS-induced alteration of redox homeostasis. In addition, it has been reported that damages to macromolecules created by ROS contribute to inflammation, tissue damage, and the activation of neurodegeneration [ 15 ]. Among the various neurodegenerative diseases, damaging influences of oxidative stress and imbalanced redox homeostasis have also been reported to contribute to the pathophysiology of LSDs [ 5 , 7 ]. The link between lysosomal accumulation and imbalanced redox homeostasis was reported in mouse models of Sandhoff disease ( Hexb-/- ), late-onset Tay-Sachs disease ( Hexa-/- ) [ 16 ], GM1 Gangliosidosis [ 17 ] and Mucopolysaccharidosis type I [ 18 ]. In addition, in vitro studies on neural progenitor cells derived from Tay-Sachs patients [ 19 ], fibroblasts from NPC patients [ 20 ], and differentiated human oligodendrocytes from Krabbe disease [ 21 ] revealed that alteration in redox homeostasis has a role in neurodegeneration. So far, no experimental evidence shows a link between early-onset TSD pathology and an imbalance in redox homeostasis. Therefore, in this study, we aimed to explore whether there is an alteration in redox homeostasis, which is related to neurodegeneration and apoptosis in the tissues from the murine model of TSD and cells from Tay-Sachs patients. Materials and methods Animals Hexa-/- , Neu3-/- , and Hexa-/-Neu3-/- mice were obtained as described in the previous studies [ 4 , 22 , 23 ]. The mice breeding and maintenance were generated in the Turkish Council on Animal Care (TCAC) accredited animal facility of Izmir Institute of Technology according to the TCAC guidelines. Mice were kept in an environment with constant humidity and temperature on a 12-hour light-dark cycle. The Animal Care and Use Committee of Izmir Institute of Technology, Izmir, Turkey, granted animal care and its use in the experiments. The mice breeding and genotyping were generated as described in the previous studies [ 4 ]. After the scarification of mice at five months old, brain samples were dissected into the cortex, cerebellum, thalamus, and hippocampus and snap-freezing in liquid nitrogen. The tissues were stored at -80 o C until used. Cell Culture Fibroblast lines from 5-month-old WT , Hexa-/- , Neu3-/- , and Hexa-/- Neu3-/- were established and performed in DMEM (Gibco™, USA) with 10% FBS (Gibco™, USA) and 1% (v/v) penicillin/streptomycin (Gibco™, USA). Primary fibroblasts were immortalized using serum-free DMEM containing viral supernatant (LXSN 16E6E7) and polybrene (400µg/ml) overnight at 37 o C. After removal of DMEM supplemented with viral supernatant and polybrene, the cells were cultivated in DMEM with 10% FBS and G418 (400µg/ml) to select immortalized cells. Immortal fibroblasts were treated for one hour with 100 µM hydrogen peroxide (Sigma, Germany). After transformation of the immortalized control neuroglia cells (NG-124) and TSD neuroglia cells (NG-125) [ 24 ] with the pCMV-HexA plasmid that encodes the α subunit of HexA and expresses the active HexA enzyme [ 25 ], cells were culturized in DMEM, including 10% (vol/vol) FBS and 1% (vol/vol) penicillin/streptomycin. Real-Time PCR Total RNA extraction from brain tissues and fibroblasts of all genotypes at five months of age, control neuroglia cells (NG-124), and TSD neuroglia cells (NG-125) were done using Trizol Reagent (GeneAid, Taiwan). Following the extraction, cDNA conversion was generated using a reverse transcription kit (Applied Biosystems, USA). Expression analyses of oxidative stress markers (SOD2, Catalase, and Ttase1) were done using LightCycler 480 SYBR Green I Master Mix by applying the manufacturer’s protocol. The endogenous control was GAPDH. The primers listed were used for expression analyses; SOD2F: 5’- GTGTCTGTGGGAGTCCAAG G-3’, SOD2R: 5’-CCCCAGTCATAGTGCTGCAA-3’, CatalaseF: 5’-TTCGTCCCGAGTCT CTCCAT-3’, CatalaseR: 5’-GAGGCCAAACCTTGGTCAGA-3’, Ttase1F: 5’-CTGCAAGAT CCAGTCTGGGAA-3’, Ttase1R: 5’-CTCTGCCTGCCACCCCTTTTAT-3’, GAPDHF: 5’-C CCCTTCATTGACCTCAACTAC-3’, GAPDHR: 5’-ATGCATTGCTGACAATCTTGAG-3’. Protein Carbonylation Analysis Protein isolation was done from brain tissues and fibroblasts of all genotypes at five months of age, control neuroglia cells (NG-124) and TSD neuroglia cells (NG-125) using cold lysis buffer (50 Mm Tris- HCI, 150 mM NaCl, 1% TritonX-100, 50 mM HEPES, 10% glycerol, protease inhibitors) including 1% β-mercaptoethanol. Later, reagents supplemented by Protein Oxidation Detection Kit (S7150 OxyBlot™ Merck-Millipore, Canada) were used by applying the manufacturer’s protocol. Immunodetection of carbonyl proteins was done by a DNP-specific primary antibody (1:150) for the carbonyl content, and an anti- \(\:\beta\:\) -Actin (1:1000, Cell Signaling, USA) antibody was used for normalization. HRP-conjugated antibody (Jackson ImmunoResearch Lab, USA) was the secondary antibody. The carbonylated proteins were visualized by chemiluminescent reagent on a digital imaging system (Fusion SL, Vilber). Band densities were measured using NIH ImageJ [ 26 ]. \(\:\beta\:\) -Actin was used as the endogenous control for band intensity normalization. Terminal dUTP nick end-labeling (TUNEL) analysis Hexa-/- and Hexa-/-Neu3-/- mice at five months of age were anesthetized for trans-cardiac perfusion of the heart with 4% paraformaldehyde (PFA, Sigma). After perfusion, the brains were removed and incubated with 4% PFA (Sigma) overnight at 4°C. Then, brains were placed sequentially into 10%, 20% in PBS at 4°C, and 30% sucrose in PBS overnight at 4°C. Brain embedding was performed using an optimal cutting temperature (OCT) compound (Sigma). The embedded brains were sectioned in a coronal plane using a Leica cryostat at ten µm thickness on HistoBond® microscope slides (Marienfeld) at -20°C. Following the manufacturer's protocol, the ApopTag Fluorescein in situ Apoptosis Detection Kit (Millipore) was used to perform Terminal dUTP Nick End-Labeling (TUNEL) analysis. Concisely, the brain sections of Hexa-/- and Hexa-/-Neu3-/- mice were fixed in 1% PFA and precooled in ethanol acetic acid (2:1) solutions at five months. The terminal deoxynucleotidyl transferase and anti-digoxygenin conjugate were applied to the brain sections. The sections were incubated with the nuclear counterstain, propidium iodide (0.5 µg/mL). The fluorescent images were taken using a fluorescent microscope (Olympus-BX53F). The cortex, thalamus, hippocampus, cerebellum, and pons regions were analyzed for TUNEL-positive green fluorescent cells as the indicator of neuronal death, and the colocalization intensity of the images was measured using NIH ImageJ [ 26 ]. Immunocytochemical Analysis The fibroblasts were cultivated on micro slides for 24 hours and fixed with 4% PFA (Sigma). Following the fixation, a blocking buffer (10% goat serum, 4% BSA 0.3M Glycine, and 0.3% TritonX in PBS) was used for blocking the fibroblasts for 1 hour after which slides were stained with primary antibody, anti-APE1/Ref-1 (1:200; Abcam, USA), overnight at 4 0 C and anti-Alexa Fluor®-488 (1:500, Abcam, UK) as the secondary antibody. The slides were covered using DAPI (Abcam, UK). Fluorescent images were taken by Fluorescent Microscopy (Olympus-BX53F), and the intensity of the images was measured using NIH ImageJ [ 26 ]. Flow Cytometry Analysis Under normal conditions, a nonpolar substance, 2’7’-dichlorodihydrofluorescein diacetate (H 2 DCFDA), is converted to a non-fluorescent polar (H 2 DCF) by cellular esterases. Suppose ROS is present in cells; oxidation of H 2 DCF results in highly fluorescent 2’7’ dichlorofluorescein (DCF). To measure intracellular ROS level, fluorescent DCF by Flow cytometry was analyzed after treating the cells with H 2 DCFDA. H 2 O 2 - and H 2 DCFDA-treated groups as the positive control, the non-treated group as the negative control, and the H 2 DCFDA-treated group as samples were studied. The positive controls were incubated for 1 hour at 37 o C with 10 µM H 2 O 2 containing serum-free media. After the incubation, positive control and sample group cells were incubated for 30 min with 5µM H 2 DCFDA containing serum-free media and then harvested with trypsin. The cells were resuspended in 600 \(\:\mu\:\) l PBS; triplicate samples of each sample were used for Flow Cytometry analysis on the guava easyCyte™, and the results were analyzed using guavaSoft™ software. Western Blot Analysis Total protein isolation from brain tissue samples and fibroblasts was done by treating the samples with cold lysis buffer (50 Mm Tris- HCI, 150 mM NaCl, 1% TritonX-100, 50 mM HEPES, 10% glycerol, protease inhibitors) for 1 hour. Following the protein loading to SDS-PAGE, it is transferred to a nitrocellulose membrane (Bio-Rad, USA). The membranes were stained with anti-Bcl2 (1:500, Santa Cruz Biotechnology, USA), anti-BclXL (1:1000, Cell Signaling, USA), anti-Bax (1:1000, Abcam, UK), anti-APE1 (1:1000, Abcam, UK), and anti- \(\:\beta\:\) -Actin (1:1000, Cell Signaling, USA) overnight at + 4 o C. Later, membranes were treated with HRP-conjugated secondary antibodies (Jackson ImmunoResearch Lab, USA). Protein visualization was performed using LuminataTM Forte WesternHRP Substrate (Millipore, USA) on a digital imaging system (Fusion SL, Vilber). Band densities were measured using NIH ImageJ (1.48v) [ 26 ]. \(\:\beta\:\) -Actin was used as the endogenous control for the normalization of band intensities. Statistical Analysis GraphPad Prism 7 (v. 7.0a, GraphPad Software, Inc) performed all statistical analyses. For all experiments, WT , Hexa-/- , Neu3-/- , and Hexa-/- Neu3-/- groups (n = 3) were compared by One-Way ANOVA. The data are shown as mean ± SEM. Results Altered levels of the anti-apoptotic markers in different brain regions but not in fibroblast of Hexa-/-Neu3-/- mice Alterations in the protein levels of anti-apoptotic Bcl2 (Fig. 1 ), Bcl-XL (Fig. 2 ), and pro-apoptotic Bax (Fig. 3 ) in the different regions of the brain were investigated using immunoblot analyses. The protein expression levels of Bcl2 were significantly reduced in the cerebellum of Neu3-/- and Hexa-/-Neu3-/- mice compared to WT and Hexa-/- mice (Fig. 1 A, B). Similarly, we identified a remarkably lower level of Bcl2 protein in the thalamus (Fig. 1 E, F) and hippocampus (Fig. 1 G, H) of Hexa-/-Neu3-/- mice compared to age-matched single knockouts. In contrast, we determined higher levels of Bcl2 protein in the cortex (Fig. 1 C, D) of Neu3-/- and Hexa-/-Neu3-/- mice. Although our data clearly shows alterations in Blc2 protein levels in different brain regions in WT , single, and double knockouts, higher levels of Bcl2 only in the cortex of Hexa-/-Neu3-/- might be related to the severe phenotype observed. In another study, we also used the fibroblasts of Hexa-/-Neu3-/- mice and showed there is a significant increase in the levels of Bcl2 protein compared to the fibroblasts of Neu3-/- mice under non-treated conditions (Fig. 1 I, J). Although there was a remarkable decrease in levels of Bcl2 protein under the same conditions only in Hexa-/- mice’s fibroblasts compared to their non-treated counterparts, we found no difference in the levels of Bcl2 protein under H 2 O 2 -treated conditions of the fibroblasts from Hexa-/-Neu3-/- mice compared to the non-treated counterparts. In parallel to in vitro studies with TSD mice fibroblast mice, we also evaluated the levels of Bcl2 protein in the neuroglia of Tay-Sachs patients under both non- and H 2 O 2 -treated conditions (Fig. 1 K, L). We demonstrated that the levels of Bcl2 protein were not affected in non-treated NG-125 cells compared to non-treated NG-124 and H 2 O 2 -treated NG-125 cells (Fig. 1 K, L). Interestingly, our data showed a significant reduction in the levels of anti-apoptotic Bcl2 protein in H 2 O 2 -treated NG-124 cells compared to their non-treated counterparts (Fig. 1 L). Moreover, the protein levels of anti-apoptotic markers, BclXL, did not show any significant changes in the cerebellum (Fig. 2 A, B) and thalamus (Fig. 2 E, F) regions of Hexa-/-Neu3-/- mice. Remarkably, in the cortex (Fig. 2 C, D) and hippocampus (Fig. 2 G, H) of Hexa-/-Neu3-/- mice, we identified lower BclXL protein levels than WT and Hexa-/- mice. Hexa-/-Neu3-/- fibroblasts consistently displayed a significant reduction in the levels of BclXL protein under non-treated conditions (Fig. 2 I, J). Surprisingly, our results showed that H 2 O 2 treatment did not