Hepatocyte-intrinsic SMN deficiency drives metabolic dysfunction and liver steatosis in spinal muscular atrophy.

Spinal Muscular Atrophy (SMA) is typically characterized as a motor neuron disease, but extra-neuronal phenotypes are present in almost every organ in severely affected patients and animal models. Extra-neuronal phenotypes were previously underappreciated as patients with severe SMA phenotypes usually died in infancy; however, with current treatments for motor neurons increasing patient lifespan, impaired function of peripheral organs may develop into significant future comorbidities and lead to new treatment-modified phenotypes. Fatty liver is seen in SMA animal models , but generalizability to patients and whether this is due to hepatocyte-intrinsic Survival Motor Neuron (SMN) protein deficiency and/or subsequent to skeletal muscle denervation is unknown. If liver pathology in SMA is SMN-dependent and hepatocyte-intrinsic, this suggests SMN repleting therapies must target extra-neuronal tissues and motor neurons for optimal patient outcome. Here we showed that fatty liver is present in SMA and that SMA patient-specific iHeps were susceptible to steatosis. Using proteomics, functional studies and CRISPR/Cas9 gene editing, we confirmed that fatty liver in SMA is a primary SMN-dependent hepatocyte-intrinsic liver defect associated with mitochondrial and other hepatic metabolism implications. These pathologies require monitoring and indicate need for systematic clinical surveillance and additional and/or combinatorial therapies to ensure continued SMA patient health.

Hyaluronidase-1-mediated glycocalyx impairment underlies endothelial abnormalities in polypoidal choroidal vasculopathy.

BACKGROUND: Polypoidal choroidal vasculopathy (PCV), a subtype of age-related macular degeneration (AMD), is a global leading cause of vision loss in older populations. Distinct from typical AMD, PCV is characterized by polyp-like dilatation of blood vessels and turbulent blood flow in the choroid of the eye. Gold standard anti-vascular endothelial growth factor (anti-VEGF) therapy often fails to regress polypoidal lesions in patients. Current animal models have also been hampered by their inability to recapitulate such vascular lesions. These underscore the need to identify VEGF-independent pathways in PCV pathogenesis. RESULTS: We cultivated blood outgrowth endothelial cells (BOECs) from PCV patients and normal controls to serve as our experimental disease models. When BOECs were exposed to heterogeneous flow, single-cell transcriptomic analysis revealed that PCV BOECs preferentially adopted migratory-angiogenic cell state, while normal BOECs undertook proinflammatory cell state. PCV BOECs also had a repressed protective response to flow stress by demonstrating lower mitochondrial functions. We uncovered that elevated hyaluronidase-1 in PCV BOECs led to increased degradation of hyaluronan, a major component of glycocalyx that interfaces between flow stress and vascular endothelium. Notably, knockdown of hyaluronidase-1 in PCV BOEC improved mechanosensitivity, as demonstrated by a significant 1.5-fold upregulation of Krüppel-like factor 2 (KLF2) expression, a flow-responsive transcription factor. Activation of KLF2 might in turn modulate PCV BOEC migration. Barrier permeability due to glycocalyx impairment in PCV BOECs was also reversed by hyaluronidase-1 knockdown. Correspondingly, hyaluronidase-1 was detected in PCV patient vitreous humor and plasma samples. CONCLUSIONS: Hyaluronidase-1 inhibition could be a potential therapeutic modality in preserving glycocalyx integrity and endothelial stability in ocular diseases with vascular origin.

Mitchell-Riley syndrome iPSCs exhibit reduced pancreatic endoderm differentiation due to a mutation in RFX6.

Mitchell-Riley syndrome (MRS) is caused by recessive mutations in the regulatory factor X6 gene (RFX6) and is characterised by pancreatic hypoplasia and neonatal diabetes. To determine why individuals with MRS specifically lack pancreatic endocrine cells, we micro-CT imaged a 12-week-old foetus homozygous for the nonsense mutation RFX6 c.1129C>T, which revealed loss of the pancreas body and tail. From this foetus, we derived iPSCs and show that differentiation of these cells in vitro proceeds normally until generation of pancreatic endoderm, which is significantly reduced. We additionally generated an RFX6HA reporter allele by gene targeting in wild-type H9 cells to precisely define RFX6 expression and in parallel performed in situ hybridisation for RFX6 in the dorsal pancreatic bud of a Carnegie stage 14 human embryo. Both in vitro and in vivo, we find that RFX6 specifically labels a subset of PDX1-expressing pancreatic endoderm. In summary, RFX6 is essential for efficient differentiation of pancreatic endoderm, and its absence in individuals with MRS specifically impairs formation of endocrine cells of the pancreas head and tail.

Loss-of-function mutations in UDP-Glucose 6-Dehydrogenase cause recessive developmental epileptic encephalopathy.

Developmental epileptic encephalopathies are devastating disorders characterized by intractable epileptic seizures and developmental delay. Here, we report an allelic series of germline recessive mutations in UGDH in 36 cases from 25 families presenting with epileptic encephalopathy with developmental delay and hypotonia. UGDH encodes an oxidoreductase that converts UDP-glucose to UDP-glucuronic acid, a key component of specific proteoglycans and glycolipids. Consistent with being loss-of-function alleles, we show using patients' primary fibroblasts and biochemical assays, that these mutations either impair UGDH stability, oligomerization, or enzymatic activity. In vitro, patient-derived cerebral organoids are smaller with a reduced number of proliferating neuronal progenitors while mutant ugdh zebrafish do not phenocopy the human disease. Our study defines UGDH as a key player for the production of extracellular matrix components that are essential for human brain development. Based on the incidence of variants observed, UGDH mutations are likely to be a frequent cause of recessive epileptic encephalopathy.

