Efficacy of Adeno-Associated Virus Serotype 9-Mediated Gene Therapy for AB-Variant GM2 Gangliosidosis.

GM2 gangliosidoses are a group of neurodegenerative lysosomal storage disorders that are characterized by the accumulation of GM2 gangliosides (GM2), leading to rapid neurological decline and death. The hydrolysis of GM2 requires the specific synthesis, processing, and combination of products of three genes-HEXA, HEXB, and GM2A-within the cell's lysosomes. Mutations in these genes result in Tay-Sachs disease, Sandhoff disease, or AB-variant GM2 gangliosidosis (ABGM2), respectively. ABGM2, the rarest of the three types, is characterized by a mutation in the GM2A gene, which encodes the GM2 activator (GM2A) protein. Being a monogenic disease, gene therapy is a plausible and likely effective method of treatment for ABGM2. This study aimed at assessing the effects of administering a one-time intravenous treatment of single-stranded Adeno-associated virus serotype 9 (ssAAV9)-GM2A viral vector at a dose of 1 × 1014 vector genomes (vg) per kilogram per mouse in an ABGM2 mouse model (Gm2a-/-). ssAAV9-GM2A was administered at 1-day (neonatal) or 6-weeks of age (adult-stage). The results demonstrated that, in comparison to Gm2a-/- mice that received a vehicle injection, the treated mice had reduced GM2 accumulation within the central nervous system and had long-term persistence of vector genomes in the brain and liver. This proof-of-concept study is a step forward towards the development of a clinically therapeutic approach for the treatment of patients with ABGM2.

Life-Limiting Peripheral Organ Dysfunction in Feline Sandhoff Disease Emerges after Effective CNS Gene Therapy.

OBJECTIVE: GM2 gangliosidosis is usually fatal by 5 years of age in its 2 major subtypes, Tay-Sachs and Sandhoff disease. First reported in 1881, GM2 gangliosidosis has no effective treatment today, and children succumb to the disease after a protracted neurodegenerative course and semi-vegetative state. This study seeks to further develop adeno-associated virus (AAV) gene therapy for human translation. METHODS: Cats with Sandhoff disease were treated by intracranial injection of vectors expressing feline ß-N-acetylhexosaminidase, the enzyme deficient in GM2 gangliosidosis. RESULTS: Hexosaminidase activity throughout the brain and spinal cord was above normal after treatment, with highest activities at the injection sites (thalamus and deep cerebellar nuclei). Ganglioside storage was reduced throughout the brain and spinal cord, with near complete clearance in many regions. While untreated cats with Sandhoff disease lived for 4.4 ± 0.6 months, AAV-treated cats lived to 19.1 ± 8.6 months, and 3 of 9 cats lived >21 months. Correction of the central nervous system was so effective that significant increases in lifespan led to the emergence of otherwise subclinical peripheral disease, including megacolon, enlarged stomach and urinary bladder, soft tissue spinal cord compression, and patellar luxation. Throughout the gastrointestinal tract, neurons of the myenteric and submucosal plexuses developed profound pathology, demonstrating that the enteric nervous system was inadequately treated. INTERPRETATION: The vector formulation in the current study effectively treats neuropathology in feline Sandhoff disease, but whole-body targeting will be an important consideration in next-generation approaches. ANN NEUROL 2023;94:969-986.

Biochemical Correction of GM2 Ganglioside Accumulation in AB-Variant GM2 Gangliosidosis.