alter the levels of BclXL protein in the fibroblasts from WT , Hexa-/- , Neu3-/- , and Hexa-/-Neu3-/- mice compared to their non-treated counterparts (Fig. 2 I, J). Similarly, we analyzed the levels of BclXL protein in the neuroglia cells of Tay-Sachs patients under H 2 O 2 -treated conditions (Fig. 2 K, L). Our results indicated that there were also no significant changes in the levels of BclXL protein in non-treated NG-125 cells compared to NG-124 cells (Fig. 2 K, L). Besides, the levels of BclXL protein also did not show any difference in NG-125 cells under H 2 O 2 -treated oxidative stress conditions compared to non-treated NG-125 (Fig. 2 L). The significantly elevated protein levels of pro-apoptotic Bax were only shown in the cortex (Fig. 3 C, D) of Hexa-/-Neu3-/- mice compared to WT mice but not in other brain regions (Fig. 3 ). Besides, we evaluated levels of Bax protein in the fibroblasts of Hexa-/-Neu3-/- mice both in treated and untreated conditions, and we found there was a remarkable elevation of antiapoptotic Bax protein in the fibroblasts of Hexa-/-Neu3-/- mice compared to the WT and Neu3-/- under non-treated conditions (Fig. 3 I, J). Under H 2 O 2 -treated conditions, Bax protein levels significantly decreased in the fibroblasts of Hexa-/- and Hexa-/-Neu3-/- mice compared to their non-treated counterparts (Fig. 3 I, J). On the other hand, we identified significantly higher protein levels of Bax in the H 2 O 2 -treated fibroblasts of WT and Neu3-/- mice compared to non-treated WT and Neu3-/- , respectively (Fig. 3 I, J). In addition to the Western Blot analysis of Bax protein in fibroblasts of Hexa-/-Neu3-/- mice, we also evaluated levels of Bax protein in the neuroglia cells of Tay-Sachs patients in the context of our study (Fig. 3 K, L). Our results revealed a slight increase in the protein levels of Bax in H 2 O 2 -treated NG-125 cells compared to non-treated NG-125 cells, while there were no alterations in NG-125 cells compared to NG-124 cells under non-treated conditions (Fig. 3 L). We further evaluated mRNA levels of anti-apoptotic Bcl2 (Fig. S1 A, D, G, J, M) and Bcl-XL (Fig. S1 B, E, H, K, N), and pro-apoptotic Bax gene (Fig. S1 C, F, I, L, O) in the cerebellum, cortex, thalamus, and hippocampus of 5-month-old WT , Hexa-/-, Neu3-/- , and Hexa-/-Neu3-/- mice using qRT-PCR analyses. We showed that the mRNA levels of Bcl2 were significantly reduced in the cerebellum of Hexa-/-Neu3-/- mice compared to WT and Neu3-/- mice (Fig. S1 A). In the cortex region, Bcl2 mRNA was significantly down-regulated in Hexa-/-Neu3-/- mice compared to Hexa-/- counterparts (Fig. S1 D). Additionally, a significant decrease was detected in the hippocampal region of Hexa-/-Neu3-/- mice compared to WT and Neu3-/- mice (Fig. S1 J). Surprisingly, we observed no significant changes in the mRNA expression of Bcl2, Bcl-XL, and Bax genes in other brain regions of Hexa-/-Neu3-/- mice compared to age-matched WT , Hexa-/- , and Neu3-/- mice (Fig. S1 ). We also analyzed levels of the same genes in the fibroblasts derived from Hexa-/-Neu3-/- mice in normal and oxidative stress conditions; however, we did not find any significant differences with other mice groups (Fig. S1 M-O). Interestingly, only Hexa-/- mice fibroblasts showed slightly higher Bcl2 mRNA expression under H 2 O 2 -induced oxidative stress conditions (Fig. S1 M). Increased levels of cell death in the brain of Hexa-/-Neu3-/- mice We performed Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay for visualization of neuronal death in situ [ 27 ] and for showing TUNEL-positive cells in the cerebellum, pons, cortex, thalamus, and hippocampus sections of 5-month-old Hexa-/-Neu3-/- mice compared to age-matched Hexa-/- mice. TUNEL assay indicated significantly elevated levels of TUNEL-positive neurons and neuronal death in all brain regions of 5-month-old Hexa-/-Neu3-/- mice compared to the age-matched Hexa-/- mice group (Fig. 4 ). Elevated transcriptional levels of the oxidative stress-related markers in the fibroblast but not in the cerebellum of Hexa-/-Neu3-/- mice In this study, we also analyzed the mRNA expression levels of oxidative stress markers SOD2, Catalase, and Ttase1 in five brain regions of 5-month-old mice and age-matched counterparts using qRT-PCR (Fig. 5 ). Our data showed that the expression levels of SOD2 (Fig. 5 A) and Ttase1 mRNAs (Fig. 5 C) in the cerebellum of Hexa-/-Neu3-/- mice exhibited a significant reduction compared to WT, Hexa-/- , and Neu3-/- mice. Similarly, the mRNA expression levels of Catalase in the cerebellum of Hexa-/-Neu3-/- mice significantly decreased compared to WT and Hexa-/- mice (Fig. 5 B). Interestingly, there were no apparent differences in the mRNA expression levels of SOD2, Catalase, and Ttase1 in other brain regions of Hexa-/-Neu3-/- mice compared to WT, Hexa-/- , and Neu3-/- mice (Fig. 5 ). Furthermore, the mRNA expression levels of SOD2, Catalase, and Ttase1 were analyzed in fibroblasts derived from 5-month-old Hexa-/-Neu3-/- mice and WT as well as single knockouts (Fig. 5 M, O). In this experiment, one group of fibroblasts was treated with H 2 O 2 to induce oxidative stress in vitro . Our results revealed that levels of SOD2 mRNA in the fibroblasts of Hexa-/-Neu3-/- mice were significantly upregulated compared to the fibroblasts of Neu3-/- mice under normal and oxidative stress conditions (Fig. 5 M). Unexpectedly, there were no significant changes in the mRNA expression levels of SOD2 in fibroblasts of Hexa-/-Neu3-/- mice compared with WT and Hexa-/- mice (Fig. 5 M). Besides, under H 2 O 2 -treated conditions, the mRNA levels of SOD2 were not significantly changed in the fibroblasts of Hexa-/-Neu3-/- mice compared to their non-treated counterparts (Fig. 5 M). The mRNA expression levels of Catalase in the fibroblasts of Hexa-/-Neu3-/- mice under non-treated conditions did not show any significant difference compared to WT, Hexa-/- , and Neu3-/- counterparts (Fig. 5 N). Under H 2 O 2 -treated conditions, the mRNA levels of Catalase in the fibroblasts of Hexa-/-Neu3-/- mice did not change compared to the non-treated fibroblasts of Hexa-/-Neu3-/- mice (Fig. 5 N). Ttase1 mRNA expression levels displayed significantly increased levels in Hexa-/-Neu3-/- fibroblasts compared to other genotypes under non-treated and oxidative stress conditions (Fig. 5 O). Analysis of human neuroglia cells also showed no significant change in oxidative stress markers in Tay-Sachs patient neuroglia (NG-125) compared to healthy neuroglia under both non-treated and oxidative stress conditions (Fig. 5 P-S). Treatment with H 2 O 2 upregulated the mRNA expression levels of SOD2 and Ttase1 in both NG-124 and NG-125, except for the levels of catalase mRNA in NG-124 (Fig. 5 P-S). Higher levels of carbonylated proteins in the cerebellum and fibroblast of Hexa-/-Neu3-/- mice To show the detrimental effects of imbalanced redox homeostasis, we measured carbonyl protein content, an irreversible protein modification, using protein carbonylation analysis in the cerebellum, cortex, thalamus, and hippocampus of 5-month-old WT , Hexa-/- , Neu3-/- , and Hexa-/-Neu3-/- mice (Fig. 6 ). Our results indicated that in the thalamus and cerebellum of Hexa-/-Neu3-/- mice, there were significantly increased levels of carbonylated proteins compared to WT and Hexa-/- mice (Fig. 6 A, C). In fibroblasts of 5-month-old WT , Hexa-/- , Neu3-/- , and Hexa-/-Neu3-/- mice, the levels of carbonylated proteins were also analyzed. Our results indicated a significant increase in Hexa-/-Neu3-/- fibroblasts compared to their single knock-out counterparts (Fig. 6 E). Similarly, in Tay-Sachs patient neuroglia cells (NG-125), significantly increased levels of carbonylated proteins were observed when compared to normal neuroglia cells (NG-124) (Fig. 6 F). Elevated levels of APE1 protein in brain regions of Hexa-/-Neu3-/- mice APE1 protein has essential roles in repairing oxidative damage on DNA and mediating the activity of specific transcription factors [ 28 ]. To understand the response to oxidative DNA damage in the brain, we analyzed the levels of APE1 protein in four different brain regions of 5-month-old mice (Fig. 7 ). Cerebellum (Fig. 7 A), cortex (Fig. 7 B), and thalamus (Fig. 7 C) regions displayed higher levels of APE1 protein in Hexa-/-Neu3-/- mice. Interestingly, we observed no difference in the hippocampus region (Fig. 7 D). We also studied APE1 protein levels in fibroblasts under normal and H 2 O 2 -induced conditions (Fig. 7 E). There was no significant difference in APE1 protein expression in the fibroblasts for both normal and H 2 O 2 -induced conditions (Fig. 7 E). To determine the levels and localization of cellular APE1 protein, we also performed immunocytochemical analyses in the fibroblasts of 5-month-old WT , Hexa-/- , Neu3-/- , and Hexa-/-Neu3-/- mice (Fig. 7 F). Even if it is not statistically significant, fluorescent intensity demonstrated that fibroblasts of Hexa-/-Neu3-/- mice displayed higher levels of APE1 protein compared to control groups however it is down-regulated in increased oxidative stress condition (Fig. 7 G). When we analyzed the nuclear and cytoplasmic APE1 protein intensity separately, we observed that the fibroblasts of Hexa-/-Neu3-/- mice displayed a decreased level of nuclear APE1 protein; however, a significantly increased level of cytoplasmic APE1 protein in both non- and H 2 O 2 -treated groups compared to the fibroblasts of WT and Neu3-/- (Fig. 7 H, I). Induced oxidative stress using H 2 O 2 increased the cytoplasmic APE1 protein intensity level compared to non-treated conditions, but the difference was not statistically significant (Fig. 7 I). Intracellular ROS level was higher in fibroblasts of Hexa-/-Neu3-/- mice The reactive oxygen species (ROS) comprising radicals and non-radical molecules in cells become detrimental when neutralizing systems cannot overcome their production [ 29 ]. The fluorescence signal of H 2 DCFDA detected by flow cytometry reflects the oxidized dye content and is used to measure intracellular ROS level. By combining H 2 DCFDA with H 2 O 2 , we aimed to show whether there is a difference between the standard and induced oxidative stress conditions (Fig. 8 ). Our data shows WT and Hexa-/- mice showed significantly more severe oxidative stress than Neu3-/- and Hexa-/-Neu3-/- mice under induced conditions. On the other hand, Hexa-/-Neu3-/- and Neu3-/- mice displayed a significantly higher level of ROS compared to WT and Hexa-/- mice under non-treated conditions (Fig. 8 B). Similarly, H 2 O 2 -treated NG-125 cells exhibited decreased intracellular ROS levels compared to other groups; however, under normal conditions, there was no difference between intracellular ROS levels in NG-124 and NG-125 cells (Fig. S2 ). Discussion In many lysosomal storage disorders, progressive neurodegeneration and susceptibility of neurons to these disorders, including GM2 gangliosidosis, have been shown to affect the central nervous system [ 30 , 31 ]. Recently, we reported a severe neuronal death, especially in the cerebella and hippocampi of Hexa-/-Neu3-/- mice, the murine model of TSD, related to the accumulating GM2 ganglioside [ 4 ]. In addition, gene expression levels of several anti- and pro-apoptotic genes implied increased apoptotic regulation in the Hexa-/-Neu3-/- mouse model [ 3 , 4 ]. On the other hand, the mechanism of neurodegeneration relevant to imbalance in redox homeostasis has not yet been clarified in Hexa-/-Neu3-/- mice. Here, we further analyzed the other regulators of the apoptosis mechanism in Hexa-/-Neu3-/- mice under oxidative stress conditions using fibroblasts and brain samples of Hexa-/-Neu3-/- mice and neuroglia cells of TSD patients. Consistent with our previous data, we demonstrated decreased levels of Bcl2, an anti-apoptotic apoptosis regulator, particularly in the cerebella and hippocampi of Hexa-/-Neu3-/- mice. Surprisingly, elevated levels of pro-apoptotic Bax and significantly decreased levels of anti-apoptotic BclXL were shown in the cortex and fibroblasts of Hexa-/-Neu3-/- mice, indicating apoptotic cell death. Moreover, drastically higher levels of TUNEL-positive cells in Hexa-/-Neu3-/- brain regions demonstrated elevated levels of apoptosis and neurodegeneration. The balance between ROS production and the antioxidant defense mechanism provides the redox