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 β-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.

Genome-scale metabolic network reconstruction of model animals as a platform for translational research

Genome-scale metabolic models (GEMs) are used extensively for analysis of mechanisms underlying human diseases and metabolic malfunctions. However, the lack of comprehensive and high-quality GEMs for model organisms restricts translational utilization of omics data accumulating from the use of various disease models. Here we present a unified platform of GEMs that covers five major model animals, including Mouse1 (Mus musculus), Rat1 (Rattus norvegicus), Zebrafish1 (Danio rerio), Fruitfly1 (Drosophila melanogaster), and Worm1 (Caenorhabditis elegans). These GEMs represent the most comprehensive coverage of the metabolic network by considering both orthology-based pathways and species-specific reactions. All GEMs can be interactively queried via the accompanying web portal Metabolic Atlas. Specifically, through integrative analysis of Mouse1 with RNA-sequencing data from brain tissues of transgenic mice we identified a coordinated up-regulation of lysosomal GM2 ganglioside and peptide degradation pathways which appears to be a signature metabolic alteration in Alzheimer’s disease (AD) mouse models with a phenotype of amyloid precursor protein overexpression. This metabolic shift was further validated with proteomics data from transgenic mice and cerebrospinal fluid samples from human patients. The elevated lysosomal enzymes thus hold potential to be used as a biomarker for early diagnosis of AD. Taken together, we foresee that this evolving open-source platform will serve as an important resource to facilitate the development of systems medicines and translational biomedical applications.

In Cellulo Examination of a Beta-Alpha Hybrid Construct of Beta-Hexosaminidase A Subunits, Reported to Interact with the GM2 Activator Protein and Hydrolyze GM2 Ganglioside

The hydrolysis in lysosomes of GM2 ganglioside to GM3 ganglioside requires the correct synthesis, intracellular assembly and transport of three separate gene products; i.e., the alpha and beta subunits of heterodimeric beta-hexosaminidase A, E.C. # 3.2.1.52 (encoded by the HEXA and HEXB genes, respectively), and the GM2-activator protein (GM2AP, encoded by the GM2A gene). Mutations in any one of these genes can result in one of three neurodegenerative diseases collectively known as GM2 gangliosidosis (HEXA, Tay-Sachs disease, MIM # 272800; HEXB, Sandhoff disease, MIM # 268800; and GM2A, AB-variant form, MIM # 272750). Elements of both of the hexosaminidase A subunits are needed to productively interact with the GM2 ganglioside-GM2AP complex in the lysosome. Some of these elements have been predicted from the crystal structures of hexosaminidase and the activator. Recently a hybrid of the two subunits has been constructed and reported to be capable of forming homodimers that can perform this reaction in vivo, which could greatly simplify vector-mediated gene transfer approaches for Tay-Sachs or Sandhoff diseases. A cDNA encoding a hybrid hexosaminidase subunit capable of dimerizing and hydrolyzing GM2 ganglioside could be incorporated into a single vector, whereas packaging both subunits of hexosaminidase A into vectors, such as adeno-associated virus, would be impractical due to size constraints. In this report we examine the previously published hybrid construct (H1) and a new more extensive hybrid (H2), with our documented in cellulo (live cell- based) assay utilizing a fluorescent GM2 ganglioside derivative. Unfortunately when Tay-Sachs cells were transfected with either the H1 or H2 hybrid construct and then were fed the GM2 derivative, no significant increase in its turnover was detected. In vitro assays with the isolated H1 or H2 homodimers confirmed that neither was capable of human GM2AP-dependent hydrolysis of GM2 ganglioside.

In Cellulo Examination of a Beta-Alpha Hybrid Construct of Beta-Hexosaminidase A Subunits, Reported to Interact with the GM2 Activator Protein and Hydrolyze GM2 Ganglioside