GM2 gangliosidosis is a group of genetic disorders that result in the accumulation of GM2 ganglioside (GM2) in brain cells, leading to progressive central nervous system (CNS) atrophy and premature death in patients. AB-variant GM2 gangliosidosis (ABGM2) arises from loss-of-function mutations in the GM2 activator protein (GM2AP), which is essential for the breakdown of GM2 in a key catabolic pathway required for CNS lipid homeostasis. In this study, we show that intrathecal delivery of self-complementary adeno-associated virus serotype-9 (scAAV9) harbouring a functional human GM2A transgene (scAAV9.hGM2A) can prevent GM2 accumulation in in GM2AP-deficient mice (Gm2a-/- mice). Additionally, scAAV9.hGM2A efficiently distributes to all tested regions of the CNS within 14 weeks post-injection and remains detectable for the lifespan of these animals (up to 104 weeks). Remarkably, GM2AP expression from the transgene scales with increasing doses of scAAV9.hGM2A (0.5, 1.0 and 2.0 × 1011 vector genomes (vg) per mouse), and this correlates with dose-dependent correction of GM2 accumulation in the brain. No severe adverse events were observed, and comorbidities in treated mice were comparable to those in disease-free cohorts. Lastly, all doses yielded corrective outcomes. These data indicate that scAAV9.hGM2A treatment is relatively non-toxic and tolerable, and biochemically corrects GM2 accumulation in the CNS-the main cause of morbidity and mortality in patients with ABGM2. Importantly, these results constitute proof-of-principle for treating ABGM2 with scAAV9.hGM2A by means of a single intrathecal administration and establish a foundation for future preclinical research.

Treatment of GM2 Gangliosidosis in Adult Sandhoff Mice Using an Intravenous Self-Complementary Hexosaminidase Vector.

BACKGROUND: GM2 gangliosidosis is a neurodegenerative, lysosomal storage disease caused by the deficiency of ß-hexosaminidase A enzyme (Hex A), an α/ß-subunit heterodimer. A novel variant of the human hexosaminidase α-subunit, coded by HEX M, has previously been shown to form a stable homodimer, Hex M, that hydrolyzes GM2 gangliosides (GM2) in vivo. MATERIALS & METHODS: The current study assessed the efficacy of intravenous (IV) delivery of a self-complementary adeno-associated virus serotype 9 (scAAV9) vector incorporating the HEXM transgene, scAAV9/HEXM, including the outcomes based on the dosages provided to the Sandhoff (SD) mice. Six-week-old SD mice were injected with either 2.5E+12 vector genomes (low dose, LD) or 1.0E+13 vg (high dose, HD). We hypothesized that when examining the dosage comparison for scAAV9/HEXM in adult SD mice, the HD group would have more beneficial outcomes than the LD cohort. Assessments included survival, behavioral outcomes, vector biodistribution, and enzyme activity within the central nervous system. RESULTS: Toxicity was observed in the HD cohort, with 8 of 14 mice dying within one month of the injection. As compared to untreated SD mice, which have typical survival of 16 weeks, the LD cohort and the remaining HD mice had a significant survival benefit with an average/median survival of 40.6/34.5 and 55.9/56.7 weeks, respectively. Significant behavioral, biochemical and molecular benefits were also observed. The second aim of the study was to investigate the effects of IV mannitol infusions on the biodistribution of the LD scAAV9/HEXM vector and the survival of the SD mice. Increases in both the biodistribution of the vector as well as the survival benefit (average/median of 41.6/49.3 weeks) were observed. CONCLUSION: These results demonstrate the potential benefit and critical limitations of the treatment of GM2 gangliosidosis using IV delivered AAV vectors.

L-Arginine Ameliorates Defective Autophagy in GM2 Gangliosidoses by mTOR Modulation.