Phenotypic and molecular features underlying neurodegeneration of motor neurons derived from spinal and bulbar muscular atrophy patients.

Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disease caused by the expansion of polyglutamine region in the androgen receptor. To gain insights into mechanisms of SBMA, four wild-type and five SBMA iPSC lines were differentiated to spinal motor neurons (sMNs) with high efficiency. SBMA sMNs showed neurite defects, reduced sMN survival and decreased protein synthesis levels. Microarray analysis revealed a dysregulation in various neuronal-related signalling pathways in SBMA sMNs. Strikingly, FAM135B a novel gene of unknown function, was found drastically downregulated in SBMA sMNs. Knockdown of FAM135B in wild-type sMNs reduced their survival and contributed to neurite defects, similar to SBMA sMNs, suggesting a functional role of FAM135B in SBMA. The degenerative phenotypes and dysregulated genes revealed could be potential therapeutic targets for SBMA.

Generation of sibling-matched induced pluripotent stem cell lines from spinal and bulbar muscular atrophy patients.

Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disease caused by the expansion of CAG repeats in the Androgen Receptor gene (AR). We report the generation of induced pluripotent stem cell (iPSC) lines from two SBMA patients and their healthy siblings. The SBMA and healthy iPSC lines retain the number of AR CAG repeats, express pluripotency markers and are able to differentiate into the three germ layers. The iPSC lines are also free of Sendai virus transgenes and have normal karyotypes. The SBMA iPSC lines with their sibling-matched controls would serve as useful tools to study SBMA disease mechanism.

Enumeration of Neural Stem Cells Using Clonal Assays.

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages - astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Purification, Visualization, and Molecular Signature of Neural Stem Cells.

Neural stem cells (NSCs) are isolated from primary brain tissue and propagated as a heterogeneous mix of cells, including neural progenitors. To date, NSCs have not been purified in vitro to allow study of their biology and utility in regenerative medicine. In this study, we identify C1qR1 as a novel marker for NSCs and show that it can be used along with Lewis-X (LeX) to yield a highly purified population of NSCs. Using time-lapse microscopy, we are able to follow NSCs forming neurospheres, allowing their visualization. Finally, using single-cell polymerase chain reaction (PCR), we determine the molecular signature of NSCs. The single-cell PCR data suggest that along with the Notch and Shh pathways, the Hippo pathway plays an important role in NSC activity.

A fluorescent probe for imaging symmetric and asymmetric cell division in neurosphere formation.

We report here a novel fluorescent chemical probe which stains distinct neural stem/progenitor cells (NSPCs) by binding to acid ceramidase in mouse neurospheres. is distributed evenly or unevenly to the daughter cells during multiple mitoses enabling the live imaging of symmetric and asymmetric divisions of isolated NSPCs.

Neural stem cell survival factors.

Neural stem and progenitor cells (NSCs and NPs) give rise to the central nervous system (CNS) during embryonic development. NSCs and NPs differentiate into three main cell-types of the CNS; astrocytes, oligodendrocytes, and neurons. NSCs are present in the adult CNS and are important in maintenance and repair. Adult NSCs hold great promise for endogenous or self-repair of the CNS. Intriguingly, NSCs have been implicated as the cells that give rise to brain tumors. Thus, the balance between survival, growth and differentiation is a critical aspect of NSC biology, during development, in the adult, and in disease processes. In this review, we survey what is known about survival factors that control both embryonic and adult NSCs. We discuss the neurosphere culture system as this is widely used to measure NSC activity and behavior in vitro and emphasize the importance of clonality. We define here NSC survival factors in their broadest sense to include any factor that influences survival and proliferation of NSCs and NPs. NSC survival factors identified to date include growth factors, morphogens, proteoglycans, cytokines, hormones, and neurotransmitters. Understanding NSC and NP interaction in response to these survival factors will provide insight to CNS development, disease and repair.

Single-cell mRNA profiling identifies progenitor subclasses in neurospheres.

Neurospheres are widely used to propagate and investigate neural stem cells (NSCs) and neural progenitors (NPs). However, the exact cell types present within neurospheres are still unknown. To identify cell types, we used single-cell mRNA profiling of 48 genes in 187 neurosphere cells. Using a clustering algorithm, we identified 3 discrete cell populations within neurospheres. One cell population [cluster unsorted (US) 1] expresses high Bmi1 and Hes5 and low Myc and Klf12. Cluster US2 shows intermediate expression of most of the genes analyzed. Cluster US3 expresses low Bmi1 and Hes5 and high Myc and Klf12. The mRNA profiles of these 3 cell populations correlate with a developmental timeline of early, intermediate, and late NPs, as seen in vivo from the mouse brain. We enriched the cell population for neurosphere-forming cells (NFCs) using morphological criteria of forward scatter (FSC) and side scatter (SSC). FSC/SSC(high) cells generated 2.29-fold more neurospheres than FSC/SSC(low) cells at clonal density. FSC/SSC(high) cells were enriched for NSCs and Lewis-X(+ve) cells, possessed higher phosphacan levels, and were of a larger cell size. Clustering of both FSC/SSC(high) and FSC/SSC(low) cells identified an NFC cluster. Significantly, the mRNA profile of the NFC cluster drew close resemblance to that of early NPs. Taken together, data suggest that the neurosphere culture system can be used to model central nervous system development, and that early NPs are the cell population that gives rise to neurospheres. In future work, it may be possible to further dissect the NFCs and reveal the molecular signature for NSCs.