The hydrolysis in lysosomes of GM2 ganglioside to GM3 ganglioside requires the correct synthesis, intracellular assembly and transport of three separate gene products; i.e., the alpha and beta subunits of heterodimeric beta-hexosaminidase A, E.C. # 3.2.1.52 (encoded by the HEXA and HEXB genes, respectively), and the GM2-activator protein (GM2AP, encoded by the GM2A gene). Mutations in any one of these genes can result in one of three neurodegenerative diseases collectively known as GM2 gangliosidosis (HEXA, Tay-Sachs disease, MIM # 272800; HEXB, Sandhoff disease, MIM # 268800; and GM2A, AB-variant form, MIM # 272750). Elements of both of the hexosaminidase A subunits are needed to productively interact with the GM2 ganglioside-GM2AP complex in the lysosome. Some of these elements have been predicted from the crystal structures of hexosaminidase and the activator. Recently a hybrid of the two subunits has been constructed and reported to be capable of forming homodimers that can perform this reaction in vivo, which could greatly simplify vector-mediated gene transfer approaches for Tay-Sachs or Sandhoff diseases. A cDNA encoding a hybrid hexosaminidase subunit capable of dimerizing and hydrolyzing GM2 ganglioside could be incorporated into a single vector, whereas packaging both subunits of hexosaminidase A into vectors, such as adeno-associated virus, would be impractical due to size constraints. In this report we examine the previously published hybrid construct (H1) and a new more extensive hybrid (H2), with our documented in cellulo (live cell- based) assay utilizing a fluorescent GM2 ganglioside derivative. Unfortunately when Tay-Sachs cells were transfected with either the H1 or H2 hybrid construct and then were fed the GM2 derivative, no significant increase in its turnover was detected. In vitro assays with the isolated H1 or H2 homodimers confirmed that neither was capable of human GM2AP-dependent hydrolysis of GM2 ganglioside.

Infantile onset Sandhoff disease: clinical manifestation and a novel common mutation in Thai patients

Background Sandhoff disease (SD) is an autosomal recessive lysosomal storage disorder, resulting in accumulation of GM2 ganglioside, particular in neuronal cells. The disorder is caused by deficiency of β-hexosaminidase B (HEX-B), due to pathogenic variant of human HEXB gene. Method This study describes clinical features, biochemical, and genetic defects among Thai patients with infantile SD during 2008–2019. Results Five unrelated Thai patients presenting with developmental regression, axial hypotonia, seizures, exaggerated startle response to noise, and macular cherry red spot were confirmed to have infantile SD based on deficient HEX enzyme activities and biallelic variants of the HEXB gene. In addition, an uncommon presenting feature, cardiac defect, was observed in one patient. All the patients died in their early childhood. Plasma total HEX and HEX-B activities were severely deficient. Sequencing analysis of HEXB gene identified two variants including c.1652G>A (p.Cys551Tyr) and a novel variant of c.761T>C (p.Leu254Ser), in 90 and 10% of the mutant alleles found, respectively. The results from in silico analysis using multiple bioinformatics tools were in agreement that the p.Cys551Tyr and the p.Leu254Ser are likely pathogenic variants. Molecular modelling suggested that the Cys551Tyr disrupt disulfide bond, leading to protein destabilization while the Leu254Ser resulted in change of secondary structure from helix to coil and disturbing conformation of the active site of the enzyme. Genome-wide SNP array analysis showed no significant relatedness between the five affected individuals. These two variants were not present in control individuals. The prevalence of infantile SD in Thai population is estimated 1 in 1,458,521 and carrier frequency at 1 in 604. Conclusion The study suggests that SD likely represents the most common subtype of rare infantile GM2 gangliosidosis identified among Thai patients. We firstly described a potential common variant in HEXB in Thai patients with infantile onset SD. The data can aid a rapid molecular confirmation of infantile SD starting with the hotspot variant and the use of expanded carrier testing.

Clinical and Laboratory Profile of Gangliosidosis from Southern Part of India

Gangliosidoses are progressive neurodegenerative disorders caused by the deficiency of enzymes involved in the breakdown of glycosphingolipids. There are not much data about gangliosidosis in India; hence, this study was planned. The aim is to study the clinical, biochemical, and molecular profile of gangliosidosis. A retrospective chart review, in the pediatric neurology department from January 2015 to March 2020, was performed. Children diagnosed with Gangliosidosis were included. The disorder was confirmed by reduced activity of enzymes and/or pathogenic or likely pathogenic variants in associated genes. We assessed age at presentation, gender, parental consanguinity, clinical manifestations, neuroimaging findings, enzyme level, and pathogenic or likely pathogenic variants. Clinical data for 32 children with gangliosidosis were analyzed, which included 12 (37.5%) with GM1 gangliosidosis, 8 (25%) with Tay-Sachs disease (TSD), 11 (34.37%) with Sandhoff disease (SD), and 1 AB variant of GM2 gangliosidosis that occurs due to GM2 ganglioside activator protein deficiency. Twenty-four (75%) children were the offspring of consanguineous parents. Thirty-one (97%) had developmental delay. The median age at presentation was 15.5 months. Nine (28.12%) had seizures. Five children (41.6%) with GM1 gangliosidosis and two with SD had extensive Mongolian spots. Ten children with GM1 gangliosidosis (83.3%) had coarse facial features. Cherry red spot was found in 24 out of 32 children (75%). All children with GM1 gangliosidosis and none with TSD had hepato-splenomegaly. Two children (2/8; 25%) with TSD and seven (7/11; 63%) with SD had microcephaly. One child with SD had coarse facies and three did not have hepato-splenomegaly. Neuroimaging findings revealed bilateral thalamic involvement in 20 (62.5%) patients and periventricular hypomyelination in all cases. One child had a rare AB variant of GM2 gangliosidosis. GM2 Gangliosidoses are more common compared with GM1 variety. All of them had infantile onset except one child with TSD. Microcephaly can be present while usually megalencephaly is reported in the literature. The absence of hepato-splenomegaly does not rule out SD. Extensive Mongolian spots can be seen in GM2 gangliosidosis. AB variant of GM2 gangliosidosis should be considered when the enzyme is normal in the presence of strong clinical suspicion.

GM2 ganglioside accumulation causes neuroinflammation and behavioral alterations in a mouse model of early onset Tay-Sachs disease

Background Tay-Sachs disease (TSD), a type of GM2-gangliosidosis, is a progressive neurodegenerative lysosomal storage disorder caused by mutations in the α subunit of the lysosomal β-hexosaminidase enzyme. This disease is characterized by excessive accumulation of GM2 ganglioside, predominantly in the central nervous system. Although Tay-Sachs patients appear normal at birth, the progressive accumulation of undegraded GM2 gangliosides in neurons leads to death. Recently, an early onset Tay-Sachs disease mouse model, with genotype Hexa−/−Neu3−/−, was generated. Progressive accumulation of GM2 led to premature death of the double KO mice. Importantly, this double-deficient mouse model displays typical features of Tay-Sachs patients, such as cytoplasmic vacuolization of nerve cells, deterioration of Purkinje cells, neuronal death, deceleration in movement, ataxia, and tremors. GM2-gangliosidosis is characterized by acute neurodegeneration preceded by activated microglia expansion, macrophage, and astrocyte activation, along with the production of inflammatory mediators. However, the mechanism of disease progression in Hexa−/−Neu3−/− mice, relevant to neuroinflammation is poorly understood. Method In this study, we investigated the onset and progression of neuroinflammatory changes in the cortex, cerebellum, and retina of Hexa−/−Neu3−/− mice and control littermates by using a combination of molecular genetics and immunochemical procedures. Results We found elevated levels of pro-inflammatory cytokine and chemokine transcripts, such as Ccl2, Ccl3, Ccl4, and Cxcl10 and also extensive microglial and astrocyte activation and proliferation, accompanied by peripheral blood mononuclear cell infiltration in the vicinity of neurons and oligodendrocytes. Behavioral tests demonstrated a high level of anxiety, and age-dependent loss in both spatial learning and fear memory in Hexa−/−Neu3−/− mice compared with that in the controls. Conclusion Altogether, our data suggest that Hexa−/−Neu3−/− mice display a phenotype similar to Tay-Sachs patients suffering from chronic neuroinflammation triggered by GM2 accumulation. Furthermore, our work contributes to better understanding of the neuropathology in a mouse model of early onset Tay-Sachs disease.

Beneficial Effects of Acetyl-DL-Leucine (ADLL) in a Mouse Model of Sandhoff Disease

Sandhoff disease is a rare neurodegenerative lysosomal storage disease associated with the storage of GM2 ganglioside in late endosomes/lysosomes. Here, we explored the efficacy of acetyl-DL-leucine (ADLL), which has been shown to improve ataxia in observational studies in patients with Niemann–Pick Type C1 and other cerebellar ataxias. We treated a mouse model of Sandhoff disease (Hexb-/-) (0.1 g/kg/day) from 3 weeks of age with this orally available drug. ADLL produced a modest but significant increase in life span, accompanied by improved motor function and reduced glycosphingolipid (GSL) storage in the forebrain and cerebellum, in particular GA2. ADLL was also found to normalize altered glucose and glutamate metabolism, as well as increasing autophagy and the reactive oxygen species (ROS) scavenger, superoxide dismutase (SOD1). Our findings provide new insights into metabolic abnormalities in Sandhoff disease, which could be targeted with new therapeutic approaches, including ADLL.