homeostasis of the cell. Once the net ROS production reaches above the capacity of the antioxidant defense mechanism, redox homeostasis is disrupted, and oxidative stress occurs [ 32 ]. Therefore, excessively produced ROS causes progressive oxidative damage, leading to lipid peroxidation, protein oxidation, and DNA damage [ 10 ]. Even if the exact mechanism of oxidative damage in LSDs is not yet wholly explained, lysosomal accumulations create an imbalance in redox homeostasis. NPC fibroblasts of human patients showed decreased ROS generation [ 33 ]; in the human Krabbe disease cell and Pompe disease mouse model [ 21 , 34 ], ROS levels were increased. Furthermore, higher intracellular ROS levels and oxidative stress markers were identified in cultured endothelial cells of Fabry patients with accumulating Gb 3 , in the fibroblasts of Gaucher Disease patients with accumulating glucosylceramide [ 13 , 35 ], and in the fibroblasts of Tay-Sachs patients with accumulating GM2 [ 9 ]. Here, we further investigated whether there is an alteration in the levels of oxidative stress markers, SOD2, Catalase, and TTase1, in brain regions and fibroblasts of the murine model of TSD. SOD2 removes excess ROS to keep cellular redox balance, and decreased levels of SOD2 gene expression were reported in the brain tissue of the Sandhoff Disease murine model [ 36 ]. Catalase, which degrades hydrogen peroxide into water and oxygen, is decreased in Neuronal Ceroid Lipofuscinoses (NCL) patients [ 37 , 38 ]. Thioltransferase-1 (Ttase1), which belongs to the glutaredoxin family of proteins, increased in the fibroblasts of GM1 gangliosidosis, Gaucher disease, and Tay-Sachs patients [ 9 ]. Likewise, we showed decreased expression levels of SOD2 and Catalase in the cerebellum of Hexa-/-Neu3-/- mice, indicating the dysregulation of redox balance and increased oxidative stress. Besides, the increased expression of Thioltransferase-1 (Ttase1), elevated catalase expression under H 2 O 2 -treated conditions, and increased ROS levels could be correlated with the imbalance in redox homeostasis and unresponsiveness to oxidative stress in the fibroblasts of Hexa-/-Neu3-/- mice. Previously, it has been shown that the anti-apoptotic protein Bcl2 is involved in the protection of cells against ROS-mediated apoptosis. However, Bcl2 itself does not have an antioxidant effect; instead, it might upregulate the superoxide dismutase levels, including SOD2, within cells [ 39 , 40 ]. The decreased levels of SOD2 in the cerebellum of Hexa-/-Neu3-/- mice may be a response to downregulation of Bcl2 and neurodegeneration. Increased oxidative stress triggers the oxidation of biological macromolecules, including the carbonylation of proteins and DNA damage due to the accumulation of ROS molecules [ 41 ]. In the fibroblasts of Gaucher Disease patients and the plasma samples from Niemann-Pick type C patients, increased protein carbonylation and imbalanced redox homeostasis were reported [ 13 , 42 ]. Similarly, we investigated the increase in the levels of carbonylated proteins in the cerebellum and fibroblasts of Hexa-/-Neu3-/- mice. Therefore, we suggest that protein carbonylation in the tissues of the TSD murine model is correlated to protein damage and altered redox balance. APE1 is an endonuclease involved in the base excision repair mechanism and the control of nuclear redox activity. Therefore, it is a significant regulator of the cellular response to an imbalance in redox homeostasis and oxidative stress [ 43 ]. Our study observed increased protein expression levels of APE1 in all brain regions of Hexa-/-Neu3-/- mice except for the hippocampus. Moreover, we observed significantly higher cytoplasmic expression of APE1 protein in fibroblasts of Hexa-/-Neu3-/- mice; however, the exact biological role of APE1 in cytoplasm requires further investigation. Our data suggests that APE1 protein expression in the TSD murine model could be related to DNA damage caused by oxidative stress. Conclusion Little is understood about the mechanism(s) by which the GM2 ganglioside accumulation and altered redox homeostasis leads to neurodegeneration in Hexa-/-Neu3-/- mice at a molecular level, but research in the fibroblasts and brain samples of TSD murine model might increasingly point to the role of oxidative stress mediators. Here, we demonstrated the damaging effects of imbalanced redox homeostasis and its association with neurodegeneration using apoptotic regulators, protein carbonylation assays, and APE1 protein expression in fibroblast and specific brain regions of the murine model. Taken together, our results provide the first in vivo evidence that Hexa-/-Neu3-/- mice displayed an imbalance in redox homeostasis, especially in the cerebellum, which might contribute to neurodegeneration and severe phenotype in the murine model of TSD. However, further identification of oxidative stress-related players on neurodegeneration is required to evaluate possible therapeutic approaches targeting antioxidant defense mechanisms. Declarations Acknowledgment The authors thank Assoc. Prof. Dr. Ayten Nalbant Aldanmaz and Tufan Utku Çalışkan for Flow cytometer analysis, and Dr. Seçil Akyıldız Demir for technical help and support. Authors' contributions HB, NA, and VS conceived the study, designed experiments, analyzed and interpreted data, and wrote the manuscript. All authors confirm the authenticity of the raw data and have read and approved the final manuscript. Funding This study was funded by the Scientific and Technological Research Council of Turkey (TUBITAK) under Grant No:215Z083. NA was supported by the Turkish Higher Education Council’s 100/2000 Ph.D. fellowship program and the TUBITAK BIDEB National Scholarship Program for Ph.D. students (2211-A). A scholarship program under the TUBITAK-France Bosphorus 120N552 Project supported HB. Availability of data and materials All data generated or analyzed during this study are included in this published article. Conflict of interests The authors declare that they have no competing interests. Ethics approval All mice were maintained in the Turkish Council on Animal Care (TCAC) accredited animal facility of Izmir Institute of Technology according to TCAC guidelines. The Animal Care and Use Committee (Animal Ethics Committee) of Izmir Institute of Technology, Izmir, Turkey, granted animal care and use in the experiments. Consent to participate Not applicable. Consent to publish Not applicable. References Sango K, Yamanaka S, Hoffmann A et al (1995) Mouse models of Tay–Sachs and Sandhoff diseases differ in neurologic phenotype and ganglioside metabolism. 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Int J Dev Neurosci 30. https://doi.org/10.1016/j.ijdevneu.2012.07.002 Tell G, Damante G, Caldwell D, Kelley MR (2005) The intracellular localization of APE1/Ref-1: More than a passive phenomenon? Antioxid Redox Signal 7 Additional Declarations No competing interests reported. Supplementary Files FigureS1.tiff Fig. S1 Bcl2, BclXL, and Bax gene expression levels of the cerebellum (A, B, C), cortex (D, E, F), thalamus (G, H, I), hippocampus (J, K, L), non-treated (-) and H 2 O 2 -treated (+) fibroblasts (M, N, O) of 5-month-old WT, Hexa-/-, Neu3-/- and Hexa-/-Neu3-/- mice. Expression ratios were calculated by the ΔCT method. GraphPad used a one-way ANOVA analysis to determine p-values. The data are reported as mean ± SEM. (n=3; *p<0.05, **p<0.025, ***p<0.01 and ****p<0.001). FigureS2.tiff Fig. S2 Histographic (A) and graphical (B) representation of Flow Cytometry analysis for intracellular ROS level measurement of control neuroglia (NG-124) and Tay-Sachs patient neuroglia (NG-125) by using the fluorometric dye H 2 DCFDA and H 2 O 2 . One-way ANOVA by GraphPad was used to determine p-values . The data are represented as the mean ± S.E.M. (n=3; ****p<0.0001). The data on the histogram were gathered from homogenous cell populations of human neuroglia cells. The population was gated to exclude the debris. The red bar on the histogram indicates the cells with the elevated fluorescent signal in the green channel, specifically DCF-positive cells. Cite Share Download PDF Status: Published Journal Publication published 05 Mar, 2025 Read the published version in Molecular Biology Reports → Version 1 posted Editorial decision: Revision requested 18 Nov, 2024 Reviews received at journal 16 Nov, 2024 Reviews received at journal 09 Nov, 2024 Reviews received at journal 02 Nov, 2024 Reviewers agreed at journal 30 Oct, 2024 Reviewers agreed at journal 29 Oct, 2024 Reviewers agreed at journal 29 Oct, 2024 Reviewers agreed at journal 28 Oct, 2024 Reviewers invited by journal 28 Oct, 2024 Editor assigned by journal 22 Oct, 2024 Submission checks completed at journal 19 Oct, 2024 First submitted to journal 19 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5293300","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":371923408,"identity":"9d51a9ab-4157-4ddf-aff7-795e13aa243e","order_by":0,"name":"Hande Basırlı","email":"","orcid":"","institution":"Izmir Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Hande","middleName":"","lastName":"Basırlı","suffix":""},{"id":371923409,"identity":"22ca69fd-fd62-4490-964d-5052e53f8d19","order_by":1,"name":"Nurselin Ateş","email":"","orcid":"","institution":"Izmir Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Nurselin","middleName":"","lastName":"Ateş","suffix":""},{"id":371923410,"identity":"65d80cf9-73be-4a62-8d06-193ad2d933a5","order_by":2,"name":"Volkan Seyrantepe","email":"data:image/png;base64,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","orcid":"","institution":"Izmir Institute of Technology","correspondingAuthor":true,"prefix":"","firstName":"Volkan","middleName":"","lastName":"Seyrantepe","suffix":""}],"badges":[],"createdAt":"2024-10-19 08:08:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5293300/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5293300/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11033-025-10380-y","type":"published","date":"2025-03-05T15:57:24+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":67847440,"identity":"74fdb25b-77c9-4d0a-adbc-6ba7a81265c9","added_by":"auto","created_at":"2024-10-30 10:02:41","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":778648,"visible":true,"origin":"","legend":"\u003cp\u003eImmunoblot images of the anti-Bcl2 in the cerebellum (A), cortex (C), thalamus (E), hippocampus (G), and non-treated (-) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated (+) fibroblasts (I) of 5-month-old \u003cem\u003eWT, Hexa-/-, Neu3-/- \u003c/em\u003eand\u003cem\u003e Hexa-/-Neu3-/-\u003c/em\u003e mice, and non-treated (-) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated (+) control neuroglia (NG-124) and Tay-Sachs patient neuroglia (NG-125) (K). Densitometric analysis of Bcl2 in the cerebellum (B), cortex (D), thalamus (F), hippocampus (H), fibroblast (J), and neuroglia cells (L). β-actin was an internal control. ImageJ determined band intensities and \u003cem\u003ep-values\u003c/em\u003e using a one-way ANOVA analysis by GraphPad. The data are reported as mean ± SEM. (n=3; *p\u0026lt;0.05, **p\u0026lt;0.025, ***p\u0026lt;0.01 and ****p\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Figure1.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5293300/v1/a182677942cbb4e09d988a73.jpg"},{"id":67848907,"identity":"fa749b15-c014-4151-af7b-8d772881f4ee","added_by":"auto","created_at":"2024-10-30 10:10:41","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":787777,"visible":true,"origin":"","legend":"\u003cp\u003eImmunoblot images of the anti-BclXL in the cerebellum (A), cortex (C), thalamus (E), hippocampus (G), and non-treated (-) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated (+) fibroblasts (I) of 5-month-old \u003cem\u003eWT, Hexa-/-, Neu3-/- \u003c/em\u003eand\u003cem\u003e Hexa-/-Neu3-/-\u003c/em\u003e mice, and non-treated (-) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated (+) control neuroglia (NG-124) and Tay-Sachs patient neuroglia (NG-125) (K). Densitometric analysis of BclXL in the cerebellum (B), cortex (D), thalamus (F), hippocampus (H), fibroblast (J), and neuroglia cells (L). β-actin was an internal control. ImageJ determined band intensities, and p-values were determined by GraphPad using a one-way ANOVA analysis. The data are reported as mean ± SEM. (n=3; *p\u0026lt;0.05 and **p\u0026lt;0.025).