Plasma GM2 ganglioside potential biomarker for diagnosis, prognosis and disease monitoring of GM2-Gangliosidosis.

GM2-Gangliosidosis are a group of inherited lysosomal storage pathologies characterized by a large accumulation of GM2 ganglioside in the lysosome. They are caused by mutation in HEXA or HEXB causing reduced or absent activity of a lysosomal ß-hexosaminidase A, or mutation in GM2A causing defect in GM2 activator protein (GM2AP), an essential protein for the activity of the enzyme. Biochemical diagnosis relies on the measurement of ß-hexosaminidases A and B activities, which is able to detect lysosomal enzyme deficiency but fails to identify defects in GM2AP. We developed a rapid, specific and sensitive liquid chromatography-mass spectrometry-based method to measure simultaneously GM1, GM2, GM3 and GD3 molecular species. Gangliosides were analysed in plasma from 19 patients with GM2-Gangliosidosis: Tay-Sachs (n = 9), Sandhoff (n = 9) and AB variant of GM2-Gangliosidosis (n = 1) and compared to 20 age-matched controls. Among patients, 12 have a late adult-juvenile-onset and 7 have an infantile early-onset of the disease. Plasma GM2 molecular species were increased in all GM2-Gangliosidosis patients (19/19), including the patient with GM2A mutation, compared to control individuals and compared to patients with different other lysosomal storage diseases. GM234:1 and GM234:1/GM334:1 ratio discriminated patients from controls with 100% sensitivity and specificity. GM234:1 and GM234:1/GM334:1 were higher in patients with early-onset compared to those with late-onset of the disease, suggesting a relationship with severity. Longitudinal analysis in one adult with Tay-Sachs disease over 9 years showed a positive correlation of GM234:1 and GM234:1/GM334:1 ratio with age at sampling. We propose that plasma GM2 34:1 and its ratio to GM3 34:1 could be sensitive and specific biochemical diagnostic biomarkers for GM2-Gangliosidosis including AB variant and could be useful as a first line diagnostic test and potential biomarkers for monitoring upcoming therapeutic efficacy.

Efficacy and Safety of N-Acetyl-l-Leucine in Children and Adults With GM2 Gangliosidoses.

BACKGROUND AND OBJECTIVES: GM2 gangliosidoses (Tay-Sachs and Sandhoff diseases) are rare, autosomal recessive, neurodegenerative diseases with no available symptomatic or disease-modifying treatments. This clinical trial investigated N-acetyl-l-leucine (NALL), an orally administered, modified amino acid in pediatric (≥6 years) and adult patients with GM2 gangliosidoses. METHODS: In this phase IIb, multinational, open-label, rater-blinded study (IB1001-202), male and female patients aged ≥6 years with a genetically confirmed diagnosis of GM2 gangliosidoses received orally administered NALL for a 6-week treatment period (4 g/d in patients ≥13 years, weight-tiered doses for patients 6-12 years), followed by a 6-week posttreatment washout period. For the primary Clinical Impression of Change in Severity analysis, patient performance on a predetermined primary anchor test (the 8-Meter Walk Test or the 9-Hole Peg Test) at baseline, after 6 weeks on NALL, and again after a 6-week washout period was videoed and evaluated centrally by blinded raters. Secondary outcomes included assessments of ataxia, clinical global impression, and quality of life. RESULTS: Thirty patients between the age of 6 and 55 years were enrolled. Twenty-nine had an on-treatment assessment and were included in the primary modified intention-to-treat analysis. The study met its CI-CS primary end point (mean difference 0.71, SD = 2.09, 90% CI 0.00, 1.50, p = 0.039), as well as secondary measures of ataxia and global impression. NALL was safe and well tolerated, with no serious adverse reactions. DISCUSSION: Treatment with NALL was associated with statistically significant and clinically relevant changes in functioning and quality of life in patients with GM2 gangliosidosis. NALL was safe and well tolerated, contributing to an overall favorable risk:benefit profile. NALL is a promising, easily administered (oral) therapeutic option for these rare, debilitating diseases with immense unmet medical needs. TRIAL REGISTRATION INFORMATION: The trial is registered with ClinicalTrials.gov (NCT03759665; registered on November 30, 2018), EudraCT (2018-004406-25), and DRKS (DRKS00017539). The first patient was enrolled on June 7, 2019. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that NALL improves outcomes for patients with GM2 gangliosidoses.

