Defining RNA oligonucleotides that reverse deleterious phase transitions of RNA-binding proteins with prion-like domains.

RNA-binding proteins with prion-like domains, such as FUS and TDP-43, condense into functional liquids, which can transform into pathological fibrils that underpin fatal neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). Here, we define short RNAs (24-48 nucleotides) that prevent FUS fibrillization by promoting liquid phases, and distinct short RNAs that prevent and, remarkably, reverse FUS condensation and fibrillization. These activities require interactions with multiple RNA-binding domains of FUS and are encoded by RNA sequence, length, and structure. Importantly, we define a short RNA that dissolves aberrant cytoplasmic FUS condensates, restores nuclear FUS, and mitigates FUS proteotoxicity in optogenetic models and human motor neurons. Another short RNA dissolves aberrant cytoplasmic TDP-43 condensates, restores nuclear TDP-43, and mitigates TDP-43 proteotoxicity. Since short RNAs can be effectively delivered to the human brain, these oligonucleotides could have therapeutic utility for ALS/FTD and related disorders.

Loss of function of the ALS-associated NEK1 kinase disrupts microtubule homeostasis and nuclear import.

Loss-of-function variants in NIMA-related kinase 1 (NEK1) constitute a major genetic cause of amyotrophic lateral sclerosis (ALS), accounting for 2 to 3% of all cases. However, how NEK1 mutations cause motor neuron (MN) dysfunction is unknown. Using mass spectrometry analyses for NEK1 interactors and NEK1-dependent expression changes, we find functional enrichment for proteins involved in the microtubule cytoskeleton and nucleocytoplasmic transport. We show that α-tubulin and importin-ß1, two key proteins involved in these processes, are phosphorylated by NEK1 in vitro. NEK1 is essential for motor control and survival in Drosophila models in vivo, while using several induced pluripotent stem cell (iPSC)-MN models, including NEK1 knockdown, kinase inhibition, and a patient mutation, we find evidence for disruptions in microtubule homeostasis and nuclear import. Notably, stabilizing microtubules with two distinct classes of drugs restored NEK1-dependent deficits in both pathways. The capacity of NEK1 to modulate these processes that are critically involved in ALS pathophysiology renders this kinase a formidable therapeutic candidate.

NUP62 localizes to ALS/FTLD pathological assemblies and contributes to TDP-43 insolubility.

A G4C2 hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of ALS and FTLD (C9-ALS/FTLD) with cytoplasmic TDP-43 inclusions observed in regions of neurodegeneration. The accumulation of repetitive RNAs and dipeptide repeat protein (DPR) are two proposed mechanisms of toxicity in C9-ALS/FTLD and linked to impaired nucleocytoplasmic transport. Nucleocytoplasmic transport is regulated by the phenylalanine-glycine nucleoporins (FG nups) that comprise the nuclear pore complex (NPC) permeability barrier. However, the relationship between FG nups and TDP-43 pathology remains elusive. Our studies show that nuclear depletion and cytoplasmic mislocalization of one FG nup, NUP62, is linked to TDP-43 mislocalization in C9-ALS/FTLD iPSC neurons. Poly-glycine arginine (GR) DPR accumulation initiates the formation of cytoplasmic RNA granules that recruit NUP62 and TDP-43. Cytoplasmic NUP62 and TDP-43 interactions promotes their insolubility and NUP62:TDP-43 inclusions are frequently found in C9orf72 ALS/FTLD as well as sporadic ALS/FTLD postmortem CNS tissue. Our findings indicate NUP62 cytoplasmic mislocalization contributes to TDP-43 proteinopathy in ALS/FTLD.

RNA modulates physiological and neuropathological protein phase transitions.

Aggregation of RNA-binding proteins (RBPs) is a pathological hallmark of neurodegenerative disorders like amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In these diseases, TDP-43 and FUS RBPs are depleted from the nuclear compartment, where they are normally localized, and found within cytoplasmic inclusions in degenerating regions of affected individuals' postmortem tissue. The mechanisms responsible for aggregation of these proteins has remained elusive, but recent studies suggest liquid-liquid phase separation (LLPS) might serve as a critical nucleation step in formation of pathological inclusions. The process of phase separation also underlies the formation and maintenance of several functional membraneless organelles (MLOs) throughout the cell, some of which contain TDP-43, FUS, and other disease-linked RBPs. One common ligand of disease-linked RBPs, RNA, is a major component of MLOs containing RBPs and has been demonstrated to be a strong modulator of RBP phase transitions. Although early evidence suggested a largely synergistic effect of RNA on RBP phase separation and MLO assembly, recent work indicates that RNA can also antagonize RBP phase behavior under certain physiological and pathological conditions. In this review, we describe the mechanisms underlying RNA-mediated phase transitions of RBPs and examine the molecular properties of these interactions, such as RNA length, sequence, and secondary structure, that mediate physiological or pathological LLPS.

RNA dependent suppression of C9orf72 ALS/FTD associated neurodegeneration by Matrin-3.

