Creation of an in vitro model of GM1 gangliosidosis by CRISPR/Cas9 knocking-out the GLB1 gene in SH-SY5Y human neuronal cell line.

GM1 gangliosidosis is one type of hereditary error of metabolism that occurs due to the absence or reduction of ß-galactosidase enzyme content in the lysosome of cells, including neurons. In vitro, the use of neural cell lines could facilitate the study of this disease. By creating a cell model of GM1 gangliosidosis on the SH-SY5Y human nerve cell line, it is possible to understand the main role of this enzyme in breaking down lipid substrate and other pathophysiologic phenomena this disease. To knock-out the human GLB1 gene, guides targeting exons 14 and 16 of the GLB1 gene were designed using the CRISPOR and CHOP-CHOP websites, and high-efficiency guides were selected for cloning in the PX458 vector. After confirming the cloning, the vectors were transformed into DH5α bacteria and then the target vector was extracted and transfected into human nerve cells (SH-SY5Y cell line) by electroporation. After 48 h, GFP+ cells were sorted using the FACS technique and homozygous (compound heterozygous) single cells were isolated using the serial dilution method and sequencing was done to confirm them. Finally, gap PCR tests, X-gal and Periodic acid-Schiff (PAS) staining, and qPCR were used to confirm the knock-out of the human GLB1 gene. Additionally, RNA sequencing data analysis from existing data of the Gene Expression Omnibus (GEO) was used to find the correlation of GLB1 with other genes, and then the top correlated genes were tested for further evaluation of knock-out effects. The nonviral introduction of two guides targeting exons 14 and 16 of the GLB1 gene into SH-SY5Y cells led to the deletion of a large fragment with a size of 4.62 kb. In contrast to the non-transfected cell, X-gal staining resulted in no blue color in GLB1 gene knock-out cells indicating the absence of ß-galactosidase enzyme activity in these cells. Real-time PCR (qPCR) results confirmed the RNA-Seq analysis outcomes on the GEO data set and following the GLB1 gene knock-out, the expression of its downstream genes, NEU1 and CTSA, has been decreased. It has been also shown that the downregulation of GLB1-NEU1-CTSA complex gene was involved in suppressed proliferation and invasion ability of knock-out cells. This study proved that using dual guide RNA can be used as a simple and efficient tool for targeting the GLB1 gene in nerve cells and the knockout SH-SY5Y cells can be used as a model investigation of basic and therapeutic surveys for GM1 gangliosidosis disease.

Insights into the Pathobiology of GM1 Gangliosidosis from Single-Nucleus Transcriptomic Analysis of CNS Cells in a Mouse Model.

GM1 gangliosidosis is a lysosomal storage disorder characterized by the accumulation of GM1 ganglioside, leading to severe neurodegeneration and early mortality. The disease primarily affects the central nervous system, causing progressive neurodegeneration, including widespread neuronal loss and gliosis. To gain a deeper understanding of the neuropathology associated with GM1 gangliosidosis, we employed single-nucleus RNA sequencing to analyze brain tissues from both GM1 gangliosidosis model mice and control mice. No significant changes in cell proportions were detected between the two groups of animals. Differential expression analysis revealed cell type-specific changes in gene expression in neuronal and glial cells. Functional analysis highlighted the neurodegenerative processes, oxidative phosphorylation, and neuroactive ligand-receptor interactions as the significantly affected pathways. The contribution of the impairment of neurotransmitter system disruption and neuronal circuitry disruption was more important than neuroinflammatory responses to GM1 pathology. In 16-week-old GM1 gangliosidosis mice, no microglial or astrocyte activation or increased expression of innate immunity genes was detected. This suggested that nerve degeneration did not induce the inflammatory response but rather promoted glial cell clearance. Our findings provide a crucial foundation for understanding the cellular and molecular mechanisms of GM1 gangliosidosis, potentially guiding future therapeutic strategies.

Intracerebroventricular enzyme replacement therapy with ß-galactosidase reverses brain pathologies due to GM1 gangliosidosis in mice.

Autosomal recessive mutations in the galactosidase ß1 (GLB1) gene cause lysosomal ß-gal deficiency, resulting in accumulation of galactose-containing substrates and onset of the progressive and fatal neurodegenerative lysosomal storage disease, GM1 gangliosidosis. Here, an enzyme replacement therapy (ERT) approach in fibroblasts from GM1 gangliosidosis patients with recombinant human ß-gal (rhß-gal) produced in Chinese hamster ovary cells enabled direct and precise rhß-gal delivery to acidified lysosomes. A single, low dose (3 nm) of rhß-gal was sufficient for normalizing ß-gal activity and mediating substrate clearance for several weeks. We found that rhß-gal uptake by the fibroblasts is dose-dependent and saturable and can be competitively inhibited by mannose 6-phosphate, suggesting cation-independent, mannose 6-phosphate receptor-mediated endocytosis from the cell surface. A single intracerebroventricularly (ICV) administered dose of rhß-gal (100 µg) resulted in broad bilateral biodistribution of rhß-gal to critical regions of pathology in a mouse model of GM1 gangliosidosis. Weekly ICV dosing of rhß-gal for 8 weeks substantially reduced brain levels of ganglioside and oligosaccharide substrates and reversed well-established secondary neuropathology. Of note, unlike with the ERT approach, chronic lentivirus-mediated GLB1 overexpression in the GM1 gangliosidosis patient fibroblasts caused accumulation of a prelysosomal pool of ß-gal, resulting in activation of the unfolded protein response and endoplasmic reticulum stress. This outcome was unsurprising in light of our in vitro biophysical findings for rhß-gal, which include pH-dependent and concentration-dependent stability and dynamic self-association. Collectively, our results highlight that ICV-ERT is an effective therapeutic intervention for managing GM1 gangliosidosis potentially more safely than with gene therapy approaches.

A computational approach to analyse the amino acid variants of GLB1 protein causing GM1 Gangliosidosis.

Lysosomal storage diseases comprise different forms of autosomal recessive disorders from which GM1 gangliosidosis has categorized by the accumulation of complex glycolipids associated with a range of progressive neurologic phenotypes. GM1 gangliosidosis is an inherited disorder that progressively destroys nerve cells (neurons) in the brain and spinal cord. GM1 has three main types of onsets, namely infantile (type I), juvenile (type II), and adult (type III) forms. This study provides a series of computational methods that examine the mutations that occurred in GLB1 protein. Initially, the mutational analysis started with 689 amino acid variants for a sequence-based screening and it was done with quite a few In-silico tools to narrow down the most significant variants by utilizing the standard tools; namely, Evolutionary analysis (77 variants), Pathogenicity prediction (44 variants), Stability predictions (30 variants), Biophysical functions (19 variants) and according to the binding site of protein structure with PDB ID 3THC, seven variants (Y83D, Y83H, Y270S, Y270D, W273R, W273D, and Y333H) were narrowed down. Structure based analysis was performed to understand the interacting profile of the native protein and variants with Miglustat; which is the currently used FDA drug as an alternative to enzyme replacement therapy. Molecular Docking study was done to analyze the protein interaction with Miglustat (ligand), as a result native (3THC) structure had a binding affinity of -8.18 kcal/mol and two variant structures had an average binding affinities of -2.61 kcal/mol (Y83D) and - 7.63 kcal/mol (Y270D). Finally, Molecular Dynamics Simulation was performed to know the mutational activity of the protein structures on Miglustat for 50,000 ps. The Y83D variant showed higher deviation than native protein and Y270D in all trajectory analysis. The analysis was done to the protein structures to check the structural variations happened through simulations. This study aids to understand the most deleterious mutants, the activity of the drug to the protein structure and also gives an insight on the stability of the drug with the native and selected variants.

The pharmacological chaperone N-n-butyl-deoxygalactonojirimycin enhances ß-galactosidase processing and activity in fibroblasts of a patient with infantile GM1-gangliosidosis.

GM1-gangliosidosis, a lysosomal storage disorder, is associated with ~ 161 missense variants in the GLB1 gene. Affected patients present with ß-galactosidase (ß-Gal) deficiency in lysosomes. Loss of function in ER-retained misfolded enzymes with missense variants is often due to subcellular mislocalization. Deoxygalactonojirimycin (DGJ) and its derivatives are pharmaceutical chaperones that directly bind to mutated ß-Gal in the ER promoting its folding and trafficking to lysosomes and thus enhancing its activity. An Emirati child has been diagnosed with infantile GM1-gangliosidosis carrying the reported p.D151Y variant. We show that p.D151Y ß-Gal in patient's fibroblasts retained < 1% residual activity due to impaired processing and trafficking. The amino acid substitution significantly affected the enzyme conformation; however, p.D151Y ß-Gal was amenable for partial rescue in the presence of glycerol or at reduced temperature where activity was enhanced with ~ 2.3 and 7 folds, respectively. The butyl (NB-DGJ) and nonyl (NN-DGJ) derivatives of DGJ chaperoning function were evaluated by measuring their IC50s and ability to stabilize the wild-type ß-Gal against thermal degradation. Although NN-DGJ showed higher affinity to ß-Gal, it did not show a significant enhancement in p.D151Y ß-Gal activity. However, NB-DGJ promoted p.D151Y ß-Gal maturation and enhanced its activity up to ~ 4.5% of control activity within 24 h which was significantly increased to ~ 10% within 6 days. NB-DGJ enhancement effect was sustained over 3 days after washing it out from culture media. We therefore conclude that NB-DGJ might be a promising therapeutic chemical chaperone in infantile GM1 amenable variants and therefore warrants further analysis for its clinical applications.

Mass spectrometry imaging of lipids: untargeted consensus spectra reveal spatial distributions in Niemann-Pick disease type C1.

Mass spectrometry imaging (MSI) is a tool to rapidly map the spatial location of analytes without the need for tagging or a reporter system. Niemann-Pick disease type C1 (NPC1) is a neurodegenerative, lysosomal storage disorder characterized by accumulation of unesterified cholesterol and sphingolipids in the endo-lysosomal system. Here, we use MSI to visualize lipids including cholesterol in cerebellar brain tissue from the NPC1 symptomatic mouse model and unaffected controls. To complement the imaging studies, a data-processing pipeline was developed to generate consensus mass spectra, thereby using both technical and biological image replicates to assess differences. The consensus spectra are used to determine true differences in lipid relative abundance; lipid distributions can be determined in an unbiased fashion without prior knowledge of location. We show the cerebellar distribution of gangliosides GM1, GM2, and GM3, including variants of lipid chain length. We also performed MALDI-MSI of cholesterol. Further analysis of lobules IV/V and X of the cerebellum gangliosides indicates regional differences. The specificity achieved highlights the power of MSI, and this new workflow demonstrates a universal approach for addressing reproducibility in imaging experiments applied to NPC1.

4-epi-Isofagomine derivatives as pharmacological chaperones for the treatment of lysosomal diseases linked to ß-galactosidase mutations: Improved synthesis and biological investigations.

(5aR)-5a-C-pentyl-4-epi-isofagomine 1 is a powerful inhibitor of lysosomal ß-galactosidase and a remarkable chaperone for mutations associated with GM1-gangliosidosis and Morquio disease type B. We report herein an improved synthesis of this compound and analogs (5a-C-methyl, pentyl, nonyl and phenylethyl derivatives), and a crystal structure of a synthetic intermediate that confirms its configuration resulting from the addition of a Grignard reagent. These compounds were evaluated as glycosidase inhibitors and their potential as chaperones for mutant lysosomal galactosidases determined. Based on these results and on docking studies, the 5-C-pentyl derivative 1 was selected as the optimal structure for further investigations: this compound induces the maturation of mutated ß-galactosidase in fibroblasts of a GM1-gangliosidosis patient and promote the decrease of keratan sulfate and oligosaccharide load in patient cells. Compound 1 is clearly capable of restoring ß-galactosidase activity and of promoting maturation of the protein, which should result in significant clinical benefit. These properties strongly support the development of compound 1 for the treatment of GM1-gangliosidosis and Morquio disease type B patients harboring ß-galactosidase mutations sensitive to pharmacological chaperoning.

Novel Biomarkers of Human GM1 Gangliosidosis Reflect the Clinical Efficacy of Gene Therapy in a Feline Model.

