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.

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.

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.

GM2-GM3 gangliosides ratio is dependent on GRP94 through down-regulation of GM2-AP cofactor in brain metastasis cells.

GRP94 is an ATP-dependent chaperone able to regulate pro-oncogenic signaling pathways. Previous studies have shown a critical role of GRP94 in brain metastasis (BrM) pathogenesis and progression. In this work, an untargeted lipidomic analysis revealed that some lipid species were altered in GRP94-deficient cells, specially GM2 and GM3 gangliosides. The catalytic pathway of GM2 is affected by the low enzymatic activity of ß-Hexosaminidase (HexA), responsible for the hydrolysis of GM2 to GM3. Moreover, a deficiency of the GM2-activator protein (GM2-AP), the cofactor of HexA, is observed without alteration of gene expression, indicating a post-transcriptional alteration of GM2-AP in the GRP94-ablated cells. One plausible explanation of these observations is that GM2-AP is a client of GRP94, resulting in defective GM2 catabolic processing and lysosomal accumulation of GM2 in GRP94-ablated cells. Overall, given the role of gangliosides in cell surface dynamics and signaling, their imbalance might be linked to modifications of cell behaviour acquired in BrM progression. This work indicates that GM2-AP could be an important factor in ganglioside balance maintenance. These findings highlight the relevance of GM3 and GM2 gangliosides in BrM and reveal GM2-AP as a promising diagnosis and therapeutic target in BrM research.

Identification and characterization of the Onchocerca volvulus Excretory Secretory Product Ov28CRP, a putative GM2 activator protein.

Onchocerca volvulus is the nematode pathogen responsible for human onchocerciasis also known as "River blindness", a neglected tropical disease that affects up to 18 million people worldwide. Helminths Excretory Secretory Products (ESPs) constitute a rich repertoire of molecules that can be exploited for host-parasite relationship, diagnosis and vaccine studies. Here, we report, using a range of molecular techniques including PCR, western blot, recombinant DNA technology, ELISA, high performance thin-layer chromatography and mass spectrometry that the 28 KDa cysteine-rich protein (Ov28CRP) is a reliable component of the O. volvulus ESPs to address the biology of this parasite. We showed that (1) Ov28CRP is a putative ganglioside GM2 Activator Protein (GM2AP) conserved in nematode; (2) OvGM2AP gene is transcriptionally activated in all investigated stages of the parasitic life cycle, including larval and adult stages; (3) The full-length OvGM2AP was detected in in-vitro O. volvulus ESPs of adult and larval stages; (4) the mass expressed and purified recombinant OvGM2AP purified from insect cell culture medium was found to be glycosylated at asparagine 173 and lacked N-terminal signal peptide sequence; (5) the recombinant OvGM2AP discriminated serum samples of infected and uninfected individuals; (6) OvGM2AP competitively inhibits MUG degradation by recombinant ß-hexosaminidase A but not MUGS, and could not hydrolyze the GM2 to GM3; (7) humoral immune responses to the recombinant OvGM2AP revealed a negative correlation with ivermectin treatment. Altogether, our findings suggest for the first time that OvGM2AP is an antigenic molecule whose biochemical and immunological features are important to gain more insight into our understanding of host-parasite relationship, as well as its function in parasite development at large.

GWAS and network analysis of co-occurring nicotine and alcohol dependence identifies significantly associated alleles and network.

Alcohol dependence (AD) and nicotine dependence (ND) co-occur frequently (AD+ND). We integrated SNP-based, gene-based, and protein-protein interaction network analyses to identify shared risk genes or gene subnetworks for AD+ND in African Americans (AAs, N = 2,094) and European Americans (EAs, N = 1,207). The DSM-IV criterion counts for AD and ND were modeled as two dependent variables in a multivariate linear mixed model, and analyzed separately for the two populations. The most significant SNP was rs6579845 in EAs (p < 1.29 × 10-8 ) in GM2A, which encodes GM2 ganglioside activator, and is a cis-expression quantitative locus that affects GM2A expression in blood and brain tissues. However, this SNP was not replicated in our another small sample (N = 678). We identified a subnetwork of 24 genes that contributed to the AD+ND criterion counts. In the gene-set analysis for the subnetwork in an independent sample, the Study of Addiction: Genetics and Environment project (predominately EAs), these 24 genes as a set differed in AD+ND versus control subjects in EAs (p = .041). Functional enrichment analysis for this subnetwork revealed that the gene enrichment involved primarily nerve growth factor pathways, and cocaine and amphetamine addiction. In conclusion, we identified a genome-wide significant variant at GM2A and a gene subnetwork underlying the genetic trait of shared AD+ND. These results increase our understanding of the shared (pleiotropic) genetic risk that underlies AD+ND.

Glycosphingolipid GM2 Induces Invasiveness in Irradiation-tolerant Lung Cancer Cells.

Glycans, including glycosphingolipids, are broadly expressed in plasma membranes and play important roles in cell-cell interactions. Recently, it has been revealed that glycans participate in the regulation of malignant phenotypes of cancer cells, e.g. growth and invasion. However, their roles in irradiation-tolerant cancer cells have not yet been elucidated. In this study, we show that specific glycosphingolipids are highly expressed in invasive, irradiation-tolerant lung cancer cells. Particularly, the glycosphingolipid GM2 contributes to the development of an invasive phenotype in these lung cancer cells. Our results suggest that glycosphingolipids, including GM2, are implicated in the regulation of invasiveness in irradiation-tolerant lung cancer cells and may therefore serve as potential therapeutic targets for lung cancers following radiotherapy.Key words: glycosphingolipids, GM2, invasion, lung cancer cells, radiotherapy.

