Modeling Lysosomal Storage Disorders in an Innovative Way: Establishment and Characterization of Stem Cell Lines from Human Exfoliated Deciduous Teeth of Mucopolysaccharidosis Type II Patients.

Among the many lysosomal storage disorders (LSDs) that would benefit from the establishment of novel cell models, either patient-derived or genetically engineered, is mucopolysaccharidosis type II (MPS II). Here, we present our results on the establishment and characterization of two MPS II patient-derived stem cell line(s) from deciduous baby teeth. To the best of our knowledge, this is the first time a stem cell population has been isolated from LSD patient samples obtained from the dental pulp. Taking into account our results on the molecular and biochemical characterization of those cells and the fact that they exhibit visible and measurable disease phenotypes, we consider these cells may qualify as a valuable disease model, which may be useful for both pathophysiological assessments and in vitro screenings. Ultimately, we believe that patient-derived dental pulp stem cells (DPSCs), particularly those isolated from human exfoliated deciduous teeth (SHEDs), may represent a feasible alternative to induced pluripotent stem cells (iPSCs) in many labs with standard cell culture conditions and limited (human and economic) resources.

Help Comes from Unexpected Places: How a Tiny Fairy and a Tropical Fish may help us Model Mucopolysaccharidoses.

INTRODUCTION: When it comes to disease modeling, countless models are available for Lysosomal Storage Diseases (LSD). Historically, two major approaches are well-established: in vitro assessments are performed in patient fibroblasts, while in vivo pre-clinical studies are performed in mouse models. Still, both platforms have a series of drawbacks. Thus, we implemented two alternative and innovative protocols to mimic a particular sub-group of LSDs, the Mucopolysaccharidoses both in vitro and in vivo. METHODS: The first one relies on a non-invasive approach using dental pulp stem cells from deciduous teeth (SHEDs). SHEDs are multipotent neuronal precursors that can easily be collected. The second uses a state-of-the-art gene editing technology (CRISPR/Cas9) to generate zebrafish disease models. RESULTS: Even though this is an ongoing project, we have already established and characterized two MPS II and one MPS VI SHED cell models. These cells self-maintain through several passages and can give rise to a variety of cells including neurons. Furthermore, all MPS-associated sub-cellular phenotypes we have assessed so far are easily observable in these cells. Regarding our zebrafish models, we have successfully knocked down both naglu and hgsnat and the first results we got from the behavioral analysis are promising ones, as we can observe altered activity and sleep patterns in the genetically modified fish. For this particular approach we chose MPS III forms as our target disorders, since their neurological features (hyperactivity, seizures and motor impairment) and lifespan decrease would be easily recognizable in zebrafish. CONCLUSION: Now that these methods are well-established in our lab, their potential is immense. On one hand, the newly developed models will be of ultimate value to understand the mechanisms underlying MPS sub-cellular pathology, which have to be further elucidated. On the other hand, they will constitute an optimal platform for drug testing in house. Also noteworthy, our models will be published as lab resources and made available for the whole LSD community.

Challenges in the Definitive Diagnosis of Niemann-Pick Type C-Leaky Variants and Alternative Transcripts.

Niemann-Pick type C (NPC, ORPHA: 646) is a neuro-visceral, psychiatric disease caused predominantly by pathogenic variants in the NPC1 gene or seldom in NPC2. The rarity of the disease, and its wide range of clinical phenotypes and ages of onset, turn the diagnosis into a significant challenge. Other than the detailed clinical history, the typical diagnostic work-up for NPC includes the quantification of pathognomonic metabolites. However, the molecular basis diagnosis is still of utmost importance to fully characterize the disorder. Here, the authors provide an overview of splicing variants in the NPC1 and NPC2 genes and propose a new workflow for NPC diagnosis. Splicing variants cover a significant part of the disease-causing variants in NPC. The authors used cDNA analysis to study the impact of such variants, including the collection of data to classify them as leaky or non-leaky pathogenic variants. However, the presence of naturally occurring spliced transcripts can misdiagnose or mask a pathogenic variant and make the analysis even more difficult. Analysis of the NPC1 cDNA in NPC patients in parallel with controls is vital to assess and detect alternatively spliced forms. Moreover, nonsense-mediated mRNA decay (NMD) analysis plays an essential role in evaluating the naturally occurring transcripts during cDNA analysis and distinguishing them from other pathogenic variants' associated transcripts.

MicroRNA Profile, Putative Diagnostic Biomarkers and RNA-Based Therapies in the Inherited Lipid Storage Disease Niemann-Pick Type C.

Lipids are essential for cellular function and are tightly controlled at the transcriptional and post-transcriptional levels. Dysregulation of these pathways is associated with vascular diseases, diabetes, cancer, and several inherited metabolic disorders. MicroRNAs (miRNAs), in particular, are a family of post-transcriptional gene repressors associated with the regulation of many genes that encode proteins involved in multiple lipid metabolism pathways, thereby influencing their homeostasis. Thus, this class of non-coding RNAs (ncRNAs) has emerged as a promising therapeutic target for the treatment of lipid-related metabolic alterations. Most of these miRNAs act at an intracellular level, but in the past few years, a role for miRNAs as intercellular signaling molecules has also been uncovered since they can be transported in bodily fluids and used as potential biomarkers of lipid metabolic alterations. In this review, we point out the current knowledge on the miRNA signature in a lysosomal storage disorder associated with lipid dysfunction, Niemann-Pick type C, and discuss the potential use of miRNAs as biomarkers and therapeutic targets for RNA-based therapies.

Neurological Disease Modeling Using Pluripotent and Multipotent Stem Cells: A Key Step towards Understanding and Treating Mucopolysaccharidoses.

Despite extensive research, the links between the accumulation of glycosaminoglycans (GAGs) and the clinical features seen in patients suffering from various forms of mucopolysaccharidoses (MPSs) have yet to be further elucidated. This is particularly true for the neuropathology of these disorders; the neurological symptoms are currently incurable, even in the cases where a disease-specific therapeutic approach does exist. One of the best ways to get insights on the molecular mechanisms driving that pathogenesis is the analysis of patient-derived cells. Yet, not every patient-derived cell recapitulates relevant disease features. For the neuronopathic forms of MPSs, for example, this is particularly evident because of the obvious inability to access live neurons. This scenario changed significantly with the advent of induced pluripotent stem cell (iPSC) technologies. From then on, a series of differentiation protocols to generate neurons from iPSC was developed and extensively used for disease modeling. Currently, human iPSC and iPSC-derived cell models have been generated for several MPSs and numerous lessons were learnt from their analysis. Here we review most of those studies, not only listing the currently available MPS iPSC lines and their derived models, but also summarizing how they were generated and the major information different groups have gathered from their analyses. Finally, and taking into account that iPSC generation is a laborious/expensive protocol that holds significant limitations, we also hypothesize on a tempting alternative to establish MPS patient-derived neuronal cells in a much more expedite way, by taking advantage of the existence of a population of multipotent stem cells in human dental pulp to establish mixed neuronal and glial cultures.

Molecular profile and peripheral markers of neurodegeneration in patients with Niemann-Pick type C: Decrease in Plasminogen Activator Inhibitor type 1 and Platelet-Derived Growth Factor type AA.

Niemann-Pick type C1 (NPC1) is a fatal inherited disease, caused by pathogenic variants in NPC1 gene, which leads to intracellular accumulation of non-esterified cholesterol and glycosphingolipids. This accumulation leads to a wide range of clinical manifestations, including neurological and cognitive impairment as well as psychiatric disorders. The pathophysiology of cerebral damage involves loss of Purkinje cells, synaptic disturbance, and demyelination. Miglustat, a reversible inhibitor of glucosylceramide synthase, is an approved treatment for NPC1 and can slow neurological damage. The aim of this study was to assess the levels of peripheric neurodegeneration biomarkers of NPC1 patients, namely brain-derived neurotrophic factor (BDNF), platelet-derived growth factors (PDGF-AA and PDGF-AB/BB), neural cell adhesion molecule (NCAM), PAI-1 Total and Cathepsin-D, as well as the levels of cholestane-3ß,5α,6ß-triol (3ß,5α,6ß-triol), a biomarker for NPC1. Molecular analysis of the NPC1 patients under study was performed by next generation sequencing (NGS) in cultured fibroblasts. We observed that NPC1 patients treated with miglustat have a significant decrease in PAI-1 total and PDGF-AA concentrations, and no alteration in BDNF, NCAM, PDGF-AB/BB and Cathepsin D. We also found that NPC1 patients treated with miglustat have normalized levels of 3ß,5α,6ß-triol. The molecular analysis showed four described mutations, and for two patients was not possible to identify the second mutated allele. Our results indicate that the decrease of PAI-1 and PDGF-AA in NPC1 patients could be involved in the pathophysiology of this disease. This is the first work to analyze those plasmatic markers of neurodegenerative processes in NPC1 patients.

NPC1 silent variant induces skipping of exon 11 (p.V562V) and unfolded protein response was found in a specific Niemann-Pick type C patient.

BACKGROUND: Niemann-Pick type C (NPC, MIM #257220) is a neuro-visceral disease, caused predominantly by pathogenic variants in the NPC1 gene. Here we studied patients with clinical diagnosis of NPC but inconclusive results regarding the molecular analysis. METHODS: We used a Next-Generation Sequencing (NGS)-panel followed by cDNA analysis. Latter, we used massively parallel single-cell RNA-seq (MARS-Seq) to address gene profiling changes and finally the effect of different variants on the protein and cellular levels. RESULTS: We identified novel variants and cDNA analysis allowed us to establish the functional effect of a silent variant, previously reported as a polymorphism. We demonstrated that this variant induces the skipping of exon 11 leading to a premature stop codon and identified it in NPC patients from two unrelated families. MARS-Seq analysis showed that a number of upregulated genes were related to the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress in one specific patient. Also, for all analyzed variants, the NPC1 protein was partially retained in the ER. CONCLUSION: We showed that the NPC1 silent polymorphism (p.V562V) is a disease-causing variant in NPC and that the UPR is upregulated in an NPC patient.

Assessing Lysosomal Disorders in the NGS Era: Identification of Novel Rare Variants.

Lysosomal storage diseases (LSDs) are a heterogeneous group of genetic disorders with variable degrees of severity and a broad phenotypic spectrum, which may overlap with a number of other conditions. While individually rare, as a group LSDs affect a significant number of patients, placing an important burden on affected individuals and their families but also on national health care systems worldwide. Here, we present our results on the use of an in-house customized next-generation sequencing (NGS) panel of genes related to lysosome function as a first-line molecular test for the diagnosis of LSDs. Ultimately, our goal is to provide a fast and effective tool to screen for virtually all LSDs in a single run, thus contributing to decrease the diagnostic odyssey, accelerating the time to diagnosis. Our study enrolled a group of 23 patients with variable degrees of clinical and/or biochemical suspicion of LSD. Briefly, NGS analysis data workflow, followed by segregation analysis allowed the characterization of approximately 41% of the analyzed patients and the identification of 10 different pathogenic variants, underlying nine LSDs. Importantly, four of those variants were novel, and, when applicable, their effect over protein structure was evaluated through in silico analysis. One of the novel pathogenic variants was identified in the GM2A gene, which is associated with an ultra-rare (or misdiagnosed) LSD, the AB variant of GM2 Gangliosidosis. Overall, this case series highlights not only the major advantages of NGS-based diagnostic approaches but also, to some extent, its limitations ultimately promoting a reflection on the role of targeted panels as a primary tool for the prompt characterization of LSD patients.

Molecular Characterization of a Novel Splicing Mutation underlying Mucopolysaccharidosis (MPS) type VI-Indirect Proof of Principle on Its Pathogenicity.

