A new patient-derived iPSC model for dystroglycanopathies validates a compound that increases glycosylation of α-dystroglycan.

Dystroglycan, an extracellular matrix receptor, has essential functions in various tissues. Loss of α-dystroglycan-laminin interaction due to defective glycosylation of α-dystroglycan underlies a group of congenital muscular dystrophies often associated with brain malformations, referred to as dystroglycanopathies. The lack of isogenic human dystroglycanopathy cell models has limited our ability to test potential drugs in a human- and neural-specific context. Here, we generated induced pluripotent stem cells (iPSCs) from a severe dystroglycanopathy patient with homozygous FKRP (fukutin-related protein gene) mutation. We showed that CRISPR/Cas9-mediated gene correction of FKRP restored glycosylation of α-dystroglycan in iPSC-derived cortical neurons, whereas targeted gene mutation of FKRP in wild-type cells disrupted this glycosylation. In parallel, we screened 31,954 small molecule compounds using a mouse myoblast line for increased glycosylation of α-dystroglycan. Using human FKRP-iPSC-derived neural cells for hit validation, we demonstrated that compound 4-(4-bromophenyl)-6-ethylsulfanyl-2-oxo-3,4-dihydro-1H-pyridine-5-carbonitrile (4BPPNit) significantly augmented glycosylation of α-dystroglycan, in part through upregulation of LARGE1 glycosyltransferase gene expression. Together, isogenic human iPSC-derived cells represent a valuable platform for facilitating dystroglycanopathy drug discovery and therapeutic development.

Mutations in B3GALNT2 cause congenital muscular dystrophy and hypoglycosylation of α-dystroglycan.

Mutations in several known or putative glycosyltransferases cause glycosylation defects in α-dystroglycan (α-DG), an integral component of the dystrophin glycoprotein complex. The hypoglycosylation reduces the ability of α-DG to bind laminin and other extracellular matrix ligands and is responsible for the pathogenesis of an inherited subset of muscular dystrophies known as the dystroglycanopathies. By exome and Sanger sequencing we identified two individuals affected by a dystroglycanopathy with mutations in ß-1,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2). B3GALNT2 transfers N-acetyl galactosamine (GalNAc) in a ß-1,3 linkage to N-acetyl glucosamine (GlcNAc). A subsequent study of a separate cohort of individuals identified recessive mutations in four additional cases that were all affected by dystroglycanopathy with structural brain involvement. We show that functional dystroglycan glycosylation was reduced in the fibroblasts and muscle (when available) of these individuals via flow cytometry, immunoblotting, and immunocytochemistry. B3GALNT2 localized to the endoplasmic reticulum, and this localization was perturbed by some of the missense mutations identified. Moreover, knockdown of b3galnt2 in zebrafish recapitulated the human congenital muscular dystrophy phenotype with reduced motility, brain abnormalities, and disordered muscle fibers with evidence of damage to both the myosepta and the sarcolemma. Functional dystroglycan glycosylation was also reduced in the b3galnt2 knockdown zebrafish embryos. Together these results demonstrate a role for B3GALNT2 in the glycosylation of α-DG and show that B3GALNT2 mutations can cause dystroglycanopathy with muscle and brain involvement.

Mutations in GDP-mannose pyrophosphorylase B cause congenital and limb-girdle muscular dystrophies associated with hypoglycosylation of α-dystroglycan.