Gene Expression Profile in the Sandhoff Mouse Brain with Progression of Age.

Sandhoff disease (SD) is a fatal neurodegenerative disorder belonging to the family of diseases called GM2 Gangliosidosis. There is no curative treatment of SD. The molecular pathogenesis of SD is still unclear though it is clear that the pathology initiates with the build-up of ganglioside followed by microglial activation, inflammation, demyelination and apoptosis, leading to massive neuronal loss. In this article, we explored the expression profile of selected immune and myelination associated transcripts (Wfdc17, Ccl3, Lyz2, Fa2h, Mog and Ugt8a) at 5-, 10- and 16-weeks, representing young, pre-symptomatic and late stages of the SD mice. We found that immune system related genes (Wfdc17, Ccl3, Lyz2) are significantly upregulated by several fold at all ages in Hexb-KO mice relative to Hexb-het mice, while the difference in the expression levels of myelination related genes is not statistically significant. There is an age-dependent significant increase in expression of microglial/pro-inflammatory genes, from 5-weeks to the near humane end-point, i.e., 16-week time point; while the expression of those genes involved in myelination decreases slightly or remains unchanged. Future studies warrant use of new high-throughput gene expression modalities (such as 10X genomics) to delineate the underlying pathogenesis in SD by detecting gene expression changes in specific neuronal cell types and thus, paving the way for rational and precise therapeutic modalities.

P. Ala278Val mutation might cause a pathogenic defect in HEXB folding leading to the Sandhoff disease.

Sandhoff disease is a rare neurodegenerative and autosomal recessive disorder, which is characterized by a defect in ganglioside metabolism. Also, it is caused by mutations in the HEXB gene for the ß-subunit isoform 1 of ß-N-acetyl hexosaminidase. In the present study, an Iranian 14- month -old girl with 8- month history of unsteady walking and involuntary movements was described. In this regard, biochemical testing showed some defects in the normal activity of beta-hexosaminidase protein. Following sequencing of HEXB gene, a homozygous c.833C > T mutation was identified in the patient's genome. After recognition of p.A278V, several different in silico methods were used to assess the mutant protein stability, ranging from mutation prediction methods to ligand docking. The p.A278V mutation might be disruptive because of changing the three-dimensional folding at the end of the 5th alpha helix. According to the medical prognosis, in silico and structural analyses, it was predicted to be disease cause.

Metabolism of Glycosphingolipids and Their Role in the Pathophysiology of Lysosomal Storage Disorders.

Glycosphingolipids (GSLs) are a specialized class of membrane lipids composed of a ceramide backbone and a carbohydrate-rich head group. GSLs populate lipid rafts of the cell membrane of eukaryotic cells, and serve important cellular functions including control of cell-cell signaling, signal transduction and cell recognition. Of the hundreds of unique GSL structures, anionic gangliosides are the most heavily implicated in the pathogenesis of lysosomal storage diseases (LSDs) such as Tay-Sachs and Sandhoff disease. Each LSD is characterized by the accumulation of GSLs in the lysosomes of neurons, which negatively interact with other intracellular molecules to culminate in cell death. In this review, we summarize the biosynthesis and degradation pathways of GSLs, discuss how aberrant GSL metabolism contributes to key features of LSD pathophysiology, draw parallels between LSDs and neurodegenerative proteinopathies such as Alzheimer's and Parkinson's disease and lastly, discuss possible therapies for patients.

Upregulating ß-hexosaminidase activity in rodents prevents α-synuclein lipid associations and protects dopaminergic neurons from α-synuclein-mediated neurotoxicity.

Sandhoff disease (SD) is a lysosomal storage disease, caused by loss of ß-hexosaminidase (HEX) activity resulting in the accumulation of ganglioside GM2. There are shared features between SD and Parkinson's disease (PD). α-synuclein (aSYN) inclusions, the diagnostic hallmark sign of PD, are frequently found in the brain in SD patients and HEX knockout mice, and HEX activity is reduced in the substantia nigra in PD. In this study, we biochemically demonstrate that HEX deficiency in mice causes formation of high-molecular weight (HMW) aSYN and ubiquitin in the brain. As expected from HEX enzymatic function requirements, overexpression in vivo of HEXA and B combined, but not either of the subunits expressed alone, increased HEX activity as evidenced by histochemical assays. Biochemically, such HEX gene expression resulted in increased conversion of GM2 to its breakdown product GM3. In a neurodegenerative model of overexpression of aSYN in rats, increasing HEX activity by AAV6 gene transfer in the substantia nigra reduced aSYN embedding in lipid compartments and rescued dopaminergic neurons from degeneration. Overall, these data are consistent with a paradigm shift where lipid abnormalities are central to or preceding protein changes typically associated with PD.

Abnormal organization during neurodevelopment in a mouse model of Sandhoff disease.

Sandhoff disease (SD) is a genetic disorder caused by a mutation of HEXB, which is the ß-subunit gene of ß-hexosaminidase A and B (HexA and HexB) in humans. HEXB mutation reduces HexA and HexB enzymatic activities, and results in the massive accumulation of ganglioside GM2 in the nervous system. Severe phenotypes of SD show progressive neurodegeneration in human infants, and lysosomal dysfunction that may affect the early development of the nervous system. In a previous study, neural stem cells (NSCs) and induced pluripotent stem cells derived from SD model mice, which are Hexb-deficient (Hexb-/-), demonstrated impaired neuronal differentiation. This study investigated early neurodevelopment in vivo using Hexb-/- mice. The structure of adult cerebral cortices of Hexb-/- mice was normal. However, the expression of Sox2, an NSC-related gene, was reduced in the embryonic cerebral cortices of Hexb-/- mice. Moreover, a reduction of early neuronal migration and differentiation was observed in the embryonic cerebral cortices of Hexb-/- mice. In addition, we showed that the production of layer-specific neurons was delayed in somatosensory cerebral cortices of Hexb-/- mice. These findings suggest that the alterations observed in embryonic Hexb-/- mice may contribute to deficits in neurodevelopment of SD.

Metabolomics profiling reveals profound metabolic impairments in mice and patients with Sandhoff disease.

Sandhoff disease (SD) results from mutations in the HEXB gene, subsequent deficiency of N-acetyl-ß-hexosaminidase (Hex) and accumulation of GM2 gangliosides. SD leads to progressive neurodegeneration and early death. However, there is a lack of established SD biomarkers, while the pathogenesis etiology remains to be elucidated. To identify potential biomarkers and unveil the pathogenic mechanisms, metabolomics analysis with reverse phase liquid chromatography (RPLC) was conducted. A total of 177, 112 and 119 metabolites were found to be significantly dysregulated in mouse liver, mouse brain and human hippocampus samples, respectively (p < .05, ID score > 0.5). Principal component analysis (PCA) analysis of the metabolites showed clear separation of metabolomics profiles between normal and diseased individuals. Among these metabolites, dipeptides, amino acids and derivatives were elevated, indicating a robust protein catabolism. Through pathway enrichment analysis, we also found alterations in metabolites associated with neurotransmission, lipid metabolism, oxidative stress and inflammation. In addition, N-acetylgalactosamine 4-sulphate, key component of glycosaminoglycans (GAG) was significantly elevated, which was also confirmed by biochemical assays. Collectively, these results indicated major shifts of energy utilization and profound metabolic impairments, contributing to the pathogenesis mechanisms of SD. Global metabolomics profiling may provide an innovative tool for better understanding the disease mechanisms, and identifying potential diagnostic biomarkers for SD.

Inhibition of astrocytic adenosine receptor A2A attenuates microglial activation in a mouse model of Sandhoff disease.

Astrocyte-microglia communication influences the onset and progression of central nervous system (CNS) disorders. In this study, we determined how chronic inflammation by activated astrocytes affected and regulated CNS functions in Sandhoff disease (SD), a CNS lysosomal storage disorder. SD triggers intense CNS inflammation such as microglial activation and astrogliosis. It is caused by mutation of the HEXB gene, which reduces ß-hexosaminidase (Hex) enzymatic activity in lysosomes, leading to accumulation of the substrate GM2 ganglioside in neuronal cells. Hexb-/- mice display a phenotype similar to human patients that suffer from chronic inflammation characterized by activation of astrocytes and microglia. In Hexb-/- mice, tremors and loss of muscle coordination begins at ~12 weeks. Interestingly, we found that reactive astrocytes expressed adenosine A2A receptor in the cerebral cortices of Hexb-/- mice at the later inflammatory phase. In cultured astrocytes, expression of A2A receptor could be induced by astrocyte defined medium, and then the activation of the A2A receptor induced ccl2 expression. In Hexb-/- mice, inhibition of the A2A receptor antagonized by istradefylline decreased the number of activated microglial cells and inflammatory cytokines/chemokines at 13 weeks. Thus, the astrocytic A2A receptor is an important sensor that regulates microglial activation in the late phase of inflammation.

Improvement in dysmyelination by the inhibition of microglial activation in a mouse model of Sandhoff disease.

Sandhoff disease (SD) is a genetic disorder caused by a mutation of the ß-subunit gene ß-hexosaminidase B (HexB) in humans, which results in the massive accumulation of the ganglioside GM2 and related glycosphingolipids in the nervous system. SD causes progressive neurodegeneration and changes in white matter in human infants. An animal model of SD has been established, Hexb-deficient (Hexb) mice, which shows abnormalities resembling the severe phenotype found in human infants. Previously, we reported that the activation state of microglia caused astrogliosis in the early stage of Hexb mouse development. To study how the symptoms of SD develop, we explored the difference in gene expression between 4-week-old Hexb and Hexb mouse cerebral cortices by microarray analysis. The data indicated not only the upregulation of immune system-related genes but also the downregulation of myelin-related genes in the 4-week-old Hexb mouse cerebral cortices. To test the correlation between inflammation and dysmyelination, we generated double-knockout mice of Hexb and the Fc receptor γ gene (Fcrγ), which is a regulator of autoimmune responses. Dysmyelination recovered in these double-knockout mice. The number of oligodendrocyte progenitors, which expressed platelet-derived growth factor receptor-α, did not change in the 2-week-old mouse brain. These results indicate that microglial activation plays an important role in the myelination process, without influencing the number of oligodendrocyte progenitors, in the development of Hexb mice.

Intravenous administration of scAAV9-Hexb normalizes lifespan and prevents pathology in Sandhoff disease mice.

Sandhoff disease (SD) is a rare inherited disorder caused by a deficiency of ß-hexosaminidase activity which is fatal because no effective treatment is available. A mouse model of Hexb deficiency reproduces the key pathognomonic features of SD patients with severe ubiquitous lysosomal dysfunction, GM2 accumulation, neuroinflammation and neurodegeneration, culminating in death at 4 months. Here, we show that a single intravenous neonatal administration of a self-complementary adeno-associated virus 9 vector (scAAV9) expressing the Hexb cDNA in SD mice is safe and sufficient to prevent disease development. Importantly, we demonstrate for the first time that this treatment results in a normal lifespan (over 700 days) and normalizes motor function assessed by a battery of behavioral tests, with scAAV9-treated SD mice being indistinguishable from wild-type littermates. Biochemical analyses in multiple tissues showed a significant increase in hexosaminidase A activity, which reached 10-15% of normal levels. AAV9 treatment was sufficient to prevent GM2 and GA2 storage almost completely in the cerebrum (less so in the cerebellum), as well as thalamic reactive gliosis and thalamocortical neuron loss in treated Hexb-/- mice. In summary, this study demonstrated a widespread protective effect throughout the entire CNS after a single intravenous administration of the scAAV9-Hexb vector to neonatal SD mice.

Alterations in endo-lysosomal function induce similar hepatic lipid profiles in rodent models of drug-induced phospholipidosis and Sandhoff disease.

Drug-induced phospholipidosis (DIPL) is characterized by an increase in the phospholipid content of the cell and the accumulation of drugs and lipids inside the lysosomes of affected tissues, including in the liver. Although of uncertain pathological significance for patients, the condition remains a major impediment for the clinical development of new drugs. Human Sandhoff disease (SD) is caused by inherited defects of the ß subunit of lysosomal ß-hexosaminidases (Hex) A and B, leading to a large array of symptoms, including neurodegeneration and ultimately death by the age of 4 in its most common form. The substrates of Hex A and B, gangliosides GM2 and GA2, accumulate inside the lysosomes of the CNS and in peripheral organs. Given that both DIPL and SD are associated with lysosomes and lipid metabolism in general, we measured the hepatic lipid profiles in rodent models of these two conditions using untargeted LC/MS to examine potential commonalities. Both model systems shared a number of perturbed lipid pathways, notably those involving metabolism of cholesteryl esters, lysophosphatidylcholines, bis(monoacylglycero)phosphates, and ceramides. We report here profound alterations in lipid metabolism in the SD liver. In addition, DIPL induced a wide range of lipid changes not previously observed in the liver, highlighting similarities with those detected in the model of SD and raising concerns that these lipid changes may be associated with underlying pathology associated with lysosomal storage disorders.

Neuronal pentraxin 1 depletion delays neurodegeneration and extends life in Sandhoff disease mice.

GM2 gangliosidoses are a group of lysosomal storage disorders which include Sandhoff disease and Tay-Sachs disease. Dysregulation of glutamate receptors has been recently postulated in the pathology of Sandhoff disease. Glutamate receptor association with neuronal pentraxins 1 and 2, and the neuronal pentraxin receptor facilitates receptor potentiation and synaptic shaping. In this study, we have observed an upregulation of a novel form of neuronal pentraxin 1 (NP1-38) in the brains of a mouse model of Sandhoff disease and Tay-Sachs disease. In order to determine the impact of NP1 on the pathophysiology of Sandhoff disease mouse models, we have generated an Np1-/-Hexb-/- double knockout mouse, and observed extended lifespan, improved righting reflex and enhanced body condition relative to Hexb-/- mice, with no effect on gliosis or apoptotic markers in the CNS. Sandhoff mouse brain slices reveals a reduction in AMPA receptor-mediated currents, and increased variability in total glutamate currents in the CA1 region of the hippocampus; Np1-/-Hexb-/- mice show a correction of this phenotype, suggesting NP1-38 may be interfering with glutamate receptor function. Indeed, some of the psychiatric aspects of Sandhoff and Tay-Sachs disease (particularly late onset) may be attributed to a dysfunctional hippocampal glutamatergic system. Our work highlights a potential role for synaptic proteins, such as NP1 and glutamate receptors in lysosomal storage diseases.

Bi-phasic gliosis drives neuropathology in a Sandhoff disease mouse model.

Microgliosis and astrogliosis are known to be exacerbating factors in the progression of the lysosomal storage disorder Sandhoff disease. We have also found evidence for excitotoxicity via glutamate receptors in Sandhoff disease. To view the interaction of these cascades, we measured cerebellar expression of markers for gliosis, apoptosis, and excitatory synapses over the disease course in a Sandhoff disease mouse model. We observe a 2-stage model, with initial activation of microgliosis as early as 60days of age, followed by a later onset of astrogliosis, caspase-mediated apoptosis, and reduction in GluR1 at approximately 100days of age. These results implicate immune cells as first responders in Sandhoff disease.

Clinical,biochemical and molecular analysis of five Chinese patients with Sandhoff disease.

Sandhoff disease (SD) is a rare autosomal recessive lysosomal storage disorder of sphingolipid metabolism resulting from the deficiency of ß-hexosaminidase (HEX). Mutations of the HEXB gene cause Sandhoff disease. In order to improve the diagnosis and expand the knowledge of the disease, we collected and analyzed relevant data of clinical diagnosis, biochemical investigation, and molecular mutational analysis in five Chinese patients with SD. The patients presented with heterogenous symptoms of neurologic deterioration. HEX activity in leukocytes was severely deficient. We identified seven different mutations, including three known mutations: IVS12-26G > A, p.T209I, p.I207V, and four novel mutations: p.P468PfsX62, p.L223P, p.Y463X, p.G549R. We also detected two different heterozygous mutations c.-122delC and c.-126C > T in the promoter which were suspected to be deleterious mutations. We attempted to correlate these mutations with the clinical presentation of the patients. Our study indicates that the mutation p.T209I and p.P468PfsX62 may link to the infantile form of SD. Our study expands the spectrum of genotype of SD in China, provides new insights into the molecular mechanism of SD and helps to the diagnosis and treatment of this disease.

TSPO in a murine model of Sandhoff disease: presymptomatic marker of neurodegeneration and disease pathophysiology.

Translocator protein (18 kDa), formerly known as the peripheral benzodiazepine receptor (PBR), has been extensively used as a biomarker of active brain disease and neuroinflammation. TSPO expression increases dramatically in glial cells, particularly in microglia and astrocytes, as a result of brain injury, and this phenomenon is a component of the hallmark response of the brain to injury. In this study, we used a mouse model of Sandhoff disease (SD) to assess the longitudinal expression of TSPO as a function of disease progression and its relationship to behavioral and neuropathological endpoints. Focusing on the presymptomatic period of the disease, we used ex vivo [(3)H]DPA-713 quantitative autoradiography and in vivo [(125)I]IodoDPA-713 small animal SPECT imaging to show that brain TSPO levels markedly increase prior to physical and behavioral manifestation of disease. We further show that TSPO upregulation coincides with early neuronal GM2 ganglioside aggregation and is associated with ongoing neurodegeneration and activation of both microglia and astrocytes. In brain regions with increased TSPO levels, there is a differential pattern of glial cell activation with astrocytes being activated earlier than microglia during the progression of disease. Immunofluorescent confocal imaging confirmed that TSPO colocalizes with both microglia and astrocyte markers, but the glial source of the TSPO response differs by brain region and age in SD mice. Notably, TSPO colocalization with the astrocyte marker GFAP was greater than with the microglia marker, Mac-1. Taken together, our findings have significant implications for understanding TSPO glial cell biology and for detecting neurodegeneration prior to clinical expression of disease.

Bis(monoacylglycero)phosphate: a secondary storage lipid in the gangliosidoses.

Bis(monoacylglycero)phosphate (BMP) is a negatively charged glycerophospholipid with an unusual sn-1;sn-1' structural configuration. BMP is primarily enriched in endosomal/lysosomal membranes. BMP is thought to play a role in glycosphingolipid degradation and cholesterol transport. Elevated BMP levels have been found in many lysosomal storage diseases (LSDs), suggesting an association with lysosomal storage material. The gangliosidoses are a group of neurodegenerative LSDs involving the accumulation of either GM1 or GM2 gangliosides resulting from inherited deficiencies in ß-galactosidase or ß-hexosaminidase, respectively. Little information is available on BMP levels in gangliosidosis brain tissue. Our results showed that the content of BMP in brain was significantly greater in humans and in animals (mice, cats, American black bears) with either GM1 or GM2 ganglioside storage diseases, than in brains of normal subjects. The storage of BMP and ganglioside GM2 in brain were reduced similarly following adeno-associated viral-mediated gene therapy in Sandhoff disease mice. We also found that C22:6, C18:0, and C18:1 were the predominant BMP fatty acid species in gangliosidosis brains. The results show that BMP accumulates as a secondary storage material in the brain of a broad range of mammals with gangliosidoses.

Biomarkers of central nervous system inflammation in infantile and juvenile gangliosidoses.

BACKGROUND: The gangliosidoses (Tay-Sachs disease, Sandhoff disease, and GM1-gangliosidosis) are progressive neurodegenerative diseases caused by lysosomal enzyme activity deficiencies and consequent accumulation of gangliosides in the central nervous system (CNS). The infantile forms are distinguished from the juvenile forms by age of onset, rate of disease progression, and age of death. There are no approved treatments for the gangliosidoses. In search of potential biomarkers of disease, we quantified 188 analytes in CSF and serum from living human patients with longitudinal (serial) measurements. Notably, several associated with inflammation were elevated in the CSF of infantile gangliosidosis patients, and less so in more slowly progressing forms of juvenile gangliosidosis, but not in MPS disease. Thirteen CSF and two serum biomarker candidates were identified. Five candidate biomarkers were distinguished by persistent elevation in the CSF of patients with the severe infantile phenotype: ENA-78, MCP-1, MIP-1α, MIP-1ß, and TNFR2. Correspondence of abnormal elevation with other variables of disease-i.e., severity of clinical phenotype, differentiation from changes in serum, and lack of abnormality in other neurodegenerative lysosomal diseases-identifies these analytes as biomarkers of neuropathology specific to the gangliosidosis diseases.

Deletion of tumor necrosis factor-α ameliorates neurodegeneration in Sandhoff disease mice.

Sandhoff disease (SD) is a lysosomal storage disorder caused by a lack of a functional ß-subunit of the ß-hexosaminidase A and B enzymes, leading to the accumulation of gangliosides in the central nervous system (CNS). The Hexb-/- mouse model of SD shows a progressive neurodegenerative phenotype similar to the human equivalent. Previous studies have revealed that Hexb-/- mice suffer from chronic neuroinflammation characterized by microglial activation and expansion. Tumor necrosis factor-α (TNFα), a key modulator of the CNS immune response in models of neurodegeneration, is a hallmark of this activation. In this study, we explore the role of TNFα in the development and progression of SD in mice, by creating a Hexb-/- Tnfα-/- double-knockout mouse. Our results revealed that the double-knockout mice have an ameliorated disease course, with an extended lifespan, enhanced sensorimotor coordination and improved neurological function. TNFα-deficient SD mice also show decreased levels of astrogliosis and reduced neuronal cell death, with no alterations in neuronal storage of gangliosides. Interestingly, temporal microglia activation appears similar between the Hexb-/- Tnfα-/- and SD mice. Evidence is provided for the TNFα activation of the JAK2/STAT3 pathway as a mechanism for astrocyte activation in the disease. Bone marrow transplantation experiments reveal that both CNS-derived and bone marrow-derived TNFα have a pathological effect in SD mouse models, with CNS-derived TNFα playing a larger role. This study reveals TNFα as a neurodegenerative cytokine mediating astrogliosis and neuronal cell death in SD and points to TNFα as a potential therapeutic target to attenuate neuropathogenesis.