Here, we present the molecular diagnosis of a patient with a general clinical suspicion of Mucopolysaccharidosis, highlighting the different tools used to perform its molecular characterization. In order to decrease the turnaround time for the final report and contribute to reduce the "diagnostic odyssey", which frequently afflicts affected families, the proband's sample was simultaneously screened for mutations in a number of lysosomal function-related genes with targeted next-generation sequencing (NGS) protocol. After variant calling, the most probable cause for disease was a novel ARSB intronic variant, c.1213+5G>T [IVS6+5G>T], detected in homozygosity. In general, homozygous or compound heterozygous mutations in the ARSB gene, underlie MPS type VI or Maroteaux-Lamy syndrome. Still, even though the novel c.1213+5G>T variant was easy to detect by both NGS and Sanger sequencing, only through indirect studies and functional analyses could we present proof of principle on its pathogenicity. Globally, this case reminds us that whenever a novel variant is detected, its pathogenicity must be carefully assessed before a definitive diagnosis is established, while highlighting alternative approaches that may be used to assess its effect in the absence RNA/cDNA sample(s) from the proband. This is particularly relevant for intronic variants such as the one here reported. Special attention will be given to the use of reporter minigene systems, which may be constructed/designed to dissect the effect of this sort of alterations, providing an insight into their consequences over the normal pre-mRNA splicing process of the affected gene.

Cholesteryl hemiesters alter lysosome structure and function and induce proinflammatory cytokine production in macrophages.

RATIONALE: Cholesteryl hemiesters are oxidation products of polyunsaturated fatty acid esters of cholesterol. Their oxo-ester precursors have been identified as important components of the "core aldehydes" of human atheromata and in oxidized lipoproteins (Ox-LDL). We had previously shown, for the first time, that a single compound of this family, cholesteryl hemisuccinate (ChS), is sufficient to cause irreversible lysosomal lipid accumulation (lipidosis), and is toxic to macrophages. These features, coupled to others such as inflammation, are typically seen in atherosclerosis. OBJECTIVE: To obtain insights into the mechanism of cholesteryl hemiester-induced pathological changes in lysosome function and induction of inflammation in vitro and assess their impact in vivo. METHODS AND RESULTS: We have examined the effects of ChS on macrophages (murine cell lines and primary cultures) in detail. Specifically, lysosomal morphology, pH, and proteolytic capacity were examined. Exposure of macrophages to sub-toxic ChS concentrations caused enlargement of the lysosomes, changes in their luminal pH, and accumulation of cargo in them. In primary mouse bone marrow-derived macrophages (BMDM), ChS-exposure increased the secretion of IL-1ß, TNF-α and IL-6. In zebrafish larvae (wild-type AB and PU.1:EGFP), fed with a ChS-enriched diet, we observed lipid accumulation, myeloid cell-infiltration in their vasculature and decrease in larval survival. Under the same conditions the effects of ChS were more profound than the effects of free cholesterol (FC). CONCLUSIONS: Our data strongly suggest that cholesteryl hemiesters are pro-atherogenic lipids able to mimic features of Ox-LDL both in vitro and in vivo.

Solving a case of allelic dropout in the GNPTAB gene: implications in the molecular diagnosis of mucolipidosis type III alpha/beta.

While being well known that the diagnosis of many genetic disorders relies on a combination of clinical suspicion and confirmatory genetic testing, not rarely, however, genetic testing needs much perseverance and cunning strategies to identify the causative mutation(s). Here we present a case of a thorny molecular diagnosis of mucolipidosis type III alpha/beta, which is an autosomal recessive lysosomal storage disorder, caused by a defect in the GNPTAB gene that codes for the α/ß-subunits of the GlcNAc-1-phosphotransferase. We used both cDNA and gDNA analyses to characterize a mucolipidosis type III alpha/beta patient whose clinical diagnosis was already confirmed biochemically. In a first stage only one causal mutation was identified in heterozygosity, the already described missense mutation c.1196C>T(p.S399F), both at cDNA and gDNA levels. Only after conducting inhibition of nonsense-mediated mRNA decay (NMD) assays and after the utilization of another pair of primers the second mutation, the c.3503_3504delTC deletion, was identified. Our findings illustrate that allelic dropout due to the presence of polymorphisms and/or of mutations that trigger the NMD pathway can cause difficulties in current molecular diagnosis tests.

A Rab3a-dependent complex essential for lysosome positioning and plasma membrane repair.

