Concomitant gain and loss of function pathomechanisms in C9ORF72 amyotrophic lateral sclerosis.

Intronic hexanucleotide repeat expansions (HREs) in C9ORF72 are the most frequent genetic cause of amyotrophic lateral sclerosis, a devastating, incurable motoneuron (MN) disease. The mechanism by which HREs trigger pathogenesis remains elusive. The discovery of repeat-associated non-ATG (RAN) translation of dipeptide repeat proteins (DPRs) from HREs along with reduced exonic C9ORF72 expression suggests gain of toxic functions (GOFs) through DPRs versus loss of C9ORF72 functions (LOFs). Through multiparametric high-content (HC) live profiling in spinal MNs from induced pluripotent stem cells and comparison to mutant FUS and TDP43, we show that HRE C9ORF72 caused a distinct, later spatiotemporal appearance of mainly proximal axonal organelle motility deficits concomitant to augmented DNA double-strand breaks (DSBs), RNA foci, DPRs, and apoptosis. We show that both GOFs and LOFs were necessary to yield the overall C9ORF72 pathology. Increased RNA foci and DPRs concurred with onset of axon trafficking defects, DSBs, and cell death, although DSB induction itself did not phenocopy C9ORF72 mutants. Interestingly, the majority of LOF-specific DEGs were shared with HRE-mediated GOF DEGs. Finally, C9ORF72 LOF was sufficient-albeit to a smaller extent-to induce premature distal axonal trafficking deficits and increased DSBs.

Knocking out C9ORF72 Exacerbates Axonal Trafficking Defects Associated with Hexanucleotide Repeat Expansion and Reduces Levels of Heat Shock Proteins.

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.

Viral Infections Exacerbate FUS-ALS Phenotypes in iPSC-Derived Spinal Neurons in a Virus Species-Specific Manner.

Amyotrophic lateral sclerosis (ALS) arises from an interplay of genetic mutations and environmental factors. ssRNA viruses are possible ALS risk factors, but testing their interaction with mutations such as in FUS, which encodes an RNA-binding protein, has been difficult due to the lack of a human disease model. Here, we use isogenic induced pluripotent stem cell (iPSC)-derived spinal neurons (SNs) to investigate the interaction between ssRNA viruses and mutant FUS. We find that rabies virus (RABV) spreads ALS phenotypes, including the formation of stress granules (SGs) with aberrant composition due to increased levels of FUS protein, as well as neurodegeneration and reduced restriction activity by FUS mutations. Consistent with this, iPSC-derived SNs harboring mutant FUS are more sensitive to human immunodeficiency virus (HIV-1) and Zika viruses (ZIKV). We demonstrate that RABV and HIV-1 exacerbate cytoplasmic mislocalization of FUS. Our results demonstrate that viral infections worsen ALS pathology in SNs with genetic risk factors, suggesting a novel role for viruses in modulating patient phenotypes.

Altered calcium dynamics and glutamate receptor properties in iPSC-derived motor neurons from ALS patients with C9orf72, FUS, SOD1 or TDP43 mutations.

The fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS) is characterized by a profound loss of motor neurons (MNs). Until now only riluzole minimally extends life expectancy in ALS, presumably by inhibiting glutamatergic neurotransmission and calcium overload of MNs. Therefore, the aim of this study was to investigate the glutamate receptor properties and key aspects of intracellular calcium dynamics in induced pluripotent stem cell (iPSC)-derived MNs from ALS patients with C9orf72 (n = 4 cell lines), fused in sarcoma (FUS) (n = 9), superoxide dismutase 1 (SOD1) (n = 3) or transactive response DNA-binding protein 43 (TDP43) (n = 3) mutations as well as healthy (n = 7 cell lines) and isogenic controls (n = 3). Using calcium imaging, we most frequently observed spontaneous transients in mutant C9orf72 MNs. Basal intracellular calcium levels and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-induced signal amplitudes were elevated in mutant TDP43 MNs. Besides, a majority of mutant TDP43 MNs responded to 3.5-dihydroxyphenylglycine as metabotropic glutamate receptor agonist. Quantitative real-time PCR demonstrated significantly increased expression levels of AMPA and kainate receptors in mutant FUS cells compared to healthy and isogenic controls. Furthermore, the expression of kainate receptors and voltage gated calcium channels in mutant C9orf72 MNs as well as metabotropic glutamate receptors in mutant SOD1 cells was markedly elevated compared to controls. Our data of iPSC-derived MNs from familial ALS patients revealed several mutation-specific alterations in glutamate receptor properties and calcium dynamics that could play a role in ALS pathogenesis and may lead to future translational strategies with individual stratification of neuroprotective ALS treatments.