The most common genetic cause of amyotrophic lateral sclerosis (ALS) is a GGGGCC (G4C2) hexanucleotide repeat expansions in first intron of the C9orf72 gene. The accumulation of repetitive RNA sequences can mediate toxicity potentially through the formation of intranuclear RNA foci that sequester key RNA-binding proteins (RBPs), and non-ATG mediated translation into toxic dipeptide protein repeats. However, the contribution of RBP sequestration to the mechanisms underlying RNA-mediated toxicity remain unknown. Here we show that the ALS-associated RNA-binding protein, Matrin-3 (MATR3), colocalizes with G4C2 RNA foci in patient tissues as well as iPSC-derived motor neurons harboring the C9orf72 mutation. Hyperexpansion of C9 repeats perturbed subcellular distribution and levels of endogenous MATR3 in C9-ALS patient-derived motor neurons. Interestingly, we observed that ectopic expression of human MATR3 strongly mitigates G4C2-mediated neurodegeneration in vivo. MATR3-mediated suppression of C9 toxicity was dependent on the RNA-binding domain of MATR3. Importantly, we found that expression of MATR3 reduced the levels of RAN-translation products in mammalian cells in an RNA-dependent manner. Finally, we have shown that knocking down endogenous MATR3 in C9-ALS patient-derived iPSC neurons decreased the presence of G4C2 RNA foci in the nucleus. Overall, these studies suggest that MATR3 genetically modifies the neuropathological and the pathobiology of C9orf72 ALS through modulating the RNA foci and RAN translation.

Optogenetic TDP-43 nucleation induces persistent insoluble species and progressive motor dysfunction in vivo.

TDP-43 is a predominantly nuclear DNA/RNA binding protein that is often mislocalized into insoluble cytoplasmic inclusions in post-mortem patient tissue in a variety of neurodegenerative disorders including Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal dementia (FTD). The underlying causes of TDP-43 proteinopathies remain unclear, but recent studies indicate the formation of these protein assemblies is driven by aberrant phase transitions of RNA deficient TDP-43. Technical limitations have prevented our ability to understand how TDP-43 proteinopathy relates to disease pathogenesis. Current animal models of TDP-43 proteinopathy often rely on overexpression of wild-type TDP-43 to non-physiological levels that may initiate neurotoxicity through nuclear gain of function mechanisms, or by the expression of disease-causing mutations found in only a fraction of ALS patients. New technologies allowing for light-responsive control of subcellular protein crowding provide a promising approach to drive intracellular protein aggregation, as we have previously demonstrated in vitro. Here we present a model for the optogenetic induction of TDP-43 proteinopathy in Drosophila that recapitulates key features of patient pathology, including detergent insoluble cytoplamsic inclusions and progressive motor dysfunction.

RNA Binding Antagonizes Neurotoxic Phase Transitions of TDP-43.

TDP-43 proteinopathy is a pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia where cytoplasmic TDP-43 inclusions are observed within degenerating regions of patient postmortem tissue. The mechanism by which TDP-43 aggregates has remained elusive due to technological limitations, which prevent the analysis of specific TDP-43 interactions in live cells. We present an optogenetic approach to reliably induce TDP-43 proteinopathy under spatiotemporal control. We show that the formation of pathologically relevant inclusions is driven by aberrant interactions between low-complexity domains of TDP-43 that are antagonized by RNA binding. Although stress granules are hypothesized to be a conduit for seeding TDP-43 proteinopathy, we demonstrate pathological inclusions outside these RNA-rich structures. Furthermore, we show that aberrant phase transitions of cytoplasmic TDP-43 are neurotoxic and that treatment with oligonucleotides composed of TDP-43 target sequences prevent inclusions and rescue neurotoxicity. Collectively, these studies provide insight into the mechanisms that underlie TDP-43 proteinopathy and present a potential avenue for therapeutic intervention.

Hypoxic-Preconditioned Bone Marrow Stem Cell Medium Significantly Improves Outcome After Retinal Ischemia in Rats.

PURPOSE: We have previously demonstrated the protective effect of bone marrow stem cell (BMSC)-conditioned medium in retinal ischemic injury. We hypothesized here that hypoxic preconditioning of stem cells significantly enhances the neuroprotective effect of the conditioned medium and thereby augments the protective effect in ischemic retina. METHODS: Rats were subjected to retinal ischemia by increasing intraocular pressure to 130 to 135 mm Hg for 55 minutes. Hypoxic-preconditioned, hypoxic unconditioned, or normoxic medium was injected into the vitreous 24 hours after ischemia ended. Recovery was assessed 7 days after injections by comparing electroretinography measurements, histologic examination, and apoptosis (TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay). To compare proteins secreted into the medium in the groups and the effect of hypoxic exposure, we used rat cytokine arrays. RESULTS: Eyes injected with hypoxic BMSC-conditioned medium 24 hours after ischemia demonstrated significantly enhanced return of retinal function, decreased retinal ganglion cell layer loss, and attenuated apoptosis compared to those administered normoxic or hypoxic unconditioned medium. Hypoxic-preconditioned medium had 21 significantly increased protein levels compared to normoxic medium. