Search

Summary: In amyotrophic lateral sclerosis (ALS) motor neurons (MNs) undergo dying-back, where the distal axon degenerates before the soma. The hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common genetic cause of ALS, but the mechanism of pathogenesis is largely unknown with both gain- and loss-of-function mechanisms being proposed. To better understand C9ORF72-ALS pathogenesis, we generated isogenic induced pluripotent stem cells. MNs with HRE in C9ORF72 showed decreased axonal trafficking compared with gene corrected MNs. However, knocking out C9ORF72 did not recapitulate these changes in MNs from healthy controls, suggesting a gain-of-function mechanism. In contrast, knocking out C9ORF72 in MNs with HRE exacerbated axonal trafficking defects and increased apoptosis as well as decreased levels of HSP70 and HSP40, and inhibition of HSPs exacerbated ALS phenotypes in MNs with HRE. Therefore, we propose that the HRE in C9ORF72 induces ALS pathogenesis via a combination of gain- and loss-of-function mechanisms. : Sterneckert and colleagues generated isogenic induced pluripotent stem cell lines and demonstrated that MNs with hexanucleotide repeat expansion (HRE) in C9ORF72 show reduced axonal trafficking, which is not recapitulated by knocking out C9ORF72 in MNs from healthy individuals. In contrast, knocking out C9ORF72 exacerbated phenotypes in MNs with HRE, suggesting that both gain- and loss-of-function mechanisms contribute to C9ORF72-ALS. Keywords: C9ORF72, induced pluripotent stem cells, amyotrophic lateral sclerosis, gene editing, disease modeling, axonal trafficking, heat shock proteins, HSP70, HSP40

Summary: Neuroinflammation is a hallmark of neurological disorders and is accompanied by the production of neurotoxic agents such as nitric oxide. We used stem cell-based phenotypic screening and identified small molecules that directly protected neurons from neuroinflammation-induced degeneration. We demonstrate that inhibition of CDK5 is involved in, but not sufficient for, neuroprotection. Instead, additional inhibition of GSK3β is required to enhance the neuroprotective effects of CDK5 inhibition, which was confirmed using short hairpin RNA-mediated knockdown of CDK5 and GSK3β. Quantitative phosphoproteomics and high-content imaging demonstrate that neurite degeneration is mediated by aberrant phosphorylation of multiple microtubule-associated proteins. Finally, we show that our hit compound protects neurons in vivo in zebrafish models of motor neuron degeneration and Alzheimer's disease. Thus, we demonstrate an overlap of CDK5 and GSK3β in mediating the regulation of the neuronal cytoskeleton and that our hit compound LDC8 represents a promising starting point for neuroprotective drugs. : Neuroinflammation is a hallmark of many neurological disorders and can be neurotoxic. Sterneckert and colleagues perform a phenotypic screening campaign identifying compounds protecting motor neurons from inflammation. Compound profiling demonstrates that dual inhibition of CDK5 and GSK3β mediates neuroprotection. Quantitative phosphoproteomics demonstrates that neuroprotection is linked to alterations in microtubule dynamics mediated by phosphorylation of microtubule-associated proteins. Keywords: neurodegeneration, neuroinflammation, stem cell-based phenotypic screening, CDK5, GSK3β, induced pluripotent stem cells, ALS

Summary: Perturbations in stress granule (SG) dynamics may be at the core of amyotrophic lateral sclerosis (ALS). Since SGs are membraneless compartments, modeling their dynamics in human motor neurons has been challenging, thus hindering the identification of effective therapeutics. Here, we report the generation of isogenic induced pluripotent stem cells carrying wild-type and P525L FUS-eGFP. We demonstrate that FUS-eGFP is recruited into SGs and that P525L profoundly alters their dynamics. With a screening campaign, we demonstrate that PI3K/AKT/mTOR pathway inhibition increases autophagy and ameliorates SG phenotypes linked to P525L FUS by reducing FUS-eGFP recruitment into SGs. Using a Drosophila model of FUS-ALS, we corroborate that induction of autophagy significantly increases survival. Finally, by screening clinically approved drugs for their ability to ameliorate FUS SG phenotypes, we identify a number of brain-penetrant anti-depressants and anti-psychotics that also induce autophagy. These drugs could be repurposed as potential ALS treatments. : Sterneckert and colleagues generate isogenic FUS-eGFP reporter iPSCs that enable the identification of stress granule (SG) phenotypes specifically induced by the ALS mutation FUS P525L. Compound screening shows that modulation of the PI3K/AKT/mTOR pathway regulating autophagy ameliorates SG phenotypes. A second screen identifies similarly acting brain-penetrant US FDA-approved drugs that could be repurposed to treat ALS. Keywords: amyotrophic lateral sclerosis, induced pluripotent stem cells, FUS, stress granules, autophagy, gene editing, CRISPR/Cas9n