Generation and characterization of induced pluripotent stem cell (iPSC) lines from patients affected with Tay-Sachs and Sandhoff disease.

Breaking barriers in lysosomal disorder screening: A novel simultaneous LC-MS/MS approach for Tay-Sachs, Sandhoff, and GM1 gangliosidosis diseases

PS gene-editing system corrects Sandhoff disease with a brain-penetrant HEXO-APOE enzyme

Venglustat in GM2 gangliosidoses and related disorders: Results of the AMETHIST randomized controlled and basket trials.

Nizubaglustat is a novel selective inhibitor of glucosylceramide synthase (GCS) and the non-lysosomal glucocerebrosidase (NLGase, GbA2) with brain penetrant properties. It is currently in clinical development as an oral treatment for rare lysosomal storage diseases with neurological involvement. One such disease group called GM2 gangliosidosis, to date, has no approved therapeutic treatment. To test the potential efficacy of nizubaglustat in a mouse model of GM2 gangliosidoses, we treated Sandhoff disease (SD) mice carrying a homozygous null mutation in the Hexb gene, as well as healthy heterozygous controls, to understand exposure versus effect under disease conditions. Oral doses of nizubaglustat from 0.2 to 6 mg/kg/day showed linear pharmacokinetics with plasma and brain concentrations sufficient to drive pharmacodynamic changes in markers of target engagement and efficacy. In the brain, an approximately 10-fold increase in GlcCer C16:0 and C18:0 was observed, which is consistent with NLGase inhibition. A statistically significant increase in survival (22%) was noted in SD mice treated at doses as low as 0.2 mg/kg/day compared to controls. Behavioral analyses, which included rotarod and open field tests, were also significantly improved. To understand the added potential mechanism of the improved survival, a subset of neuroinflammatory markers was also examined in specific brain regions. Gene expression studies showed an anti-inflammatory pattern with downregulation of Itgax, Trem2, Cxcl10 genes as an example. Brain immunohistochemistry for GFAP was decreased compared to vehicle treated control animals. These results provide proof-of-concept that nizubaglustat can be a promising therapeutic drug to treat patients with GM2 gangliosidoses.

BackgroundGM1 and GM2 (Tay–Sachs and Sandhoff diseases) gangliosidoses are rare, autosomal recessive, potentially life-threatening, disabling disorders characterized by progressive neurodegeneration, with no disease-modifying treatment. This qualitative study aimed to understand the humanistic burden of GM1 and GM2 gangliosidoses from caregivers’ perspectives by expanding knowledge on the day-to-day responsibilities of primary caregivers and the impacts experienced while providing care and support.MethodsFocus groups (90-minute duration) were conducted with caregivers (≥ 18 years) under three separate categories based on the age of the individuals with GM1/GM2 gangliosidoses either in-person (attending Annual National Tay–Sachs & Allied Diseases Association [NTSAD] Conference, Colorado, July 2022) or online (recruited through the NTSAD and Cure GM1 Foundation during November–December 2022).ResultsThis study included 29 primary caregivers (mean [range] age: 49.0 [37.0–75.0] years) of individuals (children [24.1%], adolescents [31.0%], and adults [44.8%]) diagnosed with juvenile/late-onset GM1 (41.4%) or GM2 (58.6%) gangliosidoses. The caregivers reported that most individuals required mobility aids (64.3%) and experienced speech difficulties (83.3%); they described their caregiving responsibilities as non-stop, pervasive, and often done without additional support, with marginal variance by disease type or patient age. Supporting activities of daily living was the most prominent responsibility (90.0%), followed by symptom/care management (69.0%), ensuring quality of life (45.0%), and maintaining emotional (24.0%) and physical (10.0%) well-being. Caregiving impacted every facet of life; the caregivers reported 25 different impacts, with constant psychological burden (82.8%), physical ailments/strain (62.1%), anxiety/fear/worry (58.6%), financial difficulties (58.6%), limited time for other family members (55.2%), and limitations on relationships outside family (51.7%) having the most significant effects. The caregivers relied mostly on patient advocacy organizations for resources and expressed the need for financial support, broader disease awareness, and disease-modifying treatments. Although providing care and support deleteriously impacted caregivers’ lives, they reported experiencing positive impacts on relationship building, personal development, family cohesion, community support, and life outlook.ConclusionsThis study showed a substantial humanistic burden with long-term impacts among the caregivers of individuals with GM1 and GM2 gangliosidoses. The findings provide important insights to enhance clinical care while advocating for the resources needed to improve caregivers’ and patients’ lives.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13023-025-04030-6.