\u003c/p\u003e","description":"","filename":"Figure2.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5293300/v1/811ade47df607ce77e448545.jpg"},{"id":67847442,"identity":"298d466e-f574-432c-bc72-093db2dc273b","added_by":"auto","created_at":"2024-10-30 10:02:41","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":764506,"visible":true,"origin":"","legend":"\u003cp\u003eImmunoblot images of the anti-Bax in the cerebellum (A), cortex (C), thalamus (E), hippocampus (G), and non-treated (-) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated (+) fibroblasts (I) of 5-month-old \u003cem\u003eWT, Hexa-/-, Neu3-/- \u003c/em\u003eand\u003cem\u003e Hexa-/-Neu3-/-\u003c/em\u003e mice, and non-treated (-) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated (+) control neuroglia (NG-124) and Tay-Sachs patient neuroglia (NG-125) (K). Densitometric analysis of Bax in the cerebellum (B), cortex (D), thalamus (F), hippocampus (H), fibroblast (J), and neuroglia cells (L). β-actin was an internal control. ImageJ determined band intensities, and p-values were determined by GraphPad using a one-way ANOVA analysis. The data are reported as mean ± SEM. (n=3; *p\u0026lt;0.05, **p\u0026lt;0.025, ***p\u0026lt;0.01 and ****p\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Figure3.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5293300/v1/5e9e214da067151bf39067f6.jpg"},{"id":67848910,"identity":"6b93f84c-5442-4d88-86b4-ce5a48a135d1","added_by":"auto","created_at":"2024-10-30 10:10:42","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1030818,"visible":true,"origin":"","legend":"\u003cp\u003eTerminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling\u003cstrong\u003e (\u003c/strong\u003eTUNEL) assay images (A) of the cerebellum, pons, cortex, thalamus, and hippocampus of 5-month-old \u003cem\u003eHexa-/-\u003c/em\u003eand\u003cem\u003e Hexa-/-Neu3-/-\u003c/em\u003e mice. Graphical representation of the colocalization intensities of TUNEL-positive neurons (yellow) for the cerebellum (B), pons (C), cortex (D), thalamus (E), and hippocampus (F) of 5-month-old \u003cem\u003eHexa-/- \u003c/em\u003eand\u003cem\u003e Hexa-/-Neu3-/-\u003c/em\u003e mice. Nuclei are counterstained in red. Images for each genotype were taken under the same light intensity, differing only for filter type at 40x magnification. The data are represented as the mean ± S.E.M. The data are represented as the mean ± S.E.M. x (n=3, *p\u0026lt;0.05, **p\u0026lt;0.025, and ****p\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Figure4.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5293300/v1/bbb91fbe0f418e07c55546c1.jpg"},{"id":67847443,"identity":"96108f83-aaca-47c8-be9b-59d6857a2cf0","added_by":"auto","created_at":"2024-10-30 10:02:41","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1095986,"visible":true,"origin":"","legend":"\u003cp\u003eSOD2, Catalase, and Ttase1 gene expression levels of the cerebellum (A, B, C), cortex (D, E, F), thalamus (G, H, I), hippocampus (J, K, L), and non-treated (-) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated (+)\u003cstrong\u003e \u003c/strong\u003efibroblasts (M, N, O) of 5-month-old \u003cem\u003eWT\u003c/em\u003e, \u003cem\u003eHexa-/-\u003c/em\u003e, \u003cem\u003eNeu3-/-\u003c/em\u003e and \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice, and non-treated (-) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated (+) control neuroglia (NG-124) and Tay-Sachs patient neuroglia (NG-125) (P, R, S). Expression ratios were calculated by the ΔCT method. GraphPad used a one-way ANOVA analysis to determine p-values. The data are reported as mean ± SEM. (n=3; *p\u0026lt;0.05, **p\u0026lt;0.025, ***p\u0026lt;0.01 and ****p\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Figure5.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5293300/v1/7aeb5f439edbee2b14f42658.jpg"},{"id":67847444,"identity":"f7338e25-1214-454d-8b79-c8fa5144d3b8","added_by":"auto","created_at":"2024-10-30 10:02:41","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":810010,"visible":true,"origin":"","legend":"\u003cp\u003eImmunoblot images and densitometric analysis of the carbonylated proteins in the cerebellum (A), cortex (B), thalamus (C), hippocampus (D), and fibroblasts (E) of 5-month-old \u003cem\u003eWT\u003c/em\u003e, \u003cem\u003eHexa-/-\u003c/em\u003e,\u003cem\u003e Neu3-/-\u003c/em\u003e and \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice, and\u003cstrong\u003e \u003c/strong\u003enon-treated (-) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated \u003cstrong\u003e(\u003c/strong\u003e+\u003cstrong\u003e) \u003c/strong\u003econtrol neuroglia (NG-124) and Tay-Sachs patient neuroglia (NG-125) (F). β-actin\u003cstrong\u003e \u003c/strong\u003ewas an internal control. ImageJ determined band intensities, and p-values were determined by GraphPad using a one-way ANOVA analysis. The data are reported as mean ± SEM. (n=3; *p\u0026lt;0.05 and **p\u0026lt;0.025).\u003c/p\u003e","description":"","filename":"Figure6.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5293300/v1/2535b6b4b3534eb3eaab3204.jpg"},{"id":67848908,"identity":"94c25a84-ac18-49af-acce-e360402d8e7a","added_by":"auto","created_at":"2024-10-30 10:10:42","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1486356,"visible":true,"origin":"","legend":"\u003cp\u003eImmunoblot images and densitometric analyses of the Anti\u003cstrong\u003e-\u003c/strong\u003eAPE1 in the cerebellum (A), cortex (B), thalamus (C), hippocampus (D), and\u003cstrong\u003e \u003c/strong\u003enon-treated (-) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated (+) fibroblasts (E) of 5-month-old \u003cem\u003eWT\u003c/em\u003e, \u003cem\u003eHexa-/-\u003c/em\u003e, \u003cem\u003eNeu3-/-\u003c/em\u003e and \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice. β-actin\u003cstrong\u003e \u003c/strong\u003ewas an internal control. Immunocytochemical analyses (F) of anti-APE1 in non-treated (-) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated (+) fibroblasts of 5-month-old \u003cem\u003eWT, Hexa-/-, Neu3-/- \u003c/em\u003eand\u003cem\u003e Hexa-/-Neu3-/-\u003c/em\u003e mice. Graphical representation of total (G), nuclear (H), and cytoplasmic (I) APE1 intensity level per cell. Images for each genotype were taken under the same light intensity, differing only for filter type at 20x magnification. One-way and Two-way ANOVA were used for statistical analysis. The data are represented as the mean ± S.E.M. (n=3, *p\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"Figure7.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5293300/v1/ce332e7f1dbbb8f5c1818439.jpg"},{"id":67849669,"identity":"8fd5f85c-9331-4b79-967e-8f91ba2aa2c1","added_by":"auto","created_at":"2024-10-30 10:18:42","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":737827,"visible":true,"origin":"","legend":"\u003cp\u003eHistographic (A) and graphical (B) representation of Flow Cytometry analysis for intracellular ROS level measurement of fibroblasts derived from \u003cem\u003eWT, Hexa-/-, Neu3-/- \u003c/em\u003eand\u003cem\u003e Hexa-/-Neu3-/-\u003c/em\u003e mice by using the fluorometric dye H\u003csub\u003e2\u003c/sub\u003eDCFDA. One-way ANOVA by GraphPad was used to determine \u003cem\u003ep-values\u003c/em\u003e. The data are represented as the mean ± S.E.M. (n=3; ***p\u0026lt;0.01 and ****p\u0026lt;0.001). The data on the histogram were gathered from homogenous cell populations isolated from the mice. The population was gated to exclude the debris. The red bar on the histogram indicates the cells with the elevated fluorescent signal in the green channel, specifically DCF-positive cells.\u003c/p\u003e","description":"","filename":"Figure8.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5293300/v1/5d182f27f372ac64891a2d6b.jpg"},{"id":78183905,"identity":"83b91e44-3841-4f3c-b225-97e8ad85b4cb","added_by":"auto","created_at":"2025-03-10 18:19:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8481657,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5293300/v1/9fa1d0b6-f0a9-416d-ac3f-6fc8b55d607c.pdf"},{"id":67847449,"identity":"2f58c949-0358-46f8-a028-9a3b6e5b9066","added_by":"auto","created_at":"2024-10-30 10:02:42","extension":"tiff","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":8700262,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S1 Bcl2, BclXL, and Bax gene expression levels of the cerebellum (A, B, C), cortex (D, E, F), thalamus (G, H, I), hippocampus (J, K, L), non-treated (-) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated (+) fibroblasts (M, N, O) of 5-month-old \u003cem\u003eWT, Hexa-/-, Neu3-/- \u003c/em\u003eand\u003cem\u003e Hexa-/-Neu3-/-\u003c/em\u003e mice. Expression ratios were calculated by the ΔCT method. GraphPad used a one-way ANOVA analysis to determine p-values. The data are reported as mean ± SEM. (n=3; *p\u0026lt;0.05, **p\u0026lt;0.025, ***p\u0026lt;0.01 and ****p\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"FigureS1.tiff","url":"https://assets-eu.researchsquare.com/files/rs-5293300/v1/76f2094a55dd3284c7ee165e.tiff"},{"id":67847445,"identity":"f9e7a311-68b1-4450-95e2-270f922dcd2b","added_by":"auto","created_at":"2024-10-30 10:02:42","extension":"tiff","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":26089576,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S2\u003c/strong\u003e Histographic (A) and graphical (B) representation of Flow Cytometry analysis for intracellular ROS level measurement of control neuroglia (NG-124) and Tay-Sachs patient neuroglia (NG-125) by using the fluorometric dye H\u003csub\u003e2\u003c/sub\u003eDCFDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. One-way ANOVA by GraphPad was used to determine \u003cem\u003ep-values\u003c/em\u003e. The data are represented as the mean ± S.E.M. \u0026nbsp;(n=3; ****p\u0026lt;0.0001). The data on the histogram were gathered from homogenous cell populations of human neuroglia cells. The population was gated to exclude the debris. The red bar on the histogram indicates the cells with the elevated fluorescent signal in the green channel, specifically DCF-positive cells.\u003c/p\u003e","description":"","filename":"FigureS2.tiff","url":"https://assets-eu.researchsquare.com/files/rs-5293300/v1/e6c6283dc965fe8822ca3f64.tiff"}],"financialInterests":"No competing interests reported.","formattedTitle":"Imbalance in Redox Homeostasis is Associated with Neurodegeneration in the Murine Model of Tay-Sachs Disease","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTay-Sachs disease (TSD) is categorized as a rare neurodegenerative disorder with the dysfunctionality of β-hexosaminidase A (HexA). Progressively accumulating GM2 ganglioside in the brain of TS patients results in a deteriorated central nervous system (CNS) and disrupted mental and motor functions. TS patients eventually die at 2\u0026ndash;4 years of age. Currently, no effective treatment has been provided for TS patients. Previously, the \u003cem\u003eHexa-/-\u003c/em\u003e mice were presented as a model of TSD. On the other hand, it did not show any neuropathological symptoms noticed in patients. In \u003cem\u003eHexa-/-\u003c/em\u003e mice different from humans, degradation of GA2 to GM2 facilitated by neuraminidase was proposed [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The role of neuraminidase 3 (Neu3) in the degradation of glycolipids and TSD in mice was discovered, and \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice were introduced as the murine model of TSD. At birth, \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice were healthy, but death occurs at five months of age because of progressive loss of neurons and Purkinje cells and increased active astrocytes. Additionally, \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice prominently showed notable neurological deteriorations, including movement problems, ataxia, and tremor [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePredominant pathology in lysosomal storage disorders (LSDs) is larger sizes and numbers of lysosomes due to the progressive accumulation of substrates, which may cause an imbalance in redox homeostasis and neurodegeneration [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. A bunch of studies reported increased reactive oxygen species (ROS) and altered redox homeostasis, which is one of the mediators of neurodegeneration in many LSDs [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The basal level of endogenous ROS is essential for redox homeostasis. However, the excess amount of ROS is removed by antioxidant defense systems comprising non-enzymatic antioxidants or oxidoreductases like superoxide dismutase, catalase, and Ttase1. When the net endogenous ROS levels are more significant than the capacity of antioxidant defense mechanisms to remove the excessive ROS, an imbalance in cellular redox homeostasis is generated in the cell, provoking the oxidation of biological macromolecules [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Notably, proteins are oxidized on their side chains, and thus, they become carbonylated. The detection of carbonylated protein content is generally performed to measure the levels of protein oxidations in cells of several neurodegenerative diseases [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Besides protein damage, ROS also alters DNA structure and causes oxidative DNA damage. It has been shown that the APE1 protein has a crucial activity in the DNA Base Excision Repair (BER) pathway against oxidative DNA damage. Several studies have reported that APE1 has a protective effect through the DNA BER pathway against imbalance in redox homeostasis, mediating neurodegeneration, oxidative signaling, and cellular injury [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Thus, the APE1 protein has a significant role in preventing ROS-induced alteration of redox homeostasis. In addition, it has been reported that damages to macromolecules created by ROS contribute to inflammation, tissue damage, and the activation of neurodegeneration [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Among the various neurodegenerative diseases, damaging influences of oxidative stress and imbalanced redox homeostasis have also been reported to contribute to the pathophysiology of LSDs [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The link between lysosomal accumulation and imbalanced redox homeostasis was reported in mouse models of Sandhoff disease (\u003cem\u003eHexb-/-\u003c/em\u003e), late-onset Tay-Sachs disease (\u003cem\u003eHexa-/-\u003c/em\u003e) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], GM1 Gangliosidosis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and Mucopolysaccharidosis type I [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In addition, \u003cem\u003ein vitro\u003c/em\u003e studies on neural progenitor cells derived from Tay-Sachs patients [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], fibroblasts from NPC patients [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and differentiated human oligodendrocytes from Krabbe disease [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] revealed that alteration in redox homeostasis has a role in neurodegeneration.\u003c/p\u003e \u003cp\u003eSo far, no experimental evidence shows a link between early-onset TSD pathology and an imbalance in redox homeostasis. Therefore, in this study, we aimed to explore whether there is an alteration in redox homeostasis, which is related to neurodegeneration and apoptosis in the tissues from the murine model of TSD and cells from Tay-Sachs patients.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003e\u003cem\u003eHexa-/-\u003c/em\u003e, \u003cem\u003eNeu3-/-\u003c/em\u003e, and \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice were obtained as described in the previous studies [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The mice breeding and maintenance were generated in the Turkish Council on Animal Care (TCAC) accredited animal facility of Izmir Institute of Technology according to the TCAC guidelines. Mice were kept in an environment with constant humidity and temperature on a 12-hour light-dark cycle. The Animal Care and Use Committee of Izmir Institute of Technology, Izmir, Turkey, granted animal care and its use in the experiments. The mice breeding and genotyping were generated as described in the previous studies [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. After the scarification of mice at five months old, brain samples were dissected into the cortex, cerebellum, thalamus, and hippocampus and snap-freezing in liquid nitrogen. The tissues were stored at -80\u003csup\u003eo\u003c/sup\u003eC until used.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell Culture\u003c/h3\u003e\n\u003cp\u003eFibroblast lines from 5-month-old \u003cem\u003eWT\u003c/em\u003e, \u003cem\u003eHexa-/-\u003c/em\u003e, \u003cem\u003eNeu3-/-\u003c/em\u003e, and \u003cem\u003eHexa-/- Neu3-/-\u003c/em\u003e were established and performed in DMEM (Gibco\u0026trade;, USA) with 10% FBS (Gibco\u0026trade;, USA) and 1% (v/v) penicillin/streptomycin (Gibco\u0026trade;, USA). Primary fibroblasts were immortalized using serum-free DMEM containing viral supernatant (LXSN 16E6E7) and polybrene (400\u0026micro;g/ml) overnight at 37\u003csup\u003eo\u003c/sup\u003eC. After removal of DMEM supplemented with viral supernatant and polybrene, the cells were cultivated in DMEM with 10% FBS and G418 (400\u0026micro;g/ml) to select immortalized cells. Immortal fibroblasts were treated for one hour with 100 \u0026micro;M hydrogen peroxide (Sigma, Germany).\u003c/p\u003e \u003cp\u003eAfter transformation of the immortalized control neuroglia cells (NG-124) and TSD neuroglia cells (NG-125) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] with the pCMV-HexA plasmid that encodes the α subunit of HexA and expresses the active HexA enzyme [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], cells were culturized in DMEM, including 10% (vol/vol) FBS and 1% (vol/vol) penicillin/streptomycin.\u003c/p\u003e\n\u003ch3\u003eReal-Time PCR\u003c/h3\u003e\n\u003cp\u003eTotal RNA extraction from brain tissues and fibroblasts of all genotypes at five months of age, control neuroglia cells (NG-124), and TSD neuroglia cells (NG-125) were done using Trizol Reagent (GeneAid, Taiwan). Following the extraction, cDNA conversion was generated using a reverse transcription kit (Applied Biosystems, USA). Expression analyses of oxidative stress markers (SOD2, Catalase, and Ttase1) were done using LightCycler 480 SYBR Green I Master Mix by applying the manufacturer\u0026rsquo;s protocol. The endogenous control was GAPDH. The primers listed were used for expression analyses; SOD2F: 5\u0026rsquo;- GTGTCTGTGGGAGTCCAAG G-3\u0026rsquo;, SOD2R: 5\u0026rsquo;-CCCCAGTCATAGTGCTGCAA-3\u0026rsquo;, CatalaseF: 5\u0026rsquo;-TTCGTCCCGAGTCT CTCCAT-3\u0026rsquo;, CatalaseR: 5\u0026rsquo;-GAGGCCAAACCTTGGTCAGA-3\u0026rsquo;, Ttase1F: 5\u0026rsquo;-CTGCAAGAT CCAGTCTGGGAA-3\u0026rsquo;, Ttase1R: 5\u0026rsquo;-CTCTGCCTGCCACCCCTTTTAT-3\u0026rsquo;, GAPDHF: 5\u0026rsquo;-C CCCTTCATTGACCTCAACTAC-3\u0026rsquo;, GAPDHR: 5\u0026rsquo;-ATGCATTGCTGACAATCTTGAG-3\u0026rsquo;.\u003c/p\u003e\n\u003ch3\u003eProtein Carbonylation Analysis\u003c/h3\u003e\n\u003cp\u003eProtein isolation was done from brain tissues and fibroblasts of all genotypes at five months of age, control neuroglia cells (NG-124) and TSD neuroglia cells (NG-125) using cold lysis buffer (50 Mm Tris- HCI, 150 mM NaCl, 1% TritonX-100, 50 mM HEPES, 10% glycerol, protease inhibitors) including 1% β-mercaptoethanol. Later, reagents supplemented by Protein Oxidation Detection Kit (S7150 OxyBlot\u0026trade; Merck-Millipore, Canada) were used by applying the manufacturer\u0026rsquo;s protocol. Immunodetection of carbonyl proteins was done by a DNP-specific primary antibody (1:150) for the carbonyl content, and an anti-\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\beta\\:\\)\u003c/span\u003e\u003c/span\u003e-Actin (1:1000, Cell Signaling, USA) antibody was used for normalization. HRP-conjugated antibody (Jackson ImmunoResearch Lab, USA) was the secondary antibody. The carbonylated proteins were visualized by chemiluminescent reagent on a digital imaging system (Fusion SL, Vilber). Band densities were measured using NIH ImageJ [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\beta\\:\\)\u003c/span\u003e\u003c/span\u003e-Actin was used as the endogenous control for band intensity normalization.\u003c/p\u003e\n\u003ch3\u003eTerminal dUTP nick end-labeling (TUNEL) analysis\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eHexa-/-\u003c/em\u003e and \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice at five months of age were anesthetized for trans-cardiac perfusion of the heart with 4% paraformaldehyde (PFA, Sigma). After perfusion, the brains were removed and incubated with 4% PFA (Sigma) overnight at 4\u0026deg;C. Then, brains were placed sequentially into 10%, 20% in PBS at 4\u0026deg;C, and 30% sucrose in PBS overnight at 4\u0026deg;C. Brain embedding was performed using an optimal cutting temperature (OCT) compound (Sigma). The embedded brains were sectioned in a coronal plane using a Leica cryostat at ten \u0026micro;m thickness on HistoBond\u0026reg; microscope slides (Marienfeld) at -20\u0026deg;C.\u003c/p\u003e \u003cp\u003eFollowing the manufacturer's protocol, the ApopTag Fluorescein \u003cem\u003ein situ\u003c/em\u003e Apoptosis Detection Kit (Millipore) was used to perform Terminal dUTP Nick End-Labeling (TUNEL) analysis. Concisely, the brain sections of \u003cem\u003eHexa-/-\u003c/em\u003e and \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice were fixed in 1% PFA and precooled in ethanol acetic acid (2:1) solutions at five months. The terminal deoxynucleotidyl transferase and anti-digoxygenin conjugate were applied to the brain sections. The sections were incubated with the nuclear counterstain, propidium iodide (0.5 \u0026micro;g/mL). The fluorescent images were taken using a fluorescent microscope (Olympus-BX53F). The cortex, thalamus, hippocampus, cerebellum, and pons regions were analyzed for TUNEL-positive green fluorescent cells as the indicator of neuronal death, and the colocalization intensity of the images was measured using NIH ImageJ [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunocytochemical Analysis\u003c/h2\u003e \u003cp\u003eThe fibroblasts were cultivated on micro slides for 24 hours and fixed with 4% PFA (Sigma). Following the fixation, a blocking buffer (10% goat serum, 4% BSA 0.3M Glycine, and 0.3% TritonX in PBS) was used for blocking the fibroblasts for 1 hour after which slides were stained with primary antibody, anti-APE1/Ref-1 (1:200; Abcam, USA), overnight at 4\u003csup\u003e0\u003c/sup\u003eC and anti-Alexa Fluor\u0026reg;-488 (1:500, Abcam, UK) as the secondary antibody. The slides were covered using DAPI (Abcam, UK). Fluorescent images were taken by Fluorescent Microscopy (Olympus-BX53F), and the intensity of the images was measured using NIH ImageJ [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFlow Cytometry Analysis\u003c/h3\u003e\n\u003cp\u003eUnder normal conditions, a nonpolar substance, 2\u0026rsquo;7\u0026rsquo;-dichlorodihydrofluorescein diacetate (H\u003csub\u003e2\u003c/sub\u003eDCFDA), is converted to a non-fluorescent polar (H\u003csub\u003e2\u003c/sub\u003eDCF) by cellular esterases. Suppose ROS is present in cells; oxidation of H\u003csub\u003e2\u003c/sub\u003eDCF results in highly fluorescent 2\u0026rsquo;7\u0026rsquo; dichlorofluorescein (DCF). To measure intracellular ROS level, fluorescent DCF by Flow cytometry was analyzed after treating the cells with H\u003csub\u003e2\u003c/sub\u003eDCFDA. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e- and H\u003csub\u003e2\u003c/sub\u003eDCFDA-treated groups as the positive control, the non-treated group as the negative control, and the H\u003csub\u003e2\u003c/sub\u003eDCFDA-treated group as samples were studied. The positive controls were incubated for 1 hour at 37\u003csup\u003eo\u003c/sup\u003eC with 10 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e containing serum-free media. After the incubation, positive control and sample group cells were incubated for 30 min with 5\u0026micro;M H\u003csub\u003e2\u003c/sub\u003eDCFDA containing serum-free media and then harvested with trypsin. The cells were resuspended in 600 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:\\)\u003c/span\u003e\u003c/span\u003el PBS; triplicate samples of each sample were used for Flow Cytometry analysis on the guava easyCyte\u0026trade;, and the results were analyzed using guavaSoft\u0026trade; software.