In vitro substrate reduction, chaperone and immunomodulation treatments reduce heparan sulfate in mucolipidosis III human fibroblasts.

Mucolipidosis II and III (MLII and MLIII) are autosomal recessive diseases caused by pathogenic variants in GNPTAB and GNPTG genes that lead to defects in GlcNAc-1-phosphotransferase. This enzyme adds mannose 6-phosphate residues to lysosomal hydrolases, which allows enzymes to enter lysosomes. Defective GlcNAc-1-phosphotransferase causes substrate accumulation and inflammation. These diseases have no treatment, and we hypothesized that the use of substrate reduction therapy and immunomodulation may be beneficial at the cell level and as a future therapeutic approach. Fibroblasts from two patients with MLIII alpha/beta and 2 patients with MLIII gamma as well as from one healthy control were treated with 10 µM miglustat, 20 µM genistein, and 20 µM thalidomide independently. ELISA assay and confocal immunofluorescence microscopy were used to evaluate the presence of heparan sulfate (HS) and the impact on substrate accumulation. ELISA assay showed HS reduction in all patients with the different treatments used (p=0.05). HS reduction was also observed by immunofluorescence microscopy. Our study produced encouraging results, since the reduction in substrate accumulation, even partial, may offer benefits to the phenotype of patients with inborn errors of metabolism.

Distinct Modes of Balancing Glomerular Cell Proteostasis in Mucolipidosis Type II and III Prevent Proteinuria.

BACKGROUND: The mechanisms balancing proteostasis in glomerular cells are unknown. Mucolipidosis (ML) II and III are rare lysosomal storage disorders associated with mutations of the Golgi-resident GlcNAc-1-phosphotransferase, which generates mannose 6-phosphate residues on lysosomal enzymes. Without this modification, lysosomal enzymes are missorted to the extracellular space, which results in lysosomal dysfunction of many cell types. Patients with MLII present with severe skeletal abnormalities, multisystemic symptoms, and early death; the clinical course in MLIII is less progressive. Despite dysfunction of a major degradative pathway, renal and glomerular involvement is rarely reported, suggesting organ-specific compensatory mechanisms. METHODS: MLII mice were generated and compared with an established MLIII model to investigate the balance of protein synthesis and degradation, which reflects glomerular integrity. Proteinuria was assessed in patients. High-resolution confocal microscopy and functional assays identified proteins to deduce compensatory modes of balancing proteostasis. RESULTS: Patients with MLII but not MLIII exhibited microalbuminuria. MLII mice showed lysosomal enzyme missorting and several skeletal alterations, indicating that they are a useful model. In glomeruli, both MLII and MLIII mice exhibited reduced levels of lysosomal enzymes and enlarged lysosomes with abnormal storage material. Nevertheless, neither model had detectable morphologic or functional glomerular alterations. The models rebalance proteostasis in two ways: MLII mice downregulate protein translation and increase the integrated stress response, whereas MLIII mice upregulate the proteasome system in their glomeruli. Both MLII and MLIII downregulate the protein complex mTORC1 (mammalian target of rapamycin complex 1) signaling, which decreases protein synthesis. CONCLUSIONS: Severe lysosomal dysfunction leads to microalbuminuria in some patients with mucolipidosis. Mouse models indicate distinct compensatory pathways that balance proteostasis in MLII and MLIII.

Enigmatic in vivo GlcNAc-1-phosphotransferase (GNPTG) transcript correction to wild type in two mucolipidosis III gamma siblings homozygous for nonsense mutations.

Mucolipidosis (ML) III gamma is a rare autosomal-recessive disorder caused by pathogenic mutations in the GNPTG gene. GNPTG encodes the γ-subunit of GlcNAc-1-phosphotransferase that catalyzes mannose 6-phosphate targeting signal synthesis on soluble lysosomal enzymes. ML III gamma patients are characterized by missorting of lysosomal enzymes. In this report, we describe the probable occurrence of mRNA editing in two ML III gamma patients. Patients A and B (siblings) presented at the adult age with a typical clinical picture of ML III gamma, mainly compromising bone and joints, and high levels of lysosomal enzymes in plasma and low levels in fibroblasts. Both were found to be homozygous for c.-112C>G and c.328G>T (p.Glu110Ter) mutations in genomic DNA (gDNA) analysis of GNPTG. Analysis of complementary DNA (cDNA), however, showed normal genotypes for both patients. Low GNPTG mRNA expression was observed in both patients. The mRNA editing can explain the differences found in patients A and B regarding gDNA and cDNA analysis, and the mild clinical phenotype associated with homozygosity for a nonsense mutation. Our results suggest that mRNA editing can be more frequent than expected in monogenic disorders and that GNPTG analysis should be performed on gDNA.

Study of anesthetics for euthanasia in rats and mice: A systematic review and meta-analysis on the impact upon biological outcomes (SAFE-RM).