Congenital muscular dystrophies with hypoglycosylation of α-dystroglycan (α-DG) are a heterogeneous group of disorders often associated with brain and eye defects in addition to muscular dystrophy. Causative variants in 14 genes thought to be involved in the glycosylation of α-DG have been identified thus far. Allelic mutations in these genes might also cause milder limb-girdle muscular dystrophy phenotypes. Using a combination of exome and Sanger sequencing in eight unrelated individuals, we present evidence that mutations in guanosine diphosphate mannose (GDP-mannose) pyrophosphorylase B (GMPPB) can result in muscular dystrophy variants with hypoglycosylated α-DG. GMPPB catalyzes the formation of GDP-mannose from GTP and mannose-1-phosphate. GDP-mannose is required for O-mannosylation of proteins, including α-DG, and it is the substrate of cytosolic mannosyltransferases. We found reduced α-DG glycosylation in the muscle biopsies of affected individuals and in available fibroblasts. Overexpression of wild-type GMPPB in fibroblasts from an affected individual partially restored glycosylation of α-DG. Whereas wild-type GMPPB localized to the cytoplasm, five of the identified missense mutations caused formation of aggregates in the cytoplasm or near membrane protrusions. Additionally, knockdown of the GMPPB ortholog in zebrafish caused structural muscle defects with decreased motility, eye abnormalities, and reduced glycosylation of α-DG. Together, these data indicate that GMPPB mutations are responsible for congenital and limb-girdle muscular dystrophies with hypoglycosylation of α-DG.

Missense mutations in ß-1,3-N-acetylglucosaminyltransferase 1 (B3GNT1) cause Walker-Warburg syndrome.

Several known or putative glycosyltransferases are required for the synthesis of laminin-binding glycans on alpha-dystroglycan (αDG), including POMT1, POMT2, POMGnT1, LARGE, Fukutin, FKRP, ISPD and GTDC2. Mutations in these glycosyltransferase genes result in defective αDG glycosylation and reduced ligand binding by αDG causing a clinically heterogeneous group of congenital muscular dystrophies, commonly referred to as dystroglycanopathies. The most severe clinical form, Walker-Warburg syndrome (WWS), is characterized by congenital muscular dystrophy and severe neurological and ophthalmological defects. Here, we report two homozygous missense mutations in the ß-1,3-N-acetylglucosaminyltransferase 1 (B3GNT1) gene in a family affected with WWS. Functional studies confirmed the pathogenicity of the mutations. First, expression of wild-type but not mutant B3GNT1 in human prostate cancer (PC3) cells led to increased levels of αDG glycosylation. Second, morpholino knockdown of the zebrafish b3gnt1 orthologue caused characteristic muscular defects and reduced αDG glycosylation. These functional studies identify an important role of B3GNT1 in the synthesis of the uncharacterized laminin-binding glycan of αDG and implicate B3GNT1 as a novel causative gene for WWS.

GPX2 underexpression indicates poor prognosis in patients with urothelial carcinomas of the upper urinary tract and urinary bladder.

PURPOSE: Oxidative stress is believed to be one of the important etiologies in carcinogenesis that has not been systemically investigated in urothelial carcinoma (UC). Through data mining from a published transcriptomic database of UC of urinary bladders (UBUCs) (GSE31684), glutathione peroxidase 2 (GPX2) was identified as the most significant downregulated gene among those response to oxidative stress (GO:0006979). We therefore analyze GPX2 transcript and protein expressions and its clinicopathological significance. METHODS: Real-time RT-PCR assay was used to detect GPX2 mRNA level in 20 fresh UBUC specimens. Immunohistochemistry was used to determine GPX2 protein expression in 340 urothelial carcinomas of upper tracts (UTUCs) and 295 UBUCs with mean/median follow-up of 44.7/38.9 and 30.8/23.1 months, respectively. Its expression status was further correlated with clinicopathological features and evaluated for its impact on disease-specific survival and metastasis-free survival (MeFS). RESULTS: Decrease in GPX2 transcript level was associated with both higher pT and positive nodal status in 20 UBUCs (all p < 0.05). GPX2 protein underexpression was also significantly associated with advanced pT status, nodal metastasis, high histological grade, vascular invasion, and frequent mitoses in both groups of UCs (all p < 0.05). GPX2 underexpression not only predicted dismal DDS and MeFS at univariate analysis, but also implicated worse DDS (UTUC, p = 0.002; UBUC, p = 0.029) and MeFS (UTUC, p = 0.001; UBUC, p = 0.032) in multivariate analysis. CONCLUSIONS: GPX2 underexpression is associated with advanced tumor status and implicated unfavorable clinical outcome of UCs, suggesting its role in tumor progression and may serve as a theranostic biomarker of UCs.