Decoding the Hex-GM2-MGL2 axis in microglia-neuron crosstalk.

A pathogenic alpha synuclein variant exacerbates disease progression in a neuron-specific Gba-KO mouse.

Tay-Sachs disease (TSD) and Sandhoff disease are fatal neurodegenerative diseases without an effective therapy that are caused by mutations in the HEXA and HEXB genes, respectively. Together they encode the heterodimeric isozyme of hexosaminidase, hexosaminidase A (HexA), that degrades GM2 ganglioside. This report describes a 5-year-long study using a bidirectional adeno-associated virus 9 (AAV9) vector (AAV9-Bic_HexA/HexB) encoding both HEXA and HEXB in the TSD sheep model. Bidirectional AAV9 was delivered i.v. or through various cerebrospinal fluid (CSF) delivery routes: intracerebroventricular (ICV), cisterna magna (CM), and lumbar intrathecal space (LIT). The longest survival and best distribution were achieved by multipoint CSF delivery (combined CM, ICV, and LIT) with treated animals that survived up to 5 years of age (untreated animals with TSD die after ~9 months). Extension in survival was accompanied by lasting improvement in neurological examination and maze testing. Improvement in biomarkers of efficacy, including MRI, magnetic resonance spectroscopy, diffusion tensor imaging, and CSF levels of GM2 ganglioside and HexA activity, was evident. Postmortem assessments showed broad HexA distribution, GM2 ganglioside clearance, and vector genome distribution, especially in deep brain structures. Therapeutic efficacy documented in this study supports translation of bidirectional vector and multipoint CSF delivery to a clinical trial in patients with TSD and Sandhoff disease.

Gangliosides are essential for membrane functions, cell recognition, and maintenance of the nervous system. GM2 gangliosidosis is a group of rare genetic lysosomal storage diseases that includes Tay-Sachs disease (TSD), Sandhoff disease (SD), and AB variant. TSD and SD are characterized by deficient β-N-acetyl-hexosaminidase activity. This leads to decreased catabolism of β-N-acetyl-hexosamine-containing ganglioside GM2 in the lysosomes, damage to cells and tissues, and severe neurological symptoms. GM2 is a major ganglioside accumulating in TSD and SD, and is synthesized from GM3 by β1,4-N-acetylgalactosaminyltransferase 1 (B4GALNT1, GM2 synthase). Therapies under development for GM2 gangliosidosis include adeno-associated virus gene therapy, enzyme replacement, and substrate reduction therapy (SRT). The goal of this work was to express and purify human B4GALNT1, characterize its activity, and explore its structural features by protein modeling and substrate docking. We used a panel of synthetic compounds to study their potential inhibition of B4GALNT1 activity. This work can serve to develop SRT for GM2 gangliosidosis.

Lysosomal storage disorders (LSDs) are a large disease class involving lysosomal dysfunction, often resulting in neurodegeneration. Sandhoff disease (SD) is an LSD caused by a deficiency in the β subunit of the β-hexosaminidase enzyme (Hexb). Although Hexb expression in the brain is specific to microglia, SD primarily affects neurons. To investigate how a microglial gene is involved in neuronal homeostasis, here we show that β-hexosaminidase is secreted by microglia and integrated into the lysosomal compartment of neurons. To assess therapeutic relevance, we treat the Hexb-/- SD mouse model with bone marrow transplant and colony stimulating factor 1 receptor inhibition, which broadly replaces Hexb-/- microglia with Hexb-sufficient cells. Microglial replacement reverses apoptotic gene signatures, improves behavior, restores β-hexosaminidase enzymatic activity and Hexb expression, prevents substrate buildup, and normalizes neuronal lysosomal phenotypes, underscoring the critical role of myeloid-derived β-hexosaminidase in maintaining neuronal health and establishing microglial replacement as a potential LSD therapy.

