Aberrant NOVA1 function disrupts alternative splicing in early stages of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by aberrant alternative splicing (AS). Nuclear loss and cytoplasmic accumulation of the splicing factor TDP-43 in motor neurons (MN) are hallmarks of ALS at late stages of the disease. However, it is unknown if altered AS is present before TDP-43 pathology occurs. Here, we investigate altered AS and its origins in early stages of ALS using human induced pluripotent stem cell-derived motor neurons (MNs) from sporadic and familial ALS patients. We find high levels of the RNA-binding proteins NOVA1, NOVA2, and RBFOX2 in the insoluble protein fractions and observe that AS events in ALS-associated MNs are enriched for binding sites of these proteins. Our study points to an early disrupted function of NOVA1 that drives AS changes in a complex fashion, including events caused by a consistent loss of NOVA1 function. NOVA1 exhibits increased cytoplasmic protein levels in early stage MNs without TDP-43 pathology in ALS postmortem tissue. As nuclear TDP-43 protein level depletes, NOVA1 is reduced. Potential indications for a reduction of NOVA1 also came from mice over-expressing TDP-43 lacking its nuclear localization signal and iPSC-MN stressed with puromycin. This study highlights that additional RBP-RNA perturbations in ALS occur in parallel to TDP-43.

A novel human stem cell model for Coffin-Siris syndrome-like syndrome reveals the importance of SOX11 dosage for neuronal differentiation and survival.

The SOXC transcription factors Sox4, Sox11 and Sox12, are critical neurodevelopmental regulators that are thought to function in a highly redundant fashion. Surprisingly, heterozygous missense mutations or deletions of SOX11 were recently detected in patients with Coffin-Siris syndrome-like syndrome (CSSLS), a neurodevelopmental disorder associated with intellectual disability, demonstrating that in humans SOX11 haploinsufficiency cannot be compensated and raising the question of the function of SOX11 in human neurodevelopment. Here, we describe the generation of SOX11+/- heterozygous human embryonic stem cell (hESC) lines by CRISPR/Cas9 genome engineering. SOX11 haploinsufficiency impaired the generation of neurons and resulted in a proliferation/differentiation imbalance of neural precursor cells and enhanced neuronal cell death. Using the SOX11+/- hESC model we provide for the first time experimental evidence that SOX11 haploinsufficiency is sufficient to impair key processes of human neurodevelopment, giving a first insight into the pathophysiology of CSSLS and SOX11 function in human neurodevelopment.

GSK3ß-dependent dysregulation of neurodevelopment in SPG11-patient induced pluripotent stem cell model.

OBJECTIVE: Mutations in the spastic paraplegia gene 11 (SPG11), encoding spatacsin, cause the most frequent form of autosomal-recessive complex hereditary spastic paraplegia (HSP) and juvenile-onset amyotrophic lateral sclerosis (ALS5). When SPG11 is mutated, patients frequently present with spastic paraparesis, a thin corpus callosum, and cognitive impairment. We previously delineated a neurodegenerative phenotype in neurons of these patients. In the current study, we recapitulated early developmental phenotypes of SPG11 and outlined their cellular and molecular mechanisms in patient-specific induced pluripotent stem cell (iPSC)-derived cortical neural progenitor cells (NPCs). METHODS: We generated and characterized iPSC-derived NPCs and neurons from 3 SPG11 patients and 2 age-matched controls. RESULTS: Gene expression profiling of SPG11-NPCs revealed widespread transcriptional alterations in neurodevelopmental pathways. These include changes in cell-cycle, neurogenesis, cortical development pathways, in addition to autophagic deficits. More important, the GSK3ß-signaling pathway was found to be dysregulated in SPG11-NPCs. Impaired proliferation of SPG11-NPCs resulted in a significant diminution in the number of neural cells. The decrease in mitotically active SPG11-NPCs was rescued by GSK3 modulation. INTERPRETATION: This iPSC-derived NPC model provides the first evidence for an early neurodevelopmental phenotype in SPG11, with GSK3ß as a potential novel target to reverse the disease phenotype. Ann Neurol 2016;79:826-840.

An alternative splicing modulator decreases mutant HTT and improves the molecular fingerprint in Huntington's disease patient neurons.

Huntington's disease (HD) is a neurodegenerative disorder caused by poly-Q expansion in the Huntingtin (HTT) protein. Here, we delineate elevated mutant HTT (mHTT) levels in patient-derived cells including fibroblasts and iPSC derived cortical neurons using mesoscale discovery (MSD) HTT assays. HD patients' fibroblasts and cortical neurons recapitulate aberrant alternative splicing as a molecular fingerprint of HD. Branaplam is a splicing modulator currently tested in a phase II study in HD (NCT05111249). The drug lowers total HTT (tHTT) and mHTT levels in fibroblasts, iPSC, cortical progenitors, and neurons in a dose dependent manner at an IC50 consistently below 10 nM without inducing cellular toxicity. Branaplam promotes inclusion of non-annotated novel exons. Among these Branaplam-induced exons, there is a 115 bp frameshift-inducing exon in the HTT transcript. This exon is observed upon Branaplam treatment in Ctrl and HD patients leading to a profound reduction of HTT RNA and protein levels. Importantly, Branaplam ameliorates aberrant alternative splicing in HD patients' fibroblasts and cortical neurons. These findings highlight the applicability of splicing modulators in the treatment of CAG repeat disorders and decipher their molecular effects associated with the pharmacokinetic and -dynamic properties in patient-derived cellular models.

CRISPR/Cas9 mediated generation of human ARID1B heterozygous knockout hESC lines to model Coffin-Siris syndrome.

ARID1B haploinsufficiency induced by missense or nonsense mutations of ARID1B is a cause of Coffin-Siris syndrome (CSS), a neurodevelopmental disorder associated with intellectual disability. At present, no appropriate human stem cell model for ARID1B-associated CSS has been reported. Here, we describe the generation and validation of ARID1B+/- hESCs by introducing out of frame deletions into exon 5 or 6 of ARID1B with CRISPR/Cas9 genome editing. These ARID1B+/- hESC lines allow to study the pathophysiology of ARID1B-associated CSS in 2D and 3D models of human neurodevelopment.