Zebrafish Fukutin family proteins link the unfolded protein response with dystroglycanopathies.

Allelic mutations in putative glycosyltransferase genes, fukutin and fukutin-related protein (fkrp), lead to a wide range of muscular dystrophies associated with hypoglycosylation of α-dystroglycan, commonly referred to as dystroglycanopathies. Defective glycosylation affecting dystroglycan-ligand interactions is considered to underlie the disease pathogenesis. We have modelled dystroglycanopathies in zebrafish using a novel loss-of-function dystroglycan allele and by inhibition of Fukutin family protein activities. We show that muscle pathology in embryos lacking Fukutin or FKRP is different from loss of dystroglycan. In addition to hypoglycosylated α-dystroglycan, knockdown of Fukutin or FKRP leads to a notochord defect and a perturbation of laminin expression before muscle degeneration. These are a consequence of endoplasmic reticulum stress and activation of the unfolded protein response (UPR), preceding loss of dystroglycan-ligand interactions. Together, our results suggest that Fukutin family proteins may play important roles in protein secretion and that the UPR may contribute to the phenotypic spectrum of some dystroglycanopathies in humans.

3D Compartmentalised Human Pluripotent Stem Cell-derived Neuromuscular Co-cultures.

Human neuromuscular diseases represent a diverse group of disorders with unmet clinical need, ranging from muscular dystrophies, such as Duchenne muscular dystrophy (DMD), to neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS). In many of these conditions, axonal and neuromuscular synapse dysfunction have been implicated as crucial pathological events, highlighting the need for in vitro disease models that accurately recapitulate these aspects of human neuromuscular physiology. The protocol reported here describes the co-culture of neural spheroids composed of human pluripotent stem cell (PSC)-derived motor neurons and astrocytes, and human PSC-derived myofibers in 3D compartmentalised microdevices to generate functional human neuromuscular circuits in vitro. In this microphysiological model, motor axons project from a central nervous system (CNS)-like compartment along microchannels to innervate skeletal myofibers plated in a separate muscle compartment. This mimics the spatial organization of neuromuscular circuits in vivo. Optogenetics, particle image velocimetry (PIV) analysis, and immunocytochemistry are used to control, record, and quantify functional neuromuscular transmission, axonal outgrowth, and neuromuscular synapse number and morphology. This approach has been applied to study disease-specific phenotypes for DMD and ALS by incorporating patient-derived and CRISPR-corrected human PSC-derived motor neurons and skeletal myogenic progenitors into the model, as well as testing candidate drugs for rescuing pathological phenotypes. The main advantages of this approach are: i) its simple design; ii) the in vivo-like anatomical separation between CNS and peripheral muscle; and iii) the amenability of the approach to high power imaging. This opens up the possibility for carrying out live axonal transport and synaptic imaging assays in future studies, in addition to the applications reported in this study. Graphical abstract Graphical abstract abbreviations: Channelrhodopsin-2 (CHR2+), pluripotent stem cell (PSC), motor neurons (MNs), myofibers (MFs), neuromuscular junction (NMJ).

Nuclear Respiratory Factor 1 Overexpression Inhibits Proliferation and Migration of PC3 Prostate Cancer Cells.

BACKGROUND/AIM: The role of nuclear respiratory factor 1 (NRF1) on the prostate cancer progression is controversial. We aimed to investigate the effect of NRF1 overexpression on the metastasis potential of PC3 prostate cancer cells and the associated molecular mechanisms. MATERIALS AND METHODS: Cell survival, migration capacity, mitochondrial biogenesis, the expression of TGF-ß signaling and EMT markers were examined after overexpression and silencing of NRF1 in PC3 cells. RESULTS: We found that NRF1-overexpressing cells exhibited a decreased cell viability and proliferation ability as well as a reduced migration capacity compared to control cells. Moreover, ectopic expression of NRF1 increased the mitochondrial biogenesis and inhibited the EMT characteristics, including a decrease in the mesenchymal marker, α-SMA and an increase in the epithelial cell marker, E-cadherin. We also demonstrated that overexpression of NRF1 suppressed the expression of TGF-ß signaling in PC3 cells. As expected, silencing of NRF1 reversed the abovementioned effects. CONCLUSION: This study demonstrated that upregulation of NRF1 holds the potential to inhibit the metastasis of prostate cancer, possibly through an elevation of mitochondrial biogenesis and the subsequent repression of TGF-ß-associated EMT. Therapeutic avenues that increase NRF1 expression may serve as an adjunct to conventional treatments of prostate cancer.

CRISPR-mediated correction of skeletal muscle Ca2+ handling in a novel DMD patient-derived pluripotent stem cell model.

Mutations in the dystrophin gene cause the most common and currently incurable Duchenne muscular dystrophy (DMD) characterized by progressive muscle wasting. Although abnormal Ca2+ handling is a pathological feature of DMD, mechanisms underlying defective Ca2+ homeostasis remain unclear. Here we generate a novel DMD patient-derived pluripotent stem cell (PSC) model of skeletal muscle with an isogenic control using clustered regularly interspaced short palindromic repeat (CRISPR)-mediated precise gene correction. Transcriptome analysis identifies dysregulated gene sets in the absence of dystrophin, including genes involved in Ca2+ handling, excitation-contraction coupling and muscle contraction. Specifically, analysis of intracellular Ca2+ transients and mathematical modeling of Ca2+ dynamics reveal significantly reduced cytosolic Ca2+ clearance rates in DMD-PSC derived myotubes. Pharmacological assays demonstrate Ca2+ flux in myotubes is determined by both intracellular and extracellular sources. DMD-PSC derived myotubes display significantly reduced velocity of contractility. Compared with a non-isogenic wildtype PSC line, these pathophysiological defects could be rescued by CRISPR-mediated precise gene correction. Our study provides new insights into abnormal Ca2+ homeostasis in DMD and suggests that Ca2+ signaling pathways amenable to pharmacological modulation are potential therapeutic targets. Importantly, we have established a human physiology-relevant in vitro model enabling rapid pre-clinical testing of potential therapies for DMD.

Investigating the role of dystrophin isoform deficiency in motor function in Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is caused by DMD mutations leading to dystrophin loss. Full-length Dp427 is the primary dystrophin isoform expressed in muscle and is also expressed in the central nervous system (CNS). Two shorter isoforms, Dp140 and Dp71, are highly expressed in the CNS. While a role for Dp140 and Dp71 on DMD CNS comorbidities is well known, relationships between mutations expected to disrupt Dp140 and Dp71 and motor outcomes are not. METHODS: Functional outcome data from 387 DMD boys aged 4-15 years were subdivided by DMD mutation expected effects on dystrophin isoform expression; Group 1 (Dp427 absent, Dp140/Dp71 present, n = 201); Group 2 (Dp427/Dp140 absent, Dp71 present, n = 152); and Group 3 (Dp427/Dp140/Dp71 absent, n = 34). Relationships between isoform group and North Star ambulatory assessment (NSAA) scores, 10 m walk/run velocities and rise time velocities were explored using regression analysis. Western blot analysis was used to study Dp427, Dp140 and Dp71 production in myogenic cells (control and DMD human), control skeletal muscle, DMD skeletal muscle from the three isoform groups and cerebral cortex from mice (wild-type and DMD models). Grip strength and rotarod running test were studied in wild-type mice and DMD mouse models. DMD mouse models were mdx (Dp427 absent, Dp140/Dp71 present), mdx52 (Dp427/Dp140 absent, Dp71 present) and DMD-null (lacking all isoforms). RESULTS: In DMD boys, mean NSAA scores at 5 years of age were 6.1 points lower in Group 3 than Group 1 (P < 0.01) and 4.9 points lower in Group 3 than Group 2 (P = 0.05). Mean peak NSAA scores were 4.0 points lower in Group 3 than Group 1 (P < 0.01) and 1.6 points lower in Group 2 than Group 1 (P = 0.04). Mean four-limb grip strength was 1.5 g/g lower in mdx52 than mdx mice (P = 0.003) and 1.5 g/g lower in DMD-null than mdx mice (P = 0.002). Dp71 was produced in myogenic cells (control and DMD human) and skeletal muscle from humans in Groups 1 and 2 and mdx mice, but not skeletal muscle from human controls, myogenic cells and skeletal muscle from humans in Group 3 or skeletal muscle from wild-type, mdx52 or DMD-null mice. CONCLUSIONS: Our results highlight the importance of considering expected effects of DMD mutations on dystrophin isoform production when considering patterns of DMD motor impairment and the implications for clinical practice and clinical trials. Our results suggest a complex relationship between dystrophin isoforms expressed in the brain and DMD motor function.