The two predominating subtypes of late-onset GM2 gangliosidosis are late-onset Tay-Sachs (LOTS) and late-onset Sandhoff disease (LOSD). Due to shared deficiencies of ß-hexosamindase A and significant clinical overlap, the two diseases have been considered indistinguishable. However, a growing body of evidence supports the notion of several distinctions between the two diseases. In this study, we highlight these distinctions through the cross-sectional evaluation of 27 late-onset GM2 gangliosidosis participants. Twenty-one participants with LOTS and 6 with LOSD were included in this study. We performed physical examinations alongside assessments for gait, balance, muscle strength, ataxia, nerve conduction velocities, and analyzed brain magnetic resonance imaging. Lower limb weakness (95% in LOTS, 100% in LOSD) and later development of upper limb weakness (90% in LOTS, 83% in LOSD) was highly prevalent in both cohorts. Accompanying gait disturbances, balance issues, and dysmetria (as assessed by the brief ataxia rating scale [BARS]) were also prevalent in both cohorts. Strength testing for the quadriceps and hamstrings demonstrated weakness in both cohorts, primarily impacting extensor muscles. Supratentorial gray and white matter volumes in both cohorts were similar to normative data. In contrast, BARS scores for dysarthria and oculomotor dysfunction were present and heterogenous in LOTS participants and absent in LOSD participants. 24% of LOTS participants and none of the LOSD participants had a history of neuropsychiatric symptoms. Cerebellar volume including lobules V and VI were lower in LOTS compared to LOSD and normative data. However, length dependent sensory neuropathy was present in all LOSD participants but absent in LOTS participants. Dysfunction of the posterior cerebellum (lobules VI, VII, and IX) has been shown to cause cerebellar cognitive affective syndrome (CCAS), that includes cognitive and behavioral disturbances. Furthermore, cerebellar dysfunction of lobules V and VI has been linked to dysarthric speech, and dysfunction of the posterior cerebellum has been linked to oculomotor symptoms. The finding of low cerebellar lobule volumes in LOTS, suggests the distinctive features of the LOTS phenotype are related to cerebellar dysfunction. However, the sensory symptoms unique to LOSD remains a mystery. The molecular and biochemical basis for the dichotomy between the LOTS and LOSD phenotypes requires further investigation.

As tissue-resident macrophages of the central nervous system parenchyma, microglia perform diverse essential functions during homeostasis and perturbations1. They primarily interact with neurons by means of synaptic engulfment and through the rapid elimination of apoptotic cells and non-functional synapses2. Here, by combining unbiased lipidomics and high-resolution spatial lipid imaging, deep single-cell transcriptome analysis and novel cell-type-specific mutants, we identified a previously unknown mode of microglial interaction with neurons. During homeostasis, microglia deliver the lysosomal enzyme β-hexosaminidase to neurons for the degradation of the ganglioside GM2 that is integral to maintaining cell membrane organization and function. Absence of Hexb, encoding the β subunit of β-hexosaminidase, in both mice and patients with neurodegenerative Sandhoff disease leads to a massive accumulation of GM2 derivatives in a characteristic spatiotemporal manner3. In mice, neuronal GM2 gangliosides subsequently engage the macrophage galactose-type lectin 2 receptor on microglia through N-acetylgalactosamine residues, leading to lethal neurodegeneration. Notably, replacement of microglia with peripherally derived microglia-like cells is able to break this degenerative cycle and fully restore central nervous system homeostasis. Our results reveal a mode of bidirectional microglia-neuron communication centred around GM2 ganglioside turnover, identify a microgliopathy and offer therapeutic avenues for these maladies.

Migration of transplanted allogeneic myeloid cells into the brain following systemic haematopoietic stem and progenitor cell transplantation (HCT) holds great promise as a therapeutic modality to correct genetic deficiencies in the brain such as lysosomal storage diseases1-3. However, the toxic myeloablation required for allogeneic HCT can cause serious, life-threatening side effects, limiting its applicability. Moreover, transplanted allogeneic myeloid cells are highly vulnerable to rejection even in an immune-privileged organ like the brain. Here we report a brain-restricted, high-efficiency microglia replacement approach without myeloablative preconditioning. Contrary to previous assumptions, we found that haematopoietic stem cells are not required to repopulate the myeloid compartment of the brain environment, and Sca1- committed progenitor cells were highly efficient in replacing microglia following intracerebral injection. This finding enabled the development of brain-restricted preconditioning and avoided long-term peripheral engraftment, thus eliminating complications such as graft-versus-host disease. Evaluating its therapeutic potential, we found that our allogeneic microglia replacement method rescued the mouse model of Sandhoff disease, a lysosomal storage disease caused by hexosaminidase B deficiency. In support of the translational relevance of this approach, we discovered that human embryonic stem cell-derived myeloid progenitor cells display a similar engraftment potential following brain-restricted conditioning. Our results overcome current limitations of conventional HCT and may pave the way for the development of allogeneic microglial cell therapies for the brain.

Glycosphingolipids (GSL) are important bioactive membrane components. GSLs containing sialic acids, known as gangliosides, are highly abundant in the brain and diseases of ganglioside metabolism cause severe early-onset neurodegeneration. The ganglioside GM2 is processed by β-hexosaminidase A and when non-functional GM2 accumulates causing Tay-Sachs and Sandhoff diseases. We have developed i3Neuron-based disease models demonstrating storage of GM2 and severe endolysosomal dysfunction. Additionally, the plasma membrane (PM) is significantly altered in its lipid and protein composition. These changes are driven in part by lysosomal exocytosis causing inappropriate accumulation of lysosomal proteins on the cell surface. There are also significant changes in synaptic protein abundances with direct functional impact on neuronal activity. Lysosomal proteins are also enriched at the PM in GM1 gangliosidosis supporting that lysosomal exocytosis is a conserved mechanism of PM proteome change in these diseases. This work provides mechanistic insights into neuronal dysfunction in gangliosidoses highlighting that these are severe PM disorders with implications for other lysosomal and neurodegenerative diseases.

Secondary accumulation of lyso-platelet activating factors in lysosomal storage diseases

Galactocerebrosidase (GALC) deficiency causes Krabbe disease, a severe lysosomal neurodegenerative condition. Emerging evidence suggests that heterozygous GALC variants may contribute to multiple sclerosis, attention-deficit hyperactivity disorder, and synucleinopathies. We aim to investigate the potential association between GALC heterozygous variants and neurodegenerative disorders, expanding on existing literature. We screened 110 adults with symptoms shared by lysosomal storage disorders (LSDs) and common neurodegenerative diseases, such as Parkinson's disease, Lewy body dementia, and ataxias of different etiology. We found GALC heterozygosity in this group to be notably enriched, approximately 1 in 28, compared to 1 in 150 in the general population. This led to a focus on 11 individuals with pathogenetic GALC variants and/or the disease-associated polymorphism p.(Arg184Cys). One patient, compound heterozygous for a pathogenetic variant and the p.(Arg184Cys), exhibited reduced GALC activity and a clinical course consistent with late-onset Krabbe disease. In another patient, we found the very rare synonymous variant p.(Leu238Leu) in the GALC gene. Two patients carrying known pathogenetic GALC variants were also heterozygous for other known pathogenetic variants in other LSD-associated genes, including HEXB (Sandhoff disease) and GUSB (mucopolysaccharidosis VI). All the 11 patients in the selected cohort exhibited symptoms similar to atypical Parkinson's disease and a high frequency of leukoencephalopathy, inflammatory disorders, and cancer. Our findings indicate a possible connection between the patients' neurodegenerative conditions and GALC defects, including disease-associated polymorphisms and silent variants. Additional genetic alterations affecting sphingolipid and glycosaminoglycan metabolism may act as contributing factors.

