Lysosomal and network alterations in human mucopolysaccharidosis type VII iPSC-derived neurons

Abstract

Mucopolysaccharidosis type VII (MPS VII) is a lysosomal storage disease caused by deficient β-glucuronidase (β-gluc) activity. Significantly reduced β-gluc activity leads to accumulation of glycosaminoglycans (GAGs) in many tissues, including the brain. Numerous combinations of mutations in GUSB (the gene that codes for β-gluc) cause a range of neurological features that make disease prognosis and treatment challenging. Currently, there is little understanding of the molecular basis for MPS VII brain anomalies. To identify a neuronal phenotype that could be used to complement genetic analyses, we generated two iPSC clones derived from skin fibroblasts of an MPS VII patient. We found that MPS VII neurons exhibited reduced β-gluc activity and showed previously established disease-associated phenotypes, including GAGs accumulation, expanded endocytic compartments, accumulation of lipofuscin granules, more autophagosomes, and altered lysosome function. Addition of recombinant β-gluc to MPS VII neurons, which mimics enzyme replacement therapy, restored disease-associated phenotypes to levels similar to the healthy control. MPS VII neural cells cultured as 3D neurospheroids showed upregulated GFAP gene expression, which was associated with astrocyte reactivity, and downregulation of GABAergic neuron markers. Spontaneous calcium imaging analysis of MPS VII neurospheroids showed reduced neuronal activity and altered network connectivity in patient-derived neurospheroids compared to a healthy control. These results demonstrate the interplay between reduced β-gluc activity, GAG accumulation and alterations in neuronal activity, and provide a human experimental model for elucidating the bases of MPS VII-associated cognitive defects.

Neuropathophysiology of Lysosomal Storage Diseases: Synaptic Dysfunction as a Starting Point for Disease Progression

“…[85] generated 3-dimentional neurospheres from two patient iPSCs cell lines. The MPS VII iPSC-derived neurons displayed the typical features associated with the disease phenotype like deficient β-glucuronidase activity, increased GAG accumulation, ultrastructural aberrations in the lysosome, expanded endocytic compartments, and increased autophagosomes [85]. Ratiometric calcium imaging demonstrated that MPS VII iPSC neurospheroids displayed levels of cytosolic calcium and KCl-induced depolarization similar to those of normal controls, suggesting identical synaptic vesicle trafficking process.…”

“…Reduced levels of synaptophysin were also reported in the mouse model of MPS IIIB [84], in human iPSC-derived neurons from MPS VII patients [85] and in the occipital and parietal lobes of sheep with NCL (CLN6) [86]. Two different mechanisms have been proposed to explain this reduction.…”

“…However, upon measuring spontaneous activity, a lesser number of MPS VIII neurospheroids were found to elicit calcium signals with a significantly reduced peak amplitude. A weaker neuronal connectivity pattern was also detected in MPS VII neurospheroids, indicating the possibility of an inherent altered synaptic activity [85].…”

“…They suggested that in the neurons HS oligosaccharides activate the degradation of synaptophysin by the proteasome, which leads to its reduction [84]. In contrast, Bayo-Puxan et al (2018) explained lower synaptophysin levels in MPSVII cultured neurons by the reduced gene expression [85]. In Npc1 -/mice, aggregations of synaptophysin were present in regions of the brain with reactive gliosis, including the striatum, substantia nigra, white matter tracts, and the thalamus [38].…”

“…The wide variety of methods adopted to achieve differentiation of iPSCs to a panoply of different neuronal types is another major advantage, as it allows to study mechanisms of dysfunction in the particular cell types, and detect the one most vulnerable during the disease progression. Examples of such "Achilles" heel include midbrain dopaminergic neurons in Gaucher disease [130], Purkinje cells in NPC1 and α-mannosidosis [131,132], medium spiny GABAergic neurons in G M1 gangliosidosis [133], hippocampal neurons (from the dentate gyrus and CA3) in MPS and NCL, and cortical pyramidal neurons in NCL, Tay-Sachs and MPS [65,85,134,135]. Recent advances in the above-mentioned diseases pertaining to the use of iPSC models are described in detail below.…”