AIMS: Tay-Sachs and Sandhoff diseases (GM2 gangliosidosis) are autosomal recessive disorders of lysosomal function that cause progressive neurodegeneration in infants and young children. Impaired hydrolysis catalysed by ß-hexosaminidase A (HexA) leads to the accumulation of GM2 ganglioside in neuronal lysosomes. Despite the storage phenotype, the role of autophagy and its regulation by mTOR has yet to be explored in the neuropathogenesis. Accordingly, we investigated the effects on autophagy and lysosomal integrity using skin fibroblasts obtained from patients with Tay-Sachs and Sandhoff diseases. RESULTS: Pathological autophagosomes with impaired autophagic flux, an abnormality confirmed by electron microscopy and biochemical studies revealing the accelerated release of mature cathepsins and HexA into the cytosol, indicating increased lysosomal permeability. GM2 fibroblasts showed diminished mTOR signalling with reduced basal mTOR activity. Accordingly, provision of a positive nutrient signal by L-arginine supplementation partially restored mTOR activity and ameliorated the cytopathological abnormalities. INNOVATION: Our data provide a novel molecular mechanism underlying GM2 gangliosidosis. Impaired autophagy caused by insufficient lysosomal function might represent a new therapeutic target for these diseases. CONCLUSIONS: We contend that the expression of autophagy/lysosome/mTOR-associated molecules may prove useful peripheral biomarkers for facile monitoring of treatment of GM2 gangliosidosis and neurodegenerative disorders that affect the lysosomal function and disrupt autophagy.

GM2 Gangliosidoses: Clinical Features, Pathophysiological Aspects, and Current Therapies.

GM2 gangliosidoses are a group of pathologies characterized by GM2 ganglioside accumulation into the lysosome due to mutations on the genes encoding for the ß-hexosaminidases subunits or the GM2 activator protein. Three GM2 gangliosidoses have been described: Tay-Sachs disease, Sandhoff disease, and the AB variant. Central nervous system dysfunction is the main characteristic of GM2 gangliosidoses patients that include neurodevelopment alterations, neuroinflammation, and neuronal apoptosis. Currently, there is not approved therapy for GM2 gangliosidoses, but different therapeutic strategies have been studied including hematopoietic stem cell transplantation, enzyme replacement therapy, substrate reduction therapy, pharmacological chaperones, and gene therapy. The blood-brain barrier represents a challenge for the development of therapeutic agents for these disorders. In this sense, alternative routes of administration (e.g., intrathecal or intracerebroventricular) have been evaluated, as well as the design of fusion peptides that allow the protein transport from the brain capillaries to the central nervous system. In this review, we outline the current knowledge about clinical and physiopathological findings of GM2 gangliosidoses, as well as the ongoing proposals to overcome some limitations of the traditional alternatives by using novel strategies such as molecular Trojan horses or advanced tools of genome editing.

Novel bicistronic lentiviral vectors correct ß-Hexosaminidase deficiency in neural and hematopoietic stem cells and progeny: implications for in vivo and ex vivo gene therapy of GM2 gangliosidosis.

The favorable outcome of in vivo and ex vivo gene therapy approaches in several Lysosomal Storage Diseases suggests that these treatment strategies might equally benefit GM2 gangliosidosis. Tay-Sachs and Sandhoff disease (the main forms of GM2 gangliosidosis) result from mutations in either the HEXA or HEXB genes encoding, respectively, the α- or ß-subunits of the lysosomal ß-Hexosaminidase enzyme. In physiological conditions, α- and ß-subunits combine to generate ß-Hexosaminidase A (HexA, αß) and ß-Hexosaminidase B (HexB, ßß). A major impairment to establishing in vivo or ex vivo gene therapy for GM2 gangliosidosis is the need to synthesize the α- and ß-subunits at high levels and with the correct stoichiometric ratio, and to safely deliver the therapeutic products to all affected tissues/organs. Here, we report the generation and in vitro validation of novel bicistronic lentiviral vectors (LVs) encoding for both the murine and human codon optimized Hexa and Hexb genes. We show that these LVs drive the safe and coordinate expression of the α- and ß-subunits, leading to supranormal levels of ß-Hexosaminidase activity with prevalent formation of a functional HexA in SD murine neurons and glia, murine bone marrow-derived hematopoietic stem/progenitor cells (HSPCs), and human SD fibroblasts. The restoration/overexpression of ß-Hexosaminidase leads to the reduction of intracellular GM2 ganglioside storage in transduced and in cross-corrected SD murine neural progeny, indicating that the transgenic enzyme is secreted and functional. Importantly, bicistronic LVs safely and efficiently transduce human neurons/glia and CD34+ HSPCs, which are target and effector cells, respectively, in prospective in vivo and ex vivo GT approaches. We anticipate that these bicistronic LVs may overcome the current requirement of two vectors co-delivering the α- or ß-subunits genes. Careful assessment of the safety and therapeutic potential of these bicistronic LVs in the SD murine model will pave the way to the clinical development of LV-based gene therapy for GM2 gangliosidosis.

Neurite atrophy and apoptosis mediated by PERK signaling after accumulation of GM2-ganglioside.

GM2-gangliosidosis, a subgroup of lysosomal storage disorders, is caused by deficiency of hexosaminidase activity, and comprises the closely related Tay-Sachs and Sandhoff diseases. The enzyme deficiency prevents normal metabolization of ganglioside GM2, usually resulting in progressive neurodegenerative disease. The molecular mechanisms whereby GM2 accumulation in neurons triggers neurodegeneration remain unclear. In vitro experiments, using microsomes from Sandhoff mouse model brain, showed that increase of GM2 content negatively modulates sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) (Pelled et al., 2003). Furthermore, Ca2+ depletion in endoplasmic reticulum (ER) triggers Unfolded Protein Response (UPR), which tends to restore homeostasis in the ER; however, if cellular damage persists, an apoptotic response is initiated. We found that ER GM2 accumulation in cultured neurons induces luminal Ca2+ depletion, which in turn activates PERK (protein kinase RNA [PKR]-like ER kinase), one of three UPR sensors. PERK signaling displayed biphasic activation; i.e., early upregulation of cytoprotective calcineurin (CN) and, under prolonged ER stress, enhanced expression of pro-apoptotic transcription factor C/EBP homologous protein (CHOP). Moreover, GM2 accumulation in neuronal cells induced neurite atrophy and apoptosis. Both processes were effectively modulated by treatment with the selective PERK inhibitor GSK2606414, by CN knockdown, and by CHOP knockdown. Overall, our findings demonstrate the essential role of PERK signaling pathway contributing to neurodegeneration in a model of GM2-gangliosidosis.

Direct Intracranial Injection of AAVrh8 Encoding Monkey ß-N-Acetylhexosaminidase Causes Neurotoxicity in the Primate Brain.

GM2 gangliosidoses, including Tay-Sachs disease and Sandhoff disease, are lysosomal storage disorders caused by deficiencies in ß-N-acetylhexosaminidase (Hex). Patients are afflicted primarily with progressive central nervous system (CNS) dysfunction. Studies in mice, cats, and sheep have indicated safety and widespread distribution of Hex in the CNS after intracranial vector infusion of AAVrh8 vectors encoding species-specific Hex α- or ß-subunits at a 1:1 ratio. Here, a safety study was conducted in cynomolgus macaques (cm), modeling previous animal studies, with bilateral infusion in the thalamus as well as in left lateral ventricle of AAVrh8 vectors encoding cm Hex α- and ß-subunits. Three doses (3.2 × 1012 vg [n = 3]; 3.2 × 1011 vg [n = 2]; or 1.1 × 1011 vg [n = 2]) were tested, with controls infused with vehicle (n = 1) or transgene empty AAVrh8 vector at the highest dose (n = 2). Most monkeys receiving AAVrh8-cmHexα/ß developed dyskinesias, ataxia, and loss of dexterity, with higher dose animals eventually becoming apathetic. Time to onset of symptoms was dose dependent, with the highest-dose cohort producing symptoms within a month of infusion. One monkey in the lowest-dose cohort was behaviorally asymptomatic but had magnetic resonance imaging abnormalities in the thalami. Histopathology was similar in all monkeys injected with AAVrh8-cmHexα/ß, showing severe white and gray matter necrosis along the injection track, reactive vasculature, and the presence of neurons with granular eosinophilic material. Lesions were minimal to absent in both control cohorts. Despite cellular loss, a dramatic increase in Hex activity was measured in the thalamus, and none of the animals presented with antibody titers against Hex. The high overexpression of Hex protein is likely to blame for this negative outcome, and this study demonstrates the variations in safety profiles of AAVrh8-Hexα/ß intracranial injection among different species, despite encoding for self-proteins.

Adult-onset Krabbe disease presenting with an isolated form of peripheral neuropathy.

INTRODUCTION: Adult-onset Krabbe disease is clinically rare and usually affects the pyramidal tracts in the central nervous system. Patients develop a spastic gait, and peripheral neuropathy sometimes occurs simultaneously. METHODS: A 55-year-old woman with consanguineous parents developed slowly progressive, asymmetric muscle weakness and atrophy in her forearms, while her ability to walk remained unaffected without pyramidal tract signs after onset at age 51 years. RESULTS: Nerve conduction studies demonstrated an asymmetric demyelinating-type peripheral neuropathy, and sural nerve biopsy documented reduced myelinated nerve fiber density with uniformly thin myelin sheaths, suggesting hypomyelination. Brain MRI demonstrated minor white-matter injury along the optic radiations, which was associated with asymptomatic, mild, prolonged latency on visual evoked potentials. Laboratory analysis documented low enzyme activity of galactocerebrosidase (GALC) and a known mutation of the GALC gene. CONCLUSION: Isolated peripheral neuropathy occurs very rarely in adult-onset Krabbe disease. Muscle Nerve 54: 152-157, 2016.

Polycystic kidneys and GM2 gangliosidosis-like disease in neonatal springboks (Antidorcas marsupialis).

Clinical, gross, histopathologic, electron microscopic findings and enzymatic analysis of 4 captive, juvenile springboks (Antidorcas marsupialis) showing both polycystic kidneys and a storage disease are described. Springbok offspring (4 of 34; 12%) were affected by either one or both disorders in a German zoo within a period of 5 years (2008-2013). Macroscopic findings included bilaterally severely enlarged kidneys displaying numerous cysts in 4 animals and superior brachygnathism in 2 animals. Histopathologically, kidneys of 4 animals displayed cystic dilation of the renal tubules. In addition, abundant cytoplasmic vacuoles with a diameter ranging from 2 to 10 µm in neurons of the central and peripheral nervous system, hepatocytes, thyroid follicular epithelial cells, pancreatic islets of Langerhans and renal tubular cells were found in 2 springbok neonates indicative of an additional storage disease. Ultrastructurally, round electron-lucent vacuoles, up to 4 µm in diameter, were present in neurons. Enzymatic analysis of liver and kidney tissue of 1 affected springbok revealed a reduced activity of total hexosaminidase (Hex) with relatively increased HexA activity at the same level of total Hex, suggesting a hexosaminidase defect. Pedigree analysis suggested a monogenic autosomal recessive inheritance for both diseases. In summary, related springboks showed 2 different changes resembling both polycystic kidney and a GM2 gangliosidosis similar to the human Sandhoff disease. Whether the simultaneous occurrence of these 2 entities represents an incidental finding or has a genetic link needs to be investigated in future studies.

Accelerated tumor growth mediated by sublytic levels of antibody-induced complement activation is associated with activation of the PI3K/AKT survival pathway.

PURPOSE: We addressed the possibility that low levels of tumor cell-bound antibodies targeting gangliosides might accelerate tumor growth. EXPERIMENTAL DESIGN: To test this hypothesis, we treated mice with a range of monoclonal antibody (mAb) doses against GM2, GD2, GD3, and CD20 after challenge with tumors expressing these antigens and tested the activity of the same mAbs in vitro. We also explored the mechanisms behind the complement-mediated tumor growth acceleration that we observed and an approach to overcome it. RESULTS: Serologically detectable levels of IgM-mAb against GM2 are able to delay or prevent tumor growth of high GM2 expressing cell lines both in vitro and in a SCID mouse model, whereas very low levels of this mAb resulted in slight but consistent acceleration of tumor growth in both settings. Surprisingly, this is not restricted to IgM mAb targeting GM2 but consistent against an IgG mAb targeting GD3 as well. These findings were mirrored by in vitro studies with antibodies against these antigens as well as GD2 and CD20 (with Rituxan), and shown to be complement-dependent in all cases. Complement-mediated accelerated growth of cultured tumor cell lines initiated by low mAb levels was associated with activation of the phosphoinositide 3-kinase (PI3K)/AKT survival pathway and significantly elevated levels of both p-AKT and p-PRAS40. This complement-mediated PI3K activation and accelerated tumor growth in vitro and in vivo are eliminated by PI3K inhibitors NVP-BEZ235 and Wortmannin. These PI3K inhibitors also significantly increased efficacy of high doses of these four mAbs. CONCLUSION: Our findings suggest that manipulation of the PI3K/AKT pathway and its signaling network can significantly increase the potency of passively administered mAbs and vaccine-induced antibodies targeting a variety of tumor cell surface antigens.

Gene transfer corrects acute GM2 gangliosidosis--potential therapeutic contribution of perivascular enzyme flow.

The GM2 gangliosidoses are fatal lysosomal storage diseases principally affecting the brain. Absence of ß-hexosaminidase A and B activities in the Sandhoff mouse causes neurological dysfunction and recapitulates the acute Tay-Sachs (TSD) and Sandhoff diseases (SD) in infants. Intracranial coinjection of recombinant adeno-associated viral vectors (rAAV), serotype 2/1, expressing human ß-hexosaminidase α (HEXA) and ß (HEXB) subunits into 1-month-old Sandhoff mice gave unprecedented survival to 2 years and prevented disease throughout the brain and spinal cord. Classical manifestations of disease, including spasticity-as opposed to tremor-ataxia-were resolved by localized gene transfer to the striatum or cerebellum, respectively. Abundant biosynthesis of ß-hexosaminidase isozymes and their global distribution via axonal, perivascular, and cerebrospinal fluid (CSF) spaces, as well as diffusion, account for the sustained phenotypic rescue-long-term protein expression by transduced brain parenchyma, choroid plexus epithelium, and dorsal root ganglia neurons supplies the corrective enzyme. Prolonged survival permitted expression of cryptic disease in organs not accessed by intracranial vector delivery. We contend that infusion of rAAV into CSF space and intraparenchymal administration by convection-enhanced delivery at a few strategic sites will optimally treat neurodegeneration in many diseases affecting the nervous system.

Natural history of infantile G(M2) gangliosidosis.

OBJECTIVE: G(M2) gangliosidoses are caused by an inherited deficiency of lysosomal ß-hexosaminidase and result in ganglioside accumulation in the brain. Onset during infancy leads to rapid neurodegeneration and death before 4 years of age. We set out to quantify the rate of functional decline in infantile G(M2) gangliosidosis on the basis of patient surveys and a comprehensive review of existing literature. METHODS: Patients with infantile G(M2) gangliosidosis (N = 237) were surveyed via questionnaire by the National Tay Sachs & Allied Diseases Association (NTSAD). These data were supplemented by survival data from the NTSAD database and a literature survey. Detailed retrospective surveys from 97 patients were available. Five patients who had received hematopoietic stem cell transplantation were evaluated separately. The mortality rate of the remaining 92 patients was comparable to that of the 103 patients from the NTSAD database and 121 patients reported in the literature. RESULTS: Common symptoms at onset were developmental arrest (83%), startling (65%), and hypotonia (60%). All 55 patients who had learned to sit without support lost that ability within 1 year. Individual functional measures correlated with each other but not with survival. Gastric tube placement was associated with prolonged survival. Tay Sachs and Sandhoff variants did not differ. Hematopoietic stem cell transplantation was not associated with prolonged survival. CONCLUSIONS: We studied the timing of regression in 97 cases of infantile G(M2) gangliosidosis and conclude that clinical disease progression does not correlate with survival, likely because of the impact of improved supportive care over time. However, functional measures are quantifiable and can inform power calculations and study design of future interventions.

Thymic alterations in GM2 gangliosidoses model mice.