GM1 gangliosidosis is a fatal neurodegenerative disease that affects individuals of all ages. Favorable outcomes using adeno-associated viral (AAV) gene therapy in GM1 mice and cats have prompted consideration of human clinical trials, yet there remains a paucity of objective biomarkers to track disease status. We developed a panel of biomarkers using blood, urine, cerebrospinal fluid (CSF), electrodiagnostics, 7 T MRI, and magnetic resonance spectroscopy in GM1 cats-either untreated or AAV treated for more than 5 years-and compared them to markers in human GM1 patients where possible. Significant alterations were noted in CSF and blood of GM1 humans and cats, with partial or full normalization after gene therapy in cats. Gene therapy improved the rhythmic slowing of electroencephalograms (EEGs) in GM1 cats, a phenomenon present also in GM1 patients, but nonetheless the epileptiform activity persisted. After gene therapy, MR-based analyses revealed remarkable preservation of brain architecture and correction of brain metabolites associated with microgliosis, neuroaxonal loss, and demyelination. Therapeutic benefit of AAV gene therapy in GM1 cats, many of which maintain near-normal function >5 years post-treatment, supports the strong consideration of human clinical trials, for which the biomarkers described herein will be essential for outcome assessment.

Systemic AAV9 gene transfer in adult GM1 gangliosidosis mice reduces lysosomal storage in CNS and extends lifespan.

GM1 gangliosidosis (GM1) is an autosomal recessive lysosomal storage disease where GLB1 gene mutations result in a reduction or absence of lysosomal acid ß-galactosidase (ßgal) activity. ßgal deficiency leads to accumulation of GM1-ganglioside in the central nervous system (CNS). GM1 is characterized by progressive neurological decline resulting in generalized paralysis, extreme emaciation and death. In this study, we assessed the therapeutic efficacy of an adeno-associated virus (AAV) 9-mßgal vector infused systemically in adult GM1 mice (ßGal(-/-)) at 1 × 10(11) or 3 × 10(11) vector genomes (vg). Biochemical analysis of AAV9-treated GM1 mice showed high ßGal activity in liver and serum. Moderate ßGal levels throughout CNS resulted in a 36-76% reduction in GM1-ganglioside content in the brain and 75-86% in the spinal cord. Histological analyses of the CNS of animals treated with 3 × 10(11) vg dose revealed increased presence of ßgal and clearance of lysosomal storage throughout cortex, hippocampus, brainstem and spinal cord. Storage reduction in these regions was accompanied by a marked decrease in astrogliosis. AAV9 treatment resulted in improved performance in multiple tests of motor function and behavior. Also the majority of GM1 mice in the 3 × 10(11) vg cohort retained ambulation and rearing despite reaching the humane endpoint due to weight loss. Importantly, the median survival of AAV9 treatment groups (316-576 days) was significantly increased over controls (250-264 days). This study shows that moderate widespread expression of ßgal in the CNS of GM1 gangliosidosis mice is sufficient to achieve significant biochemical impact with phenotypic amelioration and extension in lifespan.

Inhibition of complement in Guillain-Barré syndrome: the ICA-GBS study.

The outcome of Guillain-Barré syndrome (GBS) remains unchanged since plasma exchange and intravenous immunoglobulin (IVIg) were introduced over 20 years ago. Pathogenesis studies on GBS have identified the terminal component of complement cascade as a key disease mediator and therapeutic target. We report the first use of terminal complement pathway inhibition with eculizumab in humans with GBS. In a randomised, double-blind, placebo-controlled trial, 28 subjects eligible on the basis of GBS disability grade of at least 3 were screened, of whom 8 (29%) were randomised. Five received eculizumab for 4 weeks, alongside standard IVIg treatment. The safety outcomes, monitored via adverse events capture, showed eculizumab to be well-tolerated and safe when administered in conjunction with IVIg. Primary and secondary efficacy outcomes in the form of GBS disability scores (GBS DS), MRC sum scores, Rasch overall disability scores, and overall neuropathy limitation scores are reported descriptively. For the primary efficacy outcome at 4 weeks after recruitment, two of two placebo- and two of five eculizumab-treated subjects had improved by one or more grades on the GBS DS. Although the small sample size precludes a statistically meaningful analysis, these pilot data indicate further studies on complement inhibition in GBS are warranted.

GM1-ganglioside accumulation at the mitochondria-associated ER membranes links ER stress to Ca(2+)-dependent mitochondrial apoptosis.