Optogenetic modeling of human neuromuscular circuits in Duchenne muscular dystrophy with CRISPR and pharmacological corrections.

Duchenne muscular dystrophy (DMD) is caused by dystrophin gene mutations leading to skeletal muscle weakness and wasting. Dystrophin is enriched at the neuromuscular junction (NMJ), but how NMJ abnormalities contribute to DMD pathogenesis remains unclear. Here, we combine transcriptome analysis and modeling of DMD patient-derived neuromuscular circuits with CRISPR-corrected isogenic controls in compartmentalized microdevices. We show that NMJ volumes and optogenetic motor neuron­stimulated myofiber contraction are compromised in DMD neuromuscular circuits, which can be rescued by pharmacological inhibition of TGFß signaling, an observation validated in a 96-well human neuromuscular circuit coculture assay. These beneficial effects are associated with normalization of dysregulated gene expression in DMD myogenic transcriptomes affecting NMJ assembly (e.g., MUSK) and axon guidance (e.g., SLIT2 and SLIT3). Our study provides a new human microphysiological model for investigating NMJ defects in DMD and assessing candidate drugs and suggests that enhancing neuromuscular connectivity may be an effective therapeutic strategy.

Comparative epigenetic analysis of tumour initiating cells and syngeneic EPSC-derived neural stem cells in glioblastoma.

Epigenetic mechanisms which play an essential role in normal developmental processes, such as self-renewal and fate specification of neural stem cells (NSC) are also responsible for some of the changes in the glioblastoma (GBM) genome. Here we develop a strategy to compare the epigenetic and transcriptional make-up of primary GBM cells (GIC) with patient-matched expanded potential stem cell (EPSC)-derived NSC (iNSC). Using a comparative analysis of the transcriptome of syngeneic GIC/iNSC pairs, we identify a glycosaminoglycan (GAG)-mediated mechanism of recruitment of regulatory T cells (Tregs) in GBM. Integrated analysis of the transcriptome and DNA methylome of GBM cells identifies druggable target genes and patient-specific prediction of drug response in primary GIC cultures, which is validated in 3D and in vivo models. Taken together, we provide a proof of principle that this experimental pipeline has the potential to identify patient-specific disease mechanisms and druggable targets in GBM.

Molecular dissection of Drosophila Prickle isoforms distinguishes their essential and overlapping roles in planar cell polarity.