BACKGROUND: Sandhoff disease is a lysosomal storage disorder characterized by the absence of ß-hexosaminidase and storage of GM2 ganglioside and related glycolipids. We have previously found that the progressive neurologic disease induced in Hexb(-/-) mice, an animal model for Sandhoff disease, is associated with the production of pathogenic anti-glycolipid autoantibodies. METHODOLOGY/PRINCIPAL FINDINGS: In our current study, we report on the alterations in the thymus during the development of mild to severe progressive neurologic disease. The thymus from Hexb(-/-) mice of greater than 15 weeks of age showed a marked decrease in the percentage of immature CD4(+)/CD8(+) T cells and a significantly increased number of CD4(+)/CD8(-) T cells. During involution, the levels of both apoptotic thymic cells and IgG deposits to T cells were found to have increased, whilst swollen macrophages were prominently observed, particularly in the cortex. We employed cDNA microarray analysis to monitor gene expression during the involution process and found that genes associated with the immune responses were upregulated, particularly those expressed in macrophages. CXCL13 was one of these upregulated genes and is expressed specifically in the thymus. B1 cells were also found to have increased in the thy mus. It is significant that these alterations in the thymus were reduced in FcRγ additionally disrupted Hexb(-/-) mice. CONCLUSIONS/SIGNIFICANCE: These results suggest that the FcRγ chain may render the usually poorly immunogenic thymus into an organ prone to autoimmune responses, including the chemotaxis of B1 cells toward CXCL13.

Elevated levels of select gangliosides in T cells from renal cell carcinoma patients is associated with T cell dysfunction.

Increased expression of gangliosides by different tumor types including renal cell carcinoma (RCC) is thought to contribute to the immune suppression observed in cancer patients. In this study, we report an increase in apoptotic T cells from RCC patients compared with T cells from normal donors that coincided with the detection of T cells staining positive for GM2 and that the apoptosis was predominantly observed in the GM2(+) but not the GM2(-) T cell population. Ganglioside shedding from tumor rather than endogenous production accounts for GM2(+) T cells since there was no detectable level of mRNA for GM2 synthase in RCC patient T cells and in T cells from normal healthy donors after incubation with either purified GM2 or supernatant from RCC cell lines despite their staining positive for GM2. Moreover, reactive oxygen species as well as activated caspase 3, 8, and 9 were predominantly elevated in GM2(+) but not GM2(-) T cells. Similarly, increased staining for GD2 and GD3 but not GD1a was detected with patient T cells with elevated levels of apoptosis in the GD2(+) and GD3(+) cells. These findings suggest that GM2, GD2, and GD3 play a significant role in immune dysfunction observed in RCC patient T cells.

Critical role of iron in the pathogenesis of the murine gangliosidoses.

Neurodegeneration is a prominent feature of the gangliosidoses, a group of lysosomal storage diseases. Here we show altered iron homeostasis in mouse models of both GM1 and GM2 gangliosidoses, which are characterized by progressive depletion of iron in brain tissue. This finding contrasts with the findings in many other neurological disorders, where excess iron deposition has been reported. We found that key regulators of iron homeostasis, hepcidin and IL-6, were increased in gangliosidoses mice. In the brain, the principal iron transport and delivery protein transferrin was reduced, accompanied by a progressive inability of the brain to acquire iron from the circulation. Expression of the transferrin receptor was up-regulated reciprocally. Despite the deregulation of iron homeostasis administration of iron prolonged survival in the diseased mice by up to 38%, with onset of disease delayed and motor function preserved.

Glycosphingolipid disorders of the brain.