AIM: To summarize the knowledge on the effect of anesthetics employed right before euthanasia on biological outcomes. DATA SOURCE: A systematic review of the literature to find studies with isoflurane, ketamine, halothane, pentobarbital, or thiopental just before euthanasia of laboratory rats or mice. STUDY SELECTION: Controlled studies with quantitative data available. DATA EXTRACTION: The search, data extraction, and risk of bias (RoB) were performed independently by two reviewers using a structured form. For each outcome, an effect size (ES) was calculated relative to the control group. Meta-analysis was performed using robust variance meta-regression for hierarchical data structures, with adjustment for small samples. DATA SYNTHESIS: We included 20 studies with 407 biological outcomes (110 unique). RoB analysis indicated that 87.5% of the domains evaluated showed unclear risk, 2% high risk, and 10.5% low risk. The effect size for all anesthetics considered together was 0.99 (CI95% = 0.75-1.23; p < 0.0001). Sub-analyses indicate high effect sizes for pentobarbital (1.14; CI95% = 0.75-1.52; p < 0.0001), and isoflurane (1.01; CI95% = 0.58-1.44; p = 0.0005) but not for ketamine (1.49; CI95% = -7.95-10.9; p = 0.295). CONCLUSION: We showed that anesthetics interfere differently with the majority of the outcomes assessed. However, our data did not support the use of one anesthetic over others or even the killing without anesthetics. We conclude that outcomes cannot be compared among studies without considering the killing method. This protocol was registered at Prospero (CRD42019119520). FUNDING: There was no direct funding for this research.

Imbalanced cellular metabolism compromises cartilage homeostasis and joint function in a mouse model of mucolipidosis type III gamma.

Mucolipidosis type III (MLIII) gamma is a rare inherited lysosomal storage disorder caused by mutations in GNPTG encoding the γ-subunit of GlcNAc-1-phosphotransferase, the key enzyme ensuring proper intracellular location of multiple lysosomal enzymes. Patients with MLIII gamma typically present with osteoarthritis and joint stiffness, suggesting cartilage involvement. Using Gnptg knockout (Gnptgko ) mice as a model of the human disease, we showed that missorting of a number of lysosomal enzymes is associated with intracellular accumulation of chondroitin sulfate in Gnptgko chondrocytes and their impaired differentiation, as well as with altered microstructure of the cartilage extracellular matrix (ECM). We also demonstrated distinct functional and structural properties of the Achilles tendons isolated from Gnptgko and Gnptab knock-in (Gnptabki ) mice, the latter displaying a more severe phenotype resembling mucolipidosis type II (MLII) in humans. Together with comparative analyses of joint mobility in MLII and MLIII patients, these findings provide a basis for better understanding of the molecular reasons leading to joint pathology in these patients. Our data suggest that lack of GlcNAc-1-phosphotransferase activity due to defects in the γ-subunit causes structural changes within the ECM of connective and mechanosensitive tissues, such as cartilage and tendon, and eventually results in functional joint abnormalities typically observed in MLIII gamma patients. This idea was supported by a deficit of the limb motor function in Gnptgko mice challenged on a rotarod under fatigue-associated conditions, suggesting that the impaired motor performance of Gnptgko mice was caused by fatigue and/or pain at the joint.This article has an associated First Person interview with the first author of the paper.

GNPTAB missense mutations cause loss of GlcNAc-1-phosphotransferase activity in mucolipidosis type II through distinct mechanisms.

Mucolipidoses (ML) II and III alpha/beta are lysosomal storage diseases caused by pathogenic mutations in GNPTAB encoding the α/ß-subunit precursor of GlcNAc-1-phosphotransferase. To determine genotype-phenotype correlation and functional analysis of mutant GlcNAc-1-phosphotransferase, 13 Brazilian patients clinically and biochemical diagnosed for MLII or III alpha/beta were studied. By sequencing of genomic GNPTAB of the MLII and MLIII alpha/beta patients we identified six novel mutations: p.D76G, p.S385L, p.Q278Kfs*3, p.H588Qfs*27, p.N642Lfs*10 and p.Y1111*. Expression analysis by western blotting and immunofluorescence microscopy revealed that the mutant α/ß-subunit precursor p.D76G is retained in the endoplasmic reticulum whereas the mutant p.S385L is correctly transported to the cis-Golgi apparatus and proteolytically processed. Both mutations lead to complete loss of GlcNAc-1-phosphotransferase activity, consistent with the severe clinical MLII phenotype of the patients. Our study expands the genotypic spectrum of MLII and provides novel insights into structural requirements to ensure GlcNAc-1-phosphotransferase activity.

A de novo or germline mutation in a family with Mucolipidosis III gamma: Implications for molecular diagnosis and genetic counseling.

Mucolipidosis III (ML III) gamma is a very rare autosomal-recessive disorder characterized by the abnormal trafficking and subcellular localization of lysosomal enzymes due to mutations in the GNPTG gene. The present study consists of a report of a Brazilian compound heterozygote patient with ML III gamma resulting from one mutant paternal allele and one allele that had most likely undergone a de novo or maternal germline mutation. This is the first report of a de novo mutation in ML III gamma. This finding has significant implications for genetic counseling.