Prickle-Spiny-Legs (Pk) is an essential component of the planar cell polarity (PCP) pathway, together with Frizzled (Fz) and Dishevelled (Dsh). A role for Pk was proposed to mediate feedback amplification of asymmetric Fz/Dsh activity across cell boundaries, ensuring a single prehair initiates at each distal vertex. Here we show that apical localisation of Pk(Pk) and Pk(Sple) isoforms are mutually independent and regulated by the C-terminal domain. The N-terminus of Pk(Pk) is dispensable for PCP, whereas the unique N-terminal domain of Pk(Sple) contains an additional localisation function, which confers a qualitatively different activity. Our results suggest that endogenous Pk(Pk) and Pk(Sple) can affect each other's function via the C-terminal domain, yet may not form heteromeric complexes. Overexpressing PET domain-deleted Pk variants interferes with a branch of Fz/Dsh signalling that regulates the number of wing hairs, and blocks non-cell-autonomous repolarisation. We infer that Pk(Pk) is sufficient to mediate the intercellular feedback signalling. Significantly, Pk(Pk) but not Pk(Sple) is required for hexagonal cell packing in the pupal wing. We propose that Fz-dependent PCP readout reflects short-range, cell-contact based, interactions between hexagonal cells, rather than a direct response to an as yet unidentified diffusible ligand.

The functional O-mannose glycan on α-dystroglycan contains a phospho-ribitol primed for matriglycan addition.

Multiple glycosyltransferases are essential for the proper modification of alpha-dystroglycan, as mutations in the encoding genes cause congenital/limb-girdle muscular dystrophies. Here we elucidate further the structure of an O-mannose-initiated glycan on alpha-dystroglycan that is required to generate its extracellular matrix-binding polysaccharide. This functional glycan contains a novel ribitol structure that links a phosphotrisaccharide to xylose. ISPD is a CDP-ribitol (ribose) pyrophosphorylase that generates the reduced sugar nucleotide for the insertion of ribitol in a phosphodiester linkage to the glycoprotein. TMEM5 is a UDP-xylosyl transferase that elaborates the structure. We demonstrate in a zebrafish model as well as in a human patient that defects in TMEM5 result in muscular dystrophy in combination with abnormal brain development. Thus, we propose a novel structure-a ribitol in a phosphodiester linkage-for the moiety on which TMEM5, B4GAT1, and LARGE act to generate the functional receptor for ECM proteins having LG domains.

Muscle diseases in the zebrafish.

Animal models in biomedical research are important for understanding the pathological mechanisms of human diseases at a molecular and cellular level. Several aspects of mammalian animals, however, may limit their use in modelling neuromuscular disorders. Many attributes of zebrafish (Danio rerio) are complementary to mammalian experimental systems, establishing the zebrafish as a powerful model organism in disease biology. This review focuses on a number of key studies using the zebrafish to model hereditary muscle diseases with additional emphasis on recent advances in zebrafish functional genomics and drug discovery. Increasing research in zebrafish disease models, combined with knowledge from mammalian models, will bring novel insights into the disease pathogenesis of neuromuscular disorders, as well as facilitate the development of effective therapeutic strategies.

Mutations in ISPD cause Walker-Warburg syndrome and defective glycosylation of α-dystroglycan.

Walker-Warburg syndrome (WWS) is an autosomal recessive multisystem disorder characterized by complex eye and brain abnormalities with congenital muscular dystrophy (CMD) and aberrant a-dystroglycan glycosylation. Here we report mutations in the ISPD gene (encoding isoprenoid synthase domain containing) as the second most common cause of WWS. Bacterial IspD is a nucleotidyl transferase belonging to a large glycosyltransferase family, but the role of the orthologous protein in chordates is obscure to date, as this phylum does not have the corresponding non-mevalonate isoprenoid biosynthesis pathway. Knockdown of ispd in zebrafish recapitulates the human WWS phenotype with hydrocephalus, reduced eye size, muscle degeneration and hypoglycosylated a-dystroglycan. These results implicate ISPD in a-dystroglycan glycosylation in maintaining sarcolemma integrity in vertebrates.