\u003c/p\u003e\n\u003ch3\u003eWestern Blot Analysis\u003c/h3\u003e\n\u003cp\u003eTotal protein isolation from brain tissue samples and fibroblasts was done by treating the samples with cold lysis buffer (50 Mm Tris- HCI, 150 mM NaCl, 1% TritonX-100, 50 mM HEPES, 10% glycerol, protease inhibitors) for 1 hour. Following the protein loading to SDS-PAGE, it is transferred to a nitrocellulose membrane (Bio-Rad, USA). The membranes were stained with anti-Bcl2 (1:500, Santa Cruz Biotechnology, USA), anti-BclXL (1:1000, Cell Signaling, USA), anti-Bax (1:1000, Abcam, UK), anti-APE1 (1:1000, Abcam, UK), and anti-\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\beta\\:\\)\u003c/span\u003e\u003c/span\u003e-Actin (1:1000, Cell Signaling, USA) overnight at +\u0026thinsp;4\u003csup\u003eo\u003c/sup\u003eC. Later, membranes were treated with HRP-conjugated secondary antibodies (Jackson ImmunoResearch Lab, USA). Protein visualization was performed using LuminataTM Forte WesternHRP Substrate (Millipore, USA) on a digital imaging system (Fusion SL, Vilber). Band densities were measured using NIH ImageJ (1.48v) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\beta\\:\\)\u003c/span\u003e\u003c/span\u003e-Actin was used as the endogenous control for the normalization of band intensities.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eGraphPad Prism 7 (v. 7.0a, GraphPad Software, Inc) performed all statistical analyses. For all experiments, \u003cem\u003eWT\u003c/em\u003e, \u003cem\u003eHexa-/-\u003c/em\u003e, \u003cem\u003eNeu3-/-\u003c/em\u003e, and \u003cem\u003eHexa-/- Neu3-/-\u003c/em\u003e groups (n\u0026thinsp;=\u0026thinsp;3) were compared by One-Way ANOVA. The data are shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eAltered levels of the anti-apoptotic markers in different brain regions but not in fibroblast of\u003c/b\u003e \u003cb\u003eHexa-/-Neu3-/-\u003c/b\u003e \u003cb\u003emice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAlterations in the protein levels of anti-apoptotic Bcl2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), Bcl-XL (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), and pro-apoptotic Bax (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) in the different regions of the brain were investigated using immunoblot analyses. The protein expression levels of Bcl2 were significantly reduced in the cerebellum of \u003cem\u003eNeu3-/-\u003c/em\u003e and \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice compared to \u003cem\u003eWT\u003c/em\u003e and \u003cem\u003eHexa-/-\u003c/em\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). Similarly, we identified a remarkably lower level of Bcl2 protein in the thalamus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, F) and hippocampus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, H) of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice compared to age-matched single knockouts. In contrast, we determined higher levels of Bcl2 protein in the cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D) of \u003cem\u003eNeu3-/-\u003c/em\u003e and \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice. Although our data clearly shows alterations in Blc2 protein levels in different brain regions in \u003cem\u003eWT\u003c/em\u003e, single, and double knockouts, higher levels of Bcl2 only in the cortex of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e might be related to the severe phenotype observed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn another study, we also used the fibroblasts of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice and showed there is a significant increase in the levels of Bcl2 protein compared to the fibroblasts of \u003cem\u003eNeu3-/-\u003c/em\u003e mice under non-treated conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI, J). Although there was a remarkable decrease in levels of Bcl2 protein under the same conditions only in \u003cem\u003eHexa-/-\u003c/em\u003e mice\u0026rsquo;s fibroblasts compared to their non-treated counterparts, we found no difference in the levels of Bcl2 protein under H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated conditions of the fibroblasts from \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice compared to the non-treated counterparts. In parallel to \u003cem\u003ein vitro\u003c/em\u003e studies with TSD mice fibroblast mice, we also evaluated the levels of Bcl2 protein in the neuroglia of Tay-Sachs patients under both non- and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK, L). We demonstrated that the levels of Bcl2 protein were not affected in non-treated NG-125 cells compared to non-treated NG-124 and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated NG-125 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK, L). Interestingly, our data showed a significant reduction in the levels of anti-apoptotic Bcl2 protein in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated NG-124 cells compared to their non-treated counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL).\u003c/p\u003e \u003cp\u003eMoreover, the protein levels of anti-apoptotic markers, BclXL, did not show any significant changes in the cerebellum (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B) and thalamus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F) regions of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice. Remarkably, in the cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D) and hippocampus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, H) of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice, we identified lower BclXL protein levels than \u003cem\u003eWT\u003c/em\u003e and \u003cem\u003eHexa-/-\u003c/em\u003e mice. \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e fibroblasts consistently displayed a significant reduction in the levels of BclXL protein under non-treated conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, J). Surprisingly, our results showed that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment did not alter the levels of BclXL protein in the fibroblasts from \u003cem\u003eWT\u003c/em\u003e, \u003cem\u003eHexa-/-\u003c/em\u003e, \u003cem\u003eNeu3-/-\u003c/em\u003e, and \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice compared to their non-treated counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, J). Similarly, we analyzed the levels of BclXL protein in the neuroglia cells of Tay-Sachs patients under H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK, L). Our results indicated that there were also no significant changes in the levels of BclXL protein in non-treated NG-125 cells compared to NG-124 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK, L). Besides, the levels of BclXL protein also did not show any difference in NG-125 cells under H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated oxidative stress conditions compared to non-treated NG-125 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL).\u003c/p\u003e \u003cp\u003eThe significantly elevated protein levels of pro-apoptotic Bax were only shown in the cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D) of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice compared to \u003cem\u003eWT\u003c/em\u003e mice but not in other brain regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Besides, we evaluated levels of Bax protein in the fibroblasts of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice both in treated and untreated conditions, and we found there was a remarkable elevation of antiapoptotic Bax protein in the fibroblasts of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice compared to the \u003cem\u003eWT\u003c/em\u003e and \u003cem\u003eNeu3-/-\u003c/em\u003e under non-treated conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI, J). Under H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated conditions, Bax protein levels significantly decreased in the fibroblasts of \u003cem\u003eHexa-/-\u003c/em\u003e and \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice compared to their non-treated counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI, J). On the other hand, we identified significantly higher protein levels of Bax in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated fibroblasts of \u003cem\u003eWT\u003c/em\u003e and \u003cem\u003eNeu3-/-\u003c/em\u003e mice compared to non-treated \u003cem\u003eWT\u003c/em\u003e and \u003cem\u003eNeu3-/-\u003c/em\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI, J). In addition to the Western Blot analysis of Bax protein in fibroblasts of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice, we also evaluated levels of Bax protein in the neuroglia cells of Tay-Sachs patients in the context of our study (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK, L). Our results revealed a slight increase in the protein levels of Bax in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated NG-125 cells compared to non-treated NG-125 cells, while there were no alterations in NG-125 cells compared to NG-124 cells under non-treated conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL).\u003c/p\u003e \u003cp\u003eWe further evaluated mRNA levels of anti-apoptotic Bcl2 (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, D, G, J, M) and Bcl-XL (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB, E, H, K, N), and pro-apoptotic Bax gene (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC, F, I, L, O) in the cerebellum, cortex, thalamus, and hippocampus of 5-month-old \u003cem\u003eWT\u003c/em\u003e, \u003cem\u003eHexa-/-, Neu3-/-\u003c/em\u003e, and \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice using qRT-PCR analyses. We showed that the mRNA levels of Bcl2 were significantly reduced in the cerebellum of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice compared to \u003cem\u003eWT\u003c/em\u003e and \u003cem\u003eNeu3-/-\u003c/em\u003e mice (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). In the cortex region, Bcl2 mRNA was significantly down-regulated in \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice compared to \u003cem\u003eHexa-/-\u003c/em\u003e counterparts (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD). Additionally, a significant decrease was detected in the hippocampal region of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice compared to \u003cem\u003eWT\u003c/em\u003e and \u003cem\u003eNeu3-/-\u003c/em\u003e mice (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eJ). Surprisingly, we observed no significant changes in the mRNA expression of Bcl2, Bcl-XL, and Bax genes in other brain regions of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice compared to age-matched \u003cem\u003eWT\u003c/em\u003e, \u003cem\u003eHexa-/-\u003c/em\u003e, and \u003cem\u003eNeu3-/-\u003c/em\u003e mice (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). We also analyzed levels of the same genes in the fibroblasts derived from \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice in normal and oxidative stress conditions; however, we did not find any significant differences with other mice groups (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eM-O). Interestingly, only \u003cem\u003eHexa-/-\u003c/em\u003e mice fibroblasts showed slightly higher Bcl2 mRNA expression under H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress conditions (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eM).