GM2 ganglioside accumulation causes neuroinflammation and behavioral alterations in a mouse model of early-onset Tay-Sachs disease.

Background Tay-Sachs disease (TSD), a type of GM2-gangliosidosis, is a progressive neurodegenerative lysosomal storage disorder; caused by mutations in the a subunit of the lysosomal β-hexosaminidase enzyme. This disease is characterized by excessive accumulation of GM2 ganglioside, predominantly in the central nervous system. Although Tay-Sachs patients appear normal at birth, the progressive accumulation of undegraded GM2 gangliosides in neurons leads to death. Recently, an early-onset Tay-Sachs disease mouse model, with genotype Hexa -/- Neu3 -/-, was generated. Progressive accumulation of GM2 led to premature death of the double KO mice. Importantly, this double-deficient mouse model displays typical features of Tay-Sachs patients, such as cytoplasmic vacuolization of nerve cells, deterioration of Purkinje cells, neuronal death, deceleration in movement, ataxia, and tremors. GM2-gangliosidosis is characterized by acute neurodegeneration preceded by activated microglia expansion, macrophage, and astrocyte activation, along with the production of inflammatory mediators. However, the mechanism of disease progression in Hexa -/- Neu3 -/- mice, relevant to neuroinflammation is poorly understood. Method In this study we investigated the onset and progression of neuroinflammatory changes in the cortex, cerebellum, and retina of Hexa -/- Neu3 -/- mice and control littermates by using a combination of molecular genetics and immunochemical procedures. Results We found elevated levels of pro-inflammatory cytokine and chemokine transcripts, such as Ccl2, Ccl3, Ccl4, and Cxcl10 and also extensive microglial and astrocyte activation and proliferation, accompanied by peripheral blood mononuclear cell infiltration in neurons and oligodendrocytes. Behavioral tests demonstrated a high level of anxiety, and age-dependent loss in both spatial learning and fear memory in Hexa -/- Neu3 -/- mice compared with that in the controls. Conclusion Altogether, our data suggest that Hexa -/- Neu3 -/- mice display a phenotype similar to Tay-Sachs patients suffering from chronic neuroinflammation triggered by GM2 accumulation. Furthermore, our work contributes to better understanding of the neuropathology in a mouse model of early-onset Tay-Sachs disease.

Designing new diagnostic systems for the early detection of tobacco-associated chronic renal damage in patients of a primary care centre in Salamanca, Spain: an observational, prospective study protocol

IntroductionTobacco causes kidney damage that can progress to chronic kidney disease. However, the diagnostic parameters used in clinics are not effective in identifying smokers at risk. Our first objective is to more effectively detect subclinical renal damage in smokers. In addition, we hypothesise that tobacco consumption can predispose smokers to renal damage on exposure to other potentially nephrotoxic events (drugs, diagnostic procedures and so on). We will test this hypothesis in our second objective by investigating whether certain predisposition markers (GM2 ganglioside activator protein (GM2AP), transferrin and t-gelsolin) are able to detect smokers who are predisposed to kidney damage. Finally, in our third objective, we will study whether smoking cessation reduces subclinical and/or predisposition to renal damage.Methods and analysisFor our first objective, a prospective cross-sectional study will be carried out with patients from a primary healthcare centre. The influence of tobacco on renal damage, in patients both with and without additional risk factors, will be studied using a panel of early biomarkers (albuminuria, N-acetyl-beta-D-glucosaminidase, kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin). For our second objective, a prospective longitudinal study will be carried out with patients recruited for our first objective. We will study whether certain predisposition biomarkers (GM2AP, transferrin and t-gelsolin) are able to detect smokers predisposed to renal damage. For our third objective, a prospective longitudinal study will be carried out with patients from a smoking cessation unit. We will study the evolution of the markers described above following smoking cessation.Ethics and disseminationThe study has been approved by the Clinical Research Ethics Committee of the Healthcare Area of Salamanca. All study participants will sign an informed consent form in compliance with the Declaration of Helsinki and the WHO standards for observational studies. Results will be presented at conferences and submitted to peer-reviewed journals.Trial registration numberNCT03850756.