FUS pathology in ALS is linked to alterations in multiple ALS-associated proteins and rescued by drugs stimulating autophagy.

Amyotrophic lateral sclerosis (ALS) is a lethal disease characterized by motor neuron degeneration and associated with aggregation of nuclear RNA-binding proteins (RBPs), including FUS. How FUS aggregation and neurodegeneration are prevented in healthy motor neurons remain critically unanswered questions. Here, we use a combination of ALS patient autopsy tissue and induced pluripotent stem cell-derived neurons to study the effects of FUS mutations on RBP homeostasis. We show that FUS' tendency to aggregate is normally buffered by interacting RBPs, but this buffering is lost when FUS mislocalizes to the cytoplasm due to ALS mutations. The presence of aggregation-prone FUS in the cytoplasm causes imbalances in RBP homeostasis that exacerbate neurodegeneration. However, enhancing autophagy using small molecules reduces cytoplasmic FUS, restores RBP homeostasis and rescues motor function in vivo. We conclude that disruption of RBP homeostasis plays a critical role in FUS-ALS and can be treated by stimulating autophagy.

Dual Inhibition of GSK3ß and CDK5 Protects the Cytoskeleton of Neurons from Neuroinflammatory-Mediated Degeneration In Vitro and In Vivo.

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 GSK3ß is required to enhance the neuroprotective effects of CDK5 inhibition, which was confirmed using short hairpin RNA-mediated knockdown of CDK5 and GSK3ß. 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 GSK3ß in mediating the regulation of the neuronal cytoskeleton and that our hit compound LDC8 represents a promising starting point for neuroprotective drugs.

Phenotypic Screening Using Mouse and Human Stem Cell-Based Models of Neuroinflammation and Gene Expression Analysis to Study Drug Responses.

High-throughput phenotypic screening enables the identification of new therapeutic targets even when the molecular mechanism underlying the disease is unknown. In the case of neurodegenerative disease, there is a dire need to identify new targets that can ameliorate, halt, or reverse degeneration. Stem cell-based disease models are particularly powerful tools for phenotypic screening because they use the same cell type affected in patients. Here, we describe the expansion of mouse stem cells and human induced pluripotent stem cells as well as the differentiation of these cells into neural lineages that, when exposed to neuroinflammatory stress, can be used for compound screening followed by hit identification, validation, and target deconvolution.

Dynarrestin, a Novel Inhibitor of Cytoplasmic Dynein.

Aberrant hedgehog (Hh) signaling contributes to the pathogenesis of multiple cancers. Available inhibitors target Smoothened (Smo), which can acquire mutations causing drug resistance. Thus, compounds that inhibit Hh signaling downstream of Smo are urgently needed. We identified dynarrestin, a novel inhibitor of cytoplasmic dyneins 1 and 2. Dynarrestin acts reversibly to inhibit cytoplasmic dynein 1-dependent microtubule binding and motility in vitro without affecting ATP hydrolysis. It rapidly and reversibly inhibits endosome movement in living cells and perturbs mitosis by inducing spindle misorientation and pseudoprometaphase delay. Dynarrestin reversibly inhibits cytoplasmic dynein 2-dependent intraflagellar transport (IFT) of the cargo IFT88 and flux of Smo within cilia without interfering with ciliogenesis and suppresses Hh-dependent proliferation of neuronal precursors and tumor cells. As such, dynarrestin is a valuable tool for probing cytoplasmic dynein-dependent cellular processes and a promising compound for medicinal chemistry programs aimed at development of anti-cancer drugs.

Heterodimerization of Munc13 C2A domain with RIM regulates synaptic vesicle docking and priming.

The presynaptic active zone protein Munc13 is essential for neurotransmitter release, playing key roles in vesicle docking and priming. Mechanistically, it is thought that the C2A domain of Munc13 inhibits the priming function by homodimerization, and that RIM disrupts the autoinhibitory homodimerization forming monomeric priming-competent Munc13. However, it is unclear whether the C2A domain mediates other Munc13 functions in addition to this inactivation-activation switch. Here, we utilize mutations that modulate the homodimerization and heterodimerization states to define additional roles of the Munc13 C2A domain. Using electron microscopy and electrophysiology in hippocampal cultures, we show that the C2A domain is critical for additional steps of vesicular release, including vesicle docking. Optimal vesicle docking and priming is only possible when Munc13 heterodimerizes with RIM via its C2A domain. Beyond being a switching module, our data suggest that the Munc13-RIM heterodimer is an active component of the vesicle docking, priming and release complex.