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIncreased levels of cell death in the brain of\u003c/b\u003e \u003cb\u003eHexa-/-Neu3-/-\u003c/b\u003e \u003cb\u003emice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe performed Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay for visualization of neuronal death in situ [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and for showing TUNEL-positive cells in the cerebellum, pons, cortex, thalamus, and hippocampus sections of 5-month-old \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice compared to age-matched \u003cem\u003eHexa-/-\u003c/em\u003e mice. TUNEL assay indicated significantly elevated levels of TUNEL-positive neurons and neuronal death in all brain regions of 5-month-old \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice compared to the age-matched \u003cem\u003eHexa-/-\u003c/em\u003e mice group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eElevated transcriptional levels of the oxidative stress-related markers in the fibroblast but not in the cerebellum of\u003c/b\u003e \u003cb\u003eHexa-/-Neu3-/-\u003c/b\u003e \u003cb\u003emice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn this study, we also analyzed the mRNA expression levels of oxidative stress markers SOD2, Catalase, and Ttase1 in five brain regions of 5-month-old mice and age-matched counterparts using qRT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Our data showed that the expression levels of SOD2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) and Ttase1 mRNAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) in the cerebellum of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice exhibited a significant reduction compared to \u003cem\u003eWT, Hexa-/-\u003c/em\u003e, and \u003cem\u003eNeu3-/-\u003c/em\u003e mice. Similarly, the mRNA expression levels of Catalase in the cerebellum of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice significantly decreased compared to \u003cem\u003eWT\u003c/em\u003e and \u003cem\u003eHexa-/-\u003c/em\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Interestingly, there were no apparent differences in the mRNA expression levels of SOD2, Catalase, and Ttase1 in other brain regions of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice compared to \u003cem\u003eWT, Hexa-/-\u003c/em\u003e, and \u003cem\u003eNeu3-/-\u003c/em\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Furthermore, the mRNA expression levels of SOD2, Catalase, and Ttase1 were analyzed in fibroblasts derived from 5-month-old \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice and \u003cem\u003eWT\u003c/em\u003e as well as single knockouts (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eM, O). In this experiment, one group of fibroblasts was treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to induce oxidative stress \u003cem\u003ein vitro\u003c/em\u003e. Our results revealed that levels of SOD2 mRNA in the fibroblasts of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice were significantly upregulated compared to the fibroblasts of \u003cem\u003eNeu3-/-\u003c/em\u003e mice under normal and oxidative stress conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eM). Unexpectedly, there were no significant changes in the mRNA expression levels of SOD2 in fibroblasts of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice compared with \u003cem\u003eWT\u003c/em\u003e and \u003cem\u003eHexa-/-\u003c/em\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eM). Besides, under H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated conditions, the mRNA levels of SOD2 were not significantly changed in the fibroblasts of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice compared to their non-treated counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eM). The mRNA expression levels of Catalase in the fibroblasts of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice under non-treated conditions did not show any significant difference compared to \u003cem\u003eWT, Hexa-/-\u003c/em\u003e, and \u003cem\u003eNeu3-/-\u003c/em\u003e counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eN). Under H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated conditions, the mRNA levels of Catalase in the fibroblasts of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice did not change compared to the non-treated fibroblasts of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eN). Ttase1 mRNA expression levels displayed significantly increased levels in \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e fibroblasts compared to other genotypes under non-treated and oxidative stress conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eO). Analysis of human neuroglia cells also showed no significant change in oxidative stress markers in Tay-Sachs patient neuroglia (NG-125) compared to healthy neuroglia under both non-treated and oxidative stress conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eP-S). Treatment with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e upregulated the mRNA expression levels of SOD2 and Ttase1 in both NG-124 and NG-125, except for the levels of catalase mRNA in NG-124 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eP-S).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eHigher levels of carbonylated proteins in the cerebellum and fibroblast of\u003c/b\u003e \u003cb\u003eHexa-/-Neu3-/-\u003c/b\u003e \u003cb\u003emice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo show the detrimental effects of imbalanced redox homeostasis, we measured carbonyl protein content, an irreversible protein modification, using protein carbonylation analysis in the cerebellum, cortex, thalamus, and hippocampus of 5-month-old \u003cem\u003eWT\u003c/em\u003e, \u003cem\u003eHexa-/-\u003c/em\u003e, \u003cem\u003eNeu3-/-\u003c/em\u003e, and \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Our results indicated that in the thalamus and cerebellum of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice, there were significantly increased levels of carbonylated proteins compared to \u003cem\u003eWT\u003c/em\u003e and \u003cem\u003eHexa-/-\u003c/em\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn fibroblasts of 5-month-old \u003cem\u003eWT\u003c/em\u003e, \u003cem\u003eHexa-/-\u003c/em\u003e, \u003cem\u003eNeu3-/-\u003c/em\u003e, and \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice, the levels of carbonylated proteins were also analyzed. Our results indicated a significant increase in \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e fibroblasts compared to their single knock-out counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Similarly, in Tay-Sachs patient neuroglia cells (NG-125), significantly increased levels of carbonylated proteins were observed when compared to normal neuroglia cells (NG-124) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003cb\u003eElevated levels of APE1 protein in brain regions of\u003c/b\u003e \u003cb\u003eHexa-/-Neu3-/-\u003c/b\u003e \u003cb\u003emice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAPE1 protein has essential roles in repairing oxidative damage on DNA and mediating the activity of specific transcription factors [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. To understand the response to oxidative DNA damage in the brain, we analyzed the levels of APE1 protein in four different brain regions of 5-month-old mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Cerebellum (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), and thalamus (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eC) regions displayed higher levels of APE1 protein in \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice. Interestingly, we observed no difference in the hippocampus region (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). We also studied APE1 protein levels in fibroblasts under normal and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). There was no significant difference in APE1 protein expression in the fibroblasts for both normal and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). To determine the levels and localization of cellular APE1 protein, we also performed immunocytochemical analyses in the fibroblasts of 5-month-old \u003cem\u003eWT\u003c/em\u003e, \u003cem\u003eHexa-/-\u003c/em\u003e, \u003cem\u003eNeu3-/-\u003c/em\u003e, and \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). Even if it is not statistically significant, fluorescent intensity demonstrated that fibroblasts of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice displayed higher levels of APE1 protein compared to control groups however it is down-regulated in increased oxidative stress condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). When we analyzed the nuclear and cytoplasmic APE1 protein intensity separately, we observed that the fibroblasts of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice displayed a decreased level of nuclear APE1 protein; however, a significantly increased level of cytoplasmic APE1 protein in both non- and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated groups compared to the fibroblasts of \u003cem\u003eWT\u003c/em\u003e and \u003cem\u003eNeu3-/-\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eH, I). Induced oxidative stress using H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e increased the cytoplasmic APE1 protein intensity level compared to non-treated conditions, but the difference was not statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIntracellular ROS level was higher in fibroblasts of\u003c/b\u003e \u003cb\u003eHexa-/-Neu3-/-\u003c/b\u003e \u003cb\u003emice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe reactive oxygen species (ROS) comprising radicals and non-radical molecules in cells become detrimental when neutralizing systems cannot overcome their production [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The fluorescence signal of H\u003csub\u003e2\u003c/sub\u003eDCFDA detected by flow cytometry reflects the oxidized dye content and is used to measure intracellular ROS level. By combining H\u003csub\u003e2\u003c/sub\u003eDCFDA with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, we aimed to show whether there is a difference between the standard and induced oxidative stress conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Our data shows \u003cem\u003eWT\u003c/em\u003e and \u003cem\u003eHexa-/-\u003c/em\u003e mice showed significantly more severe oxidative stress than \u003cem\u003eNeu3-/-\u003c/em\u003e and \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice under induced conditions. On the other hand, \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e and \u003cem\u003eNeu3-/-\u003c/em\u003e mice displayed a significantly higher level of ROS compared to \u003cem\u003eWT\u003c/em\u003e and \u003cem\u003eHexa-/-\u003c/em\u003e mice under non-treated conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). Similarly, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated NG-125 cells exhibited decreased intracellular ROS levels compared to other groups; however, under normal conditions, there was no difference between intracellular ROS levels in NG-124 and NG-125 cells (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn many lysosomal storage disorders, progressive neurodegeneration and susceptibility of neurons to these disorders, including GM2 gangliosidosis, have been shown to affect the central nervous system [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Recently, we reported a severe neuronal death, especially in the cerebella and hippocampi of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice, the murine model of TSD, related to the accumulating GM2 ganglioside [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In addition, gene expression levels of several anti- and pro-apoptotic genes implied increased apoptotic regulation in the \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mouse model [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. On the other hand, the mechanism of neurodegeneration relevant to imbalance in redox homeostasis has not yet been clarified in \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice. Here, we further analyzed the other regulators of the apoptosis mechanism in \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice under oxidative stress conditions using fibroblasts and brain samples of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice and neuroglia cells of TSD patients. Consistent with our previous data, we demonstrated decreased levels of Bcl2, an anti-apoptotic apoptosis regulator, particularly in the cerebella and hippocampi of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice. Surprisingly, elevated levels of pro-apoptotic Bax and significantly decreased levels of anti-apoptotic BclXL were shown in the cortex and fibroblasts of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice, indicating apoptotic cell death. Moreover, drastically higher levels of TUNEL-positive cells in Hexa-/-Neu3-/- brain regions demonstrated elevated levels of apoptosis and neurodegeneration.\u003c/p\u003e \u003cp\u003eThe balance between ROS production and the antioxidant defense mechanism provides the redox homeostasis of the cell. Once the net ROS production reaches above the capacity of the antioxidant defense mechanism, redox homeostasis is disrupted, and oxidative stress occurs [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Therefore, excessively produced ROS causes progressive oxidative damage, leading to lipid peroxidation, protein oxidation, and DNA damage [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Even if the exact mechanism of oxidative damage in LSDs is not yet wholly explained, lysosomal accumulations create an imbalance in redox homeostasis. NPC fibroblasts of human patients showed decreased ROS generation [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]; in the human Krabbe disease cell and Pompe disease mouse model [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], ROS levels were increased. Furthermore, higher intracellular ROS levels and oxidative stress markers were identified in cultured endothelial cells of Fabry patients with accumulating Gb\u003csub\u003e3\u003c/sub\u003e, in the fibroblasts of Gaucher Disease patients with accumulating glucosylceramide [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], and in the fibroblasts of Tay-Sachs patients with accumulating GM2 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Here, we further investigated whether there is an alteration in the levels of oxidative stress markers, SOD2, Catalase, and TTase1, in brain regions and fibroblasts of the murine model of TSD. SOD2 removes excess ROS to keep cellular redox balance, and decreased levels of SOD2 gene expression were reported in the brain tissue of the Sandhoff Disease murine model [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Catalase, which degrades hydrogen peroxide into water and oxygen, is decreased in Neuronal Ceroid Lipofuscinoses (NCL) patients [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Thioltransferase-1 (Ttase1), which belongs to the glutaredoxin family of proteins, increased in the fibroblasts of GM1 gangliosidosis, Gaucher disease, and Tay-Sachs patients [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Likewise, we showed decreased expression levels of SOD2 and Catalase in the cerebellum of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice, indicating the dysregulation of redox balance and increased oxidative stress. Besides, the increased expression of Thioltransferase-1 (Ttase1), elevated catalase expression under H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated conditions, and increased ROS levels could be correlated with the imbalance in redox homeostasis and unresponsiveness to oxidative stress in the fibroblasts of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice. Previously, it has been shown that the anti-apoptotic protein Bcl2 is involved in the protection of cells against ROS-mediated apoptosis. However, Bcl2 itself does not have an antioxidant effect; instead, it might upregulate the superoxide dismutase levels, including SOD2, within cells [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The decreased levels of SOD2 in the cerebellum of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice may be a response to downregulation of Bcl2 and neurodegeneration.\u003c/p\u003e \u003cp\u003eIncreased oxidative stress triggers the oxidation of biological macromolecules, including the carbonylation of proteins and DNA damage due to the accumulation of ROS molecules [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In the fibroblasts of Gaucher Disease patients and the plasma samples from Niemann-Pick type C patients, increased protein carbonylation and imbalanced redox homeostasis were reported [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Similarly, we investigated the increase in the levels of carbonylated proteins in the cerebellum and fibroblasts of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice. Therefore, we suggest that protein carbonylation in the tissues of the TSD murine model is correlated to protein damage and altered redox balance. APE1 is an endonuclease involved in the base excision repair mechanism and the control of nuclear redox activity. Therefore, it is a significant regulator of the cellular response to an imbalance in redox homeostasis and oxidative stress [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Our study observed increased protein expression levels of APE1 in all brain regions of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice except for the hippocampus. Moreover, we observed significantly higher cytoplasmic expression of APE1 protein in fibroblasts of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice; however, the exact biological role of APE1 in cytoplasm requires further investigation. Our data suggests that APE1 protein expression in the TSD murine model could be related to DNA damage caused by oxidative stress.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eLittle is understood about the mechanism(s) by which the GM2 ganglioside accumulation and altered redox homeostasis leads to neurodegeneration in \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice at a molecular level, but research in the fibroblasts and brain samples of TSD murine model might increasingly point to the role of oxidative stress mediators. Here, we demonstrated the damaging effects of imbalanced redox homeostasis and its association with neurodegeneration using apoptotic regulators, protein carbonylation assays, and APE1 protein expression in fibroblast and specific brain regions of the murine model. Taken together, our results provide the first \u003cem\u003ein vivo\u003c/em\u003e evidence that \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice displayed an imbalance in redox homeostasis, especially in the cerebellum, which might contribute to neurodegeneration and severe phenotype in the murine model of TSD. However, further identification of oxidative stress-related players on neurodegeneration is required to evaluate possible therapeutic approaches targeting antioxidant defense mechanisms.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Assoc. Prof. Dr. Ayten Nalbant Aldanmaz and Tufan Utku \u0026Ccedil;alışkan for Flow cytometer analysis, and Dr. Se\u0026ccedil;il Akyıldız Demir for technical help and support.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHB, NA, and VS conceived the study, designed experiments, analyzed and interpreted data, and wrote the manuscript. All authors confirm the authenticity of the raw data and have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the Scientific and Technological Research Council of Turkey (TUBITAK) under Grant No:215Z083. NA was supported by the Turkish Higher Education Council\u0026rsquo;s 100/2000 Ph.D. fellowship program and the TUBITAK BIDEB National Scholarship Program for Ph.D. students (2211-A). A scholarship program under the TUBITAK-France Bosphorus 120N552 Project supported HB. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll mice were maintained in the Turkish Council on Animal Care (TCAC) accredited animal facility of Izmir Institute of Technology according to TCAC guidelines. The Animal Care and Use Committee (Animal Ethics Committee) of Izmir Institute of Technology, Izmir, Turkey, granted animal care and use in the experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSango K, Yamanaka S, Hoffmann A et al (1995) Mouse models of Tay\u0026ndash;Sachs and Sandhoff diseases differ in neurologic phenotype and ganglioside metabolism. 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Antioxid Redox Signal 7\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":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Tay-Sachs Disease, Neurodegeneration, Apoptosis, Oxidative stress, Reactive Oxygen Species","lastPublishedDoi":"10.21203/rs.3.rs-5293300/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5293300/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eTay-Sachs disease is a type of neurodegenerative disorder with a build-up of GM2 ganglioside in the brain, which results in progressive central nervous system dysfunction. Our group recently generated \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice, a murine model with neuropathological abnormalities similar to the infantile form of Tay-Sachs disease. Previously, we reported progressive neurodegeneration with neuronal loss in the brain sections of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice. However, the relationship of the severity of neurodegeneration to imbalance in redox homeostasis has not been clarified in \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice. Here, we evaluated whether neurodegeneration is associated with oxidative stress in the tissues and cells of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice and neuroglia cells from Tay-Sachs patients.\u003c/p\u003e\u003ch2\u003eMethods and Results\u003c/h2\u003e \u003cp\u003eIn four brain regions and fibroblasts of 5-month-old \u003cem\u003eWT\u003c/em\u003e, \u003cem\u003eHexa-/-\u003c/em\u003e, \u003cem\u003eNeu3-/-\u003c/em\u003e, and \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice and human neuroglia cells, apoptosis and oxidative stress-related markers were evaluated using Western blot, RT-PCR, and immunohistochemistry analyses. We further analyzed oxidative stress levels using flow cytometry analyses. We discovered neuronal death, alterations in intracellular ROS levels, and damaging effects of oxidative stress, especially in the cerebellum and fibroblasts of \u003cem\u003eHexa-/-Neu3-/-\u003c/em\u003e mice.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur results showed that alteration in redox homeostasis might be related to neurodegeneration in the murine model of Tay-Sachs Disease. These findings suggest that targeting the altered redox balance and increased oxidative stress might be a rational therapeutic approach for alleviating neurodegeneration and treating Tay-Sachs disease.\u003c/p\u003e","manuscriptTitle":"Imbalance in Redox Homeostasis is Associated with Neurodegeneration in the Murine Model of Tay-Sachs Disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-30 10:02:36","doi":"10.21203/rs.3.rs-5293300/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-18T09:34:09+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-16T18:43:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-09T07:02:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-02T16:03:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"200969523418091503716458622569334357046","date":"2024-10-30T16:23:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"77709537361444986228127649213417414187","date":"2024-10-29T20:10:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"125863434553782435165581852625279685957","date":"2024-10-29T06:31:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"286448448936901278586599496891195187355","date":"2024-10-28T23:36:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-28T15:46:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-22T09:09:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-19T12:50:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2024-10-19T07:52:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"eb3d2a86-a4de-46dc-9d8c-b29bf40ea4c5","owner":[],"postedDate":"October 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-03-10T17:46:44+00:00","versionOfRecord":{"articleIdentity":"rs-5293300","link":"https://doi.org/10.1007/s11033-025-10380-y","journal":{"identity":"molecular-biology-reports","isVorOnly":false,"title":"Molecular Biology Reports"},"publishedOn":"2025-03-05 15:57:24","publishedOnDateReadable":"March 5th, 2025"},"versionCreatedAt":"2024-10-30 10:02:36","video":"","vorDoi":"10.1007/s11033-025-10380-y","vorDoiUrl":"https://doi.org/10.1007/s11033-025-10380-y","workflowStages":[]},"version":"v1","identity":"rs-5293300","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5293300","identity":"rs-5293300","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}