Growing genetic and pathological evidence has identified microglial dysfunction as a key contributor to the pathogenesis and progression of various neurological disorders, positioning microglia replacement as a promising therapeutic strategy. Traditional bone marrow transplantation (BMT) methods for replenishing brain microglia have limitations, including low efficiency and the potential for brain injury because of preconditioning regimens, such as irradiation or chemotherapy. Moreover, BM-derived cells that migrate to the brain do not recapitulate the phenotypic and functional properties of resident microglia. Here, we present a microglia transplantation strategy devoid of any conditioning, termed "tricyclic microglial depletion for transplantation" (TCMDT). This approach leverages three cycles of microglial depletion using the colony stimulating factor 1 receptor (CSF1R) inhibitor PLX3397, creating an optimal window for efficient engraftment of exogenous microglia. Transplantation of primary cultured microglia by TCMDT successfully restored the identity and functions of endogenous microglia. To evaluate the therapeutic potential of TCMDT, we applied this strategy to two distinct mouse models of neurologic disorder. In a Sandhoff disease model, a neurodegenerative lysosomal storage disorder caused by hexosaminidase subunit beta (Hexb) deficiency, TCMDT effectively replaced deficient microglia, attenuating neurodegeneration and improving motor performance. Similarly, in an Alzheimer's disease (AD)-related amyloid mouse model carrying the triggering receptor expressed on myeloid cells 2 (Trem2) R47H mutation, our transplantation strategy rescued microglial dysfunction and mitigated AD-related pathology. Overall, our study introduces TCMDT as a practical, efficient, and safe approach for microglia replacement, suggesting therapeutic potential for treating neurological disorders associated with microglial dysfunction.

GM2 gangliosidosis is lysosomal storage disorder caused by deficiency of the heterodimeric enzyme β-hexosaminidase A. Tay–Sachs disease is caused by variants in HEXA encoding the α-subunit and Sandhoff disease is caused by variants in HEXB encoding the β-subunit. Due to shared clinical and biochemical findings, the two have been considered indistinguishable. We applied T1-weighted volumetric analysis, diffusion tensor imaging (DTI), and correlational fiber tractography to assess phenotypic differences in these two diseases. 51 T1-weighted and 40 DTI scans from 19 Late-Onset GM2 patients with either late-onset Sandhoff disease (LOSD), or late-onset Tay–Sachs (LOTS) were included and compared to 1033 neurotypical control volumetric MRI scans. LOTS patients had significantly smaller cerebellum volume compared to neurotypical controls (p < 0.0001) and LOSD patients (p < 0.0001). There was no statistical difference for the volume of any structure between LOSD and neurotypical controls. DTI analysis showed LOTS patients had higher mean diffusivity (MD) in the left cerebellum (p = 0.003703), right cerebellum (p = 0.003435), superior cerebellar peduncle (p = 0.007332), and vermis (p = 0.01007) compared to LOSD. LOTS patients had lower fractional anisotropy (FA) in the left cerebellum (p = 0.005537), right cerebellum (p = 0.01905), SCP (p = 0.02844), and vermis (p = 0.02469) when compared to LOSD. Correlational fiber tractography identified fiber tracts in cerebellar pathways with higher FA and lower MD in LOSD patients compared to LOTS patients. Our study shows neurobiologic differences between these two related disorders. To our knowledge, this is the first study using correlational tractography in a lysosomal storage disorder. This result indicates a greater burden of cerebellar pathology in LOTS patients compared with LOSD patients.

Late-onset Tay-Sachs (LOTS) disease and late-onset Sandhoff disease (LOSD) have long been considered indistinguishable due to similar clinical presentations and shared biochemical deficits. However, recent magnetic resonance imaging (MRI) studies have shown distinct cerebellar atrophy associated with LOTS. In this study, we furthered this investigation to determine if the cerebellar atrophy is globally uniform or preferentially targets certain cerebellar regions. We utilized DeepCERES, a deep learning cerebellar specific segmentation and cortical thickness pipeline to analyze differences between LOTS (n=20), LOSD (n=5), and neurotypical controls (n=1038). LOTS had smaller volumes of the whole cerebellum as well as cerebellar lobules IV, V, VI, VIIB, VIIIA, VIIIB, IX, and both Crus I and II compared to both LOSD and neurotypical controls. LOTS patients also had smaller cortical thickness of cerebellar lobules V, VI, VIIB, VIIIA, VIIIB, and both Crus I and II compared to both LOSD and neurotypical controls. Cerebellar functional and lesion localization studies have implicated lobules V and VI in speech articulation and execution while lobules VI, Crus I, VIIA, among others, have been implicated in a variety of behaviors and neuropsychiatric symptoms. Our observations provide a possible anatomical substrate to the higher prevalence of dysarthria and psychosis in our LOTS but not LOSD patients. Future studies are needed for direct comparisons considering phenotypic aspects such as age of symptom onset, presence and severity of dysarthria and ataxia, full characterization of neuropsychiatric profiles, molecular pathology and biochemical differences to fully understand the dichotomy observed in these two diseases.