Lysosome exocytosis plays a major role in resealing plasma membrane (PM) disruptions. This process involves two sequential steps. First, lysosomes are recruited to the periphery of the cell and then fuse with the damaged PM. However, the trafficking molecular machinery involved in lysosome exocytosis and PM repair (PMR) is poorly understood. We performed a systematic screen of the human Rab family to identify Rabs required for lysosome exocytosis and PMR. Rab3a, which partially localizes to peripheral lysosomes, was one of the most robust hits. Silencing of Rab3a or its effector, synaptotagmin-like protein 4a (Slp4-a), leads to the collapse of lysosomes to the perinuclear region and inhibition of PMR. Importantly, we have also identified a new Rab3 effector, nonmuscle myosin heavy chain IIA, as part of the complex formed by Rab3a and Slp4-a that is responsible for lysosome positioning at the cell periphery and lysosome exocytosis.

Subunit interactions of the disease-related hexameric GlcNAc-1-phosphotransferase complex.

The multimeric GlcNAc-1-phosphotransferase complex catalyzes the formation of mannose 6-phosphate recognition marker on lysosomal enzymes required for receptor-mediated targeting to lysosomes. GNPTAB and GNPTG encode the α/ß-subunit precursor membrane proteins and the soluble γ-subunits, respectively. Performing extensive mutational analysis, we identified the binding regions of γ-subunits in a previously uncharacterized domain of α-subunits comprising residues 535-698, named GNPTG binding (GB) domain. Both the deletion of GB preventing γ-subunit binding and targeted deletion of GNPTG led to significant reduction in GlcNAc-1-phosphotransferase activity. We also identified cysteine 70 in α-subunits to be involved in covalent homodimerization of α-subunits which is, however, required neither for interaction with γ-subunits nor for catalytic activity of the enzyme complex. Finally, binding assays using various γ-subunit mutants revealed that residues 130-238 interact with glycosylated α-subunits suggesting a role for the mannose 6-phosphate receptor homology domain in α-subunit binding. These studies provide new insight into the assembly of the GlcNAc-1-phosphotransferase complex, and the functions of distinct domains of the α- and γ-subunits.

Post-translational modifications of the gamma-subunit affect intracellular trafficking and complex assembly of GlcNAc-1-phosphotransferase.

GlcNAc-1-phosphotransferase plays a key role in the generation of mannose 6-phosphate, a recognition marker essential for efficient transport of lysosomal hydrolases to lysosomes. The enzyme complex is composed of six subunits (α(2)ß(2)γ(2)). The α- and ß-subunits are catalytically active, whereas the function of the γ-subunit is still unclear. We have investigated structural properties, localization, and intracellular transport of the human and mouse γ-subunits and the molecular requirements for the assembly of the phosphotransferase complex. The results showed that endogenous and overexpressed γ-subunits were localized in the cis-Golgi apparatus. Secreted forms of γ-subunits were detectable in media of cultured cells as well as in human serum. The γ-subunit contains two in vivo used N-glycosylation sites at positions 88 and 115, equipped with high mannose-type oligosaccharides. (35)S pulse-chase experiments and size exclusion chromatography revealed that the majority of non-glycosylated γ-subunit mutants were integrated in high molecular mass complexes, failed to exit the endoplasmic reticulum (ER), and were rapidly degraded. The substitution of cysteine 245 involved in dimerization of γ-subunits impaired neither ER exit nor trafficking through the secretory pathway. Monomeric γ-subunits failed, however, to associate with other GlcNAc-1-phosphotransferase subunits. The data provide evidence that assembly of the GlcNAc-1-phosphotransferase complex takes place in the ER and requires dimerization of the γ-subunits.

Proteolytic processing of the gamma-subunit is associated with the failure to form GlcNAc-1-phosphotransferase complexes and mannose 6-phosphate residues on lysosomal enzymes in human macrophages.