Mitochondria-associated ER membranes, or MAMs, define the sites of endoplasmic reticulum/mitochondria juxtaposition that control Ca(2+) flux between these organelles. We found that in a mouse model of the human lysosomal storage disease GM1-gangliosidosis, GM1-ganglioside accumulates in the glycosphingolipid-enriched microdomain (GEM) fractions of MAMs, where it interacts with the phosphorylated form of IP3 receptor-1, influencing the activity of this channel. Ca(2+) depleted from the ER is then taken up by the mitochondria, leading to Ca(2+) overload in this organelle. The latter induces mitochondrial membrane permeabilization (MMP), opening of the permeability transition pore, and activation of the mitochondrial apoptotic pathway. This study identifies the GEMs as the sites of Ca(2+) diffusion between the ER and the mitochondria. We propose a new mechanism of Ca(2+)-mediated apoptotic signaling whereby GM1 accumulation at the GEMs alters Ca(2+) dynamics and acts as a molecular effector of both ER stress-induced and mitochondria-mediated apoptosis of neuronal cells.

MRI/MRS as a surrogate marker for clinical progression in GM1 gangliosidosis.

Background GM1 gangliosidosis is a lysosomal storage disorder caused by mutations in GLB1, encoding ß-galactosidase. The range of severity is from type I infantile disease, lethal in early childhood, to type III adult onset, resulting in gradually progressive neurological symptoms in adulthood. The intermediate group of patients has been recently classified as having type II late infantile subtype with onset of symptoms at one to three years of age or type II juvenile subtype with symptom onset at 2-10 years. To characterize disease severity and progression, six Late infantile and nine juvenile patients were evaluated using magnetic resonance imaging (MRI), and MR spectroscopy (MRS). Since difficulties with ambulation (gross motor function) and speech (expressive language) are often the first reported symptoms in type II GM1, patients were also scored in these domains. Deterioration of expressive language and ambulation was more rapid in the late infantile patients. Fourteen MRI scans in six Late infantile patients identified progressive atrophy in the cerebrum and cerebellum. Twenty-six MRI scans in nine juvenile patients revealed greater variability in extent and progression of atrophy. Quantitative MRS demonstrated a deficit of N-acetylaspartate in both the late infantile and juvenile patients with greater in the late infantile patients. This correlates with clinical measures of ambulation and expressive language. The two subtypes of type II GM1 gangliosidosis have different clinical trajectories. MRI scoring, quantitative MRS and brain volume correlate with clinical disease progression and may serve as important minimally-invasive outcome measures for clinical trials.

A novel human model of the neurodegenerative disease GM1 gangliosidosis using induced pluripotent stem cells demonstrates inflammasome activation.

GM1 gangliosidosis (GM1) is an inherited neurodegenerative disorder caused by mutations in the lysosomal ß-galactosidase (ß-gal) gene. Insufficient ß-gal activity leads to abnormal accumulation of GM1 gangliosides in tissues, particularly in the central nervous system, resulting in progressive neurodegeneration. Here, we report an in vitro human GM1 model, based on induced pluripotent stem cell (iPSC) technology. Neural progenitor cells differentiated from GM1 patient-derived iPSCs (GM1-NPCs) recapitulated the biochemical and molecular phenotypes of GM1, including defective ß-gal activity and increased lysosomes. Importantly, the characterization of GM1-NPCs established that GM1 is significantly associated with the activation of inflammasomes, which play a critical role in the pathogenesis of various neurodegenerative diseases. Specific inflammasome inhibitors potently alleviated the disease-related phenotypes of GM1-NPCs in vitro and in vivo. Our data demonstrate that GM1-NPCs are a valuable in vitro human GM1 model and suggest that inflammasome activation is a novel target pathway for GM1 drug development.

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.

Gangliosides and gangliosidoses: principles of molecular and metabolic pathogenesis.

Gangliosides are the main glycolipids of neuronal plasma membranes. Their surface patterns are generated by coordinated processes, involving biosynthetic pathways of the secretory compartments, catabolic steps of the endolysosomal system, and intracellular trafficking. Inherited defects in ganglioside biosynthesis causing fatal neurodegenerative diseases have been described so far almost exclusively in mouse models, whereas inherited defects in ganglioside catabolism causing various clinical forms of GM1- and GM2-gangliosidoses have long been known. For digestion, gangliosides are endocytosed and reach intra-endosomal vesicles. At the level of late endosomes, they are depleted of membrane-stabilizing lipids like cholesterol and enriched with bis(monoacylglycero)phosphate (BMP). Lysosomal catabolism is catalyzed at acidic pH values by cationic sphingolipid activator proteins (SAPs), presenting lipids to their respective hydrolases, electrostatically attracted to the negatively charged surface of the luminal BMP-rich vesicles. Various inherited defects of ganglioside hydrolases, e.g., of ß-galactosidase and ß-hexosaminidases, and of GM2-activator protein, cause infantile (with tetraparesis, dementia, blindness) and different protracted clinical forms of GM1- and GM2-gangliosidoses. Mutations yielding proteins with small residual catabolic activities in the lysosome give rise to juvenile and adult clinical forms with a wide range of clinical symptomatology. Apart from patients' differences in their genetic background, clinical heterogeneity may be caused by rather diverse substrate specificities and functions of lysosomal hydrolases, multifunctional properties of SAPs, and the strong regulation of ganglioside catabolism by membrane lipids. Currently, there is no treatment available for neuronal ganglioside storage diseases. Therapeutic approaches in mouse models and patients with juvenile forms of gangliosidoses are discussed.

Synthesis of 1,5-dideoxy-1,5-iminoribitol C-glycosides through a nitrone-olefin cycloaddition domino strategy: identification of pharmacological chaperones of mutant human lysosomal ß-galactosidase.

We report herein a newly developed domino reaction that facilitates the synthesis of new 1,5-dideoxy-1,5-iminoribitol iminosugar C-glycosides 7a-e and 8. The key intermediate in this approach is a six-membered cyclic sugar nitrone that is generated in situ and trapped by an alkene dipolarophile via a [2 + 3] cycloaddition reaction to give the corresponding isooxazolidines 10a-e in a "one-pot" protocol. The iminoribitol C-glycosides 7a-e and 8 were found to be modest ß-galactosidase (bGal) inhibitors. However, compounds 7c and 7e showed "pharmacological chaperone" activity for mutant lysosomal bGal activity and facilitated its recovery in GM1 gangliosidosis patient fibroblasts by 2-6-fold.

Altered GM1 catabolism affects NMDAR-mediated Ca2+ signaling at ER-PM junctions and increases synaptic spine formation in a GM1-gangliosidosis model.

Endoplasmic reticulum-plasma membrane (ER-PM) junctions mediate Ca2+ flux across neuronal membranes. The properties of these membrane contact sites are defined by their lipid content, but little attention has been given to glycosphingolipids (GSLs). Here, we show that GM1-ganglioside, an abundant GSL in neuronal membranes, is integral to ER-PM junctions; it interacts with synaptic proteins/receptors and regulates Ca2+ signaling. In a model of the neurodegenerative lysosomal storage disease, GM1-gangliosidosis, pathogenic accumulation of GM1 at ER-PM junctions due to ß-galactosidase deficiency drastically alters neuronal Ca2+ homeostasis. Mechanistically, we show that GM1 interacts with the phosphorylated N-methyl D-aspartate receptor (NMDAR) Ca2+ channel, thereby increasing Ca2+ flux, activating extracellular signal-regulated kinase (ERK) signaling, and increasing the number of synaptic spines without increasing synaptic connectivity. Thus, GM1 clustering at ER-PM junctions alters synaptic plasticity and worsens the generalized neuronal cell death characteristic of GM1-gangliosidosis.

Single molecule analysis of serotonin transporter regulation using antagonist-conjugated quantum dots reveals restricted, p38 MAPK-dependent mobilization underlying uptake activation.

The presynaptic serotonin (5-HT) transporter (SERT) is targeted by widely prescribed antidepressant medications. Altered SERT expression or regulation has been implicated in multiple neuropsychiatric disorders, including anxiety, depression and autism. Here, we implement a generalizable strategy that exploits antagonist-conjugated quantum dots (Qdots) to monitor, for the first time, single SERT proteins on the surface of serotonergic cells. We document two pools of SERT proteins defined by lateral mobility, one that exhibits relatively free diffusion, and a second, localized to cholesterol and GM1 ganglioside-enriched microdomains, that displays restricted mobility. Receptor-linked signaling pathways that enhance SERT activity mobilize transporters that, nonetheless, remain confined to membrane microdomains. Mobilization of transporters arises from a p38 MAPK-dependent untethering of the SERT C terminus from the juxtamembrane actin cytoskeleton. Our studies establish the utility of ligand-conjugated Qdots for analysis of the behavior of single membrane proteins and reveal a physical basis for signaling-mediated SERT regulation.