Glycosphingolipids, comprising a ceramide lipid backbone linked to one/more saccharides, are particularly abundant on the outer leaflet of the eukaryotic plasma membrane and play a role in a wide variety of essential cellular processes. Biosynthesis and subsequently degradation of these lipids is tightly regulated via the involvement of numerous enzymes, and failure of an enzyme to participate in the metabolism results in storage of the enzyme's substrate, giving rise to a lysosomal storage disease. The characteristics, severity and onset of the disease are dependent on the enzyme deficient and the residual activity. Most lysosomal storage disorders found thus far are caused by a defect in the catabolic activity of a hydrolase, causing progressive accumulation of its substrate, predominantly in the lysosome. Storage of gangliosides, sialic acid containing glycosphingolipids, mostly found in the central nervous system, is a hallmark of neuronopathic forms of the disease, that include GM1 and GM2 gangliosidoses, Gaucher type II and III and Niemann-Pick C. Models for these diseases have provided valuable insight into the disease pathology and potential treatment methods.Treatment of these rare but severe disorders proves challenging due to restricted access of therapeutics through the blood-brain barrier. However, recent advances in enzyme replacement, bone marrow transplantation, gene transfer, substrate reduction and chaperon-mediated therapy provide great potential in treating these devastating disorders.

Establishment of subcellular fractionation techniques to monitor the intracellular fate of polymer therapeutics II. Identification of endosomal and lysosomal compartments in HepG2 cells combining single-step subcellular fractionation with fluorescent imaging.

As they are often designed for lysosomotropic, endosomotropic and/or transcellular delivery, an understanding of intracellular trafficking pathways is essential to enable optimised design of novel polymer therapeutics. Here, we describe a single-step density gradient subcellular fractionation method combined with fluorescent detection analysis that provides a new tool for characterisation of endocytic traffic of polymer therapeutics. Hepatoma (HepG2) cells were used as a model and cell breakage was optimised using a cell cracker to ensure assay of the whole cell population. After removal of unbroken cells and nuclei, the cell lysate as a post-nuclear supernatant (PNS) was layered onto an iodixanol (OptiPrep) density gradient optimised to 5-20%. Early endosomes, late endosomes and lysosomes were identified from gradient fractions by immunoblotting for marker proteins early endosome antigen 1 (EEA 1) and lysosomal associated membrane protein 1 (LAMP 1) using horseradish peroxidase or fluorescently-labelled secondary antibodies. Lysosomes were also detected using N-acetyl-beta-glucosamindase (Hex A) activity. In addition, cells were incubated with Texas-red labelled transferrin (TxR-Tf) for 5 min to specifically label early endosomes and this was directly detected from SDS-PAGE gels. Internalised macromolecules and colloidal particles can potentially alter vesicle buoyant density. To see if typical macromolecules of interest would alter vesicle density or perturb vesicle traffic, HepG2 cells were incubated with dextran or a polyethyleneglycol (PEG)-polyester dendron G4 (1 mg/ml for 24 h). The PEG-polyester dendron G4 caused a slight redistribution of endocytic structures to lower density fractions but immunofluorescence microscopy showed no obvious dendron effects. In conclusion, the combined subcellular fractionation with fluorescent imaging approach described here can be used as a tool for both fundamental cell biology research and/or the quantitative localisation of polymer therapeutics in the endocytic pathway.

Clinical and molecular analysis of GM2 gangliosidosis in two apparent littermate kittens of the Japanese domestic cat.

This case report documents clinical and molecular findings in two littermate kittens of the Japanese domestic cat with GM2 gangliosidosis variant 0. Analysis included detailed physical, magnetic resonance imaging, biochemical, pathological and genetic examinations. At first, these littermate kittens showed typical cerebellar signs at approximately 2 months of age. About 2 months later, they progressively showed other neurological signs and subsequently died at about 7 months of age. Magnetic resonance imaging just before the death showed an enlarged ventricular system, T1 hyperintensity in the internal capsule, and T2 hyperintensity in the white matter of the whole brain. Histological findings suggested a type of lysosomal storage disease. Biochemical studies demonstrated that the kittens were affected with GM2 gangliosidosis variant 0, and a DNA assay finally demonstrated that these animals were homozygous for the mutation, which the authors had identified in a different family of the Japanese domestic cat. The findings in the present cases provide useful information about GM2 gangliosidosis variant 0 in Japanese domestic cats.