GlcNAc-1-phosphotransferase is a Golgi-resident 540-kDa complex of three subunits, alpha(2)beta(2)gamma(2), that catalyze the first step in the formation of the mannose 6-phosphate (M6P) recognition marker on lysosomal enzymes. Anti-M6P antibody analysis shows that human primary macrophages fail to generate M6P residues. Here we have explored the sorting and intracellular targeting of cathepsin D as a model, and the expression of the GlcNAc-1-phosphotransferase complex in macrophages. Newly synthesized cathepsin D is transported to lysosomes in an M6P-independent manner in association with membranes whereas the majority is secreted. Realtime PCR analysis revealed a 3-10-fold higher GlcNAc-1-phosphotransferase subunit mRNA levels in macrophages than in fibroblasts or HeLa cells. At the protein level, the gamma-subunit but not the beta-subunit was found to be proteolytically cleaved into three fragments which form irregular 97-kDa disulfide-linked oligomers in macrophages. Size exclusion chromatography showed that the gamma-subunit fragments lost the capability to assemble with other GlcNAc-1-phosphotransferase subunits to higher molecular complexes. These findings demonstrate that proteolytic processing of the gamma-subunit represents a novel mechanism to regulate GlcNAc-1-phosphotransferase activity and the subsequent sorting of lysosomal enzymes.

Mannose phosphorylation in health and disease.

Lysosomal hydrolases catalyze the degradation of a variety of macromolecules including proteins, carbohydrates, nucleic acids and lipids. The biogenesis of lysosomes or lysosome-related organelles requires a continuous substitution of soluble acid hydrolases and lysosomal membrane proteins. The targeting of lysosomal hydrolases depends on mannose 6-phosphate residues (M6P) that are recognized by specific receptors mediating their transport to an endosomal/prelysosomal compartment. The key role in the formation of M6P residues plays the GlcNAc-1-phosphotransferase localized in the Golgi apparatus. Two genes have been identified recently encoding the type III alpha/beta-subunit precursor membrane protein and the soluble gamma-subunit of GlcNAc-1-phosphotransferase. Mutations in these genes result in two severe diseases, mucolipidosis type II (MLII) and III (MLIII), biochemically characterized by the missorting of multiple lysosomal hydrolases due to impaired formation of the M6P recognition marker, and general lysosomal dysfunction. This review gives an update on structural properties, localization and functions of the GlcNAc-1-phosphotransferase subunits and improvements of pre- and postnatal diagnosis of ML patients. Further, the generation of recombinant single-chain antibody fragments against M6P residues and of new mouse models of MLII and MLIII will have considerable impact to provide deeper insight into the cell biology of lysosomal dysfunctions and the pathomechanisms underlying these lysosomal disorders.

Loss of N-acetylglucosamine-1-phosphotransferase gamma subunit due to intronic mutation in GNPTG causes mucolipidosis type III gamma: Implications for molecular and cellular diagnostics.

Mucolipidosis type III gamma (MLIII, pseudo-Hurler polydystrophy) is a rare autosomal recessive disorder where the activity of the multimeric GlcNAc-1-phosphotransferase is reduced and formation of the mannose 6-phosphate (M6P) recognition marker on lysosomal enzymes is impaired. In this disease, the targeting of lysosomal enzymes is affected resulting in their hypersecretion, and an intracellular deficiency of multiple hydrolases. We report the biochemical and molecular diagnosis of MLIII in three siblings, aged 17, 15, and 14 years, who presented with joint pain and progressive joint stiffness. In addition to missorting of newly synthesized lysosomal protease cathepsin D, there were low levels of M6P-containing proteins in cell extracts and media of cultured fibroblasts of the Patients. Direct sequencing identified a novel homozygous mutation in intron 7, IVS7-10G>A, of the GNPTG gene, which encodes the gamma-subunit of the GlcNAc-1-phosphotransferase. This mutation created a cryptic 3'-splice site resulting in a frameshift and premature translational termination (p.V176GfsX18). The GNPTG mRNA levels were markedly reduced in Patients' fibroblasts indicating that the intronic mutation mediates mRNA decay, which was confirmed by absence of the gamma-subunit protein. These data contribute to an efficient diagnostic strategy to identify Patients with MLIII gamma and characterize their biochemical defect in fibroblasts.