Characterizing solution surface loop conformational flexibility of the GM2 activator protein.

GM2AP has a ß-cup topology with numerous X-ray structures showing multiple conformations for some of the surface loops, revealing conformational flexibility that may be related to function, where function is defined as either membrane binding associated with ligand binding and extraction or interaction with other proteins. Here, site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and molecular dynamic (MD) simulations are used to characterize the mobility and conformational flexibility of various structural regions of GM2AP. A series of 10 single cysteine amino acid substitutions were generated, and the constructs were chemically modified with the methanethiosulfonate spin label. Continuous wave (CW) EPR line shapes were obtained and subsequently simulated using the microscopic order macroscopic disorder (MOMD) program. Line shapes for sites that have multiple conformations in the X-ray structures required two spectral components, whereas spectra of the remaining sites were adequately fit with single-component parameters. For spin labeled sites L126C and I66C, spectra were acquired as a function of temperature, and simulations provided for the determination of thermodynamic parameters associated with conformational change. Binding to GM2 ligand did not alter the conformational flexibility of the loops, as evaluated by EPR and NMR spectroscopies. These results confirm that the conformational flexibility observed in the surface loops of GM2AP crystals is present in solution and that the exchange is slow on the EPR time scale (>ns). Furthermore, MD simulation results are presented and agree well with the conformational heterogeneity revealed by SDSL.

Integrated multiplex ligation dependent probe amplification (MLPA) assays for the detection of alterations in the HEXB, GM2A and SMARCAL1 genes to support the diagnosis of Morbus Sandhoff, M. Tay-Sachs variant AB and Schimke immuno-osseous dysplasia in humans.

Multiplex ligation dependent probe amplification (MLPA) assays were designed for the genes HEXB (OMIM: 606873), GM2A (OMIM: 613109) and SMARCAL1 (OMIM: 606622) of humans. Two sets of synthetic MLPA probes for these coding exons were tested. Changes in copy numbers were detected as well as single nucleotide polymorphisms (SNPs) by complementary DNA sequence analyses. The MLPA method was shown to be reliable for mutation detection and identified five published and 12 new mutations. In all cases from a Morbus Sandhoff cohort of patients, exclusively one variation in copy number was observed and linked to a nucleotide alteration called c.1614-14C>A. This deletion comprised exons 1-5. One of these cases is described in detail. Deletions were neither detected in the GM2A nor the SMARCAL1 genes. The MLPA assays complement routine diagnostics for M. Sandhoff (OMIM: 268800), M. Tay-Sachs variant AB (OMIM: 272750) and Schimke immuno-osseous dysplasia (OMIM: 242900).

Sequential proteolysis and high-field FTICR MS to determine disulfide connectivity and 4-maleimide TEMPO spin-label location in L126C GM2 activator protein.

The GM2 activator protein (GM2AP) is an 18 kDa nonenzymatic accessory protein involved in the degradation of neuronal gangliosides. Genetic mutations of GM2AP can disrupt ganglioside catabolism and lead to deadly lysosomal storage disorders. Crystallography of wild-type GM2AP reveals 4 disulfide bonds and multiple conformations of a flexible loop region that is thought to be involved in lipid binding. To extend the crystallography results, a cysteine construct (L126C) was expressed and modified with 4-maleimide TEMPO for electron paramagnetic resonance (EPR) studies. However, because a ninth cysteine has been added by site-directed mutagenesis and the protein was expressed in E. coli in the form of inclusion bodies, the protein could misfold during expression. To verify correct protein folding and labeling, a sequential multiple-protease digestion, nano-liquid chromatograph (LC) electrospray ionization 14.5 T Fourier transform ion cyclotron resonance mass spectrometry assay was developed. High-magnetic field and robust automatic gain control results in subppm mass accuracy for location of the spin-labeled cysteine and verification of proper connectivity of the four disulfide bonds. The sequential multiple protease digestion strategy and ultrahigh mass accuracy provided by FTICR MS allow for rapid and unequivocal assignment of relevant peptides and provide a simple pipeline for analyzing other GM2AP constructs.

Identification and tissue-specific expression of amphioxus GM2 activator protein gene from amphioxus Branchiostoma belcheri.

An amphioxus cDNA, AmphiGM2AP, encoding GM2 activator protein was isolated from the gut cDNA library of Branchiostoma belcheri. It is 907 bp long, and its longest open reading frame codes for a precursor protein consisting of 242 amino acid residues with a signal peptide of 14 amino acids. The deduced amino acid sequence includes a conserved domain typical of GM2APs between residues 53 and 224, a single N-linked glycosylation site at position 65 and 8 conserved cysteines. Phylogenetic analysis showed that amphiGM2AP forms a club together with invertebrate GM2APs, indicating that AmphiGM2AP is evolutionarily closely related to invertebrate GM2APs rather than vertebrate ones. Both Northern blotting and in situ hybridization histochemistry analyses revealed a tissue-specific expression pattern of AmphiGM2AP in adult amphioxus with the strongest expression in the digestive system, which is in contrast to the widespread expression pattern of human, mouse and sheep GM2AP genes. It is suggested that AmphiGM2AP is possibly involved in the take-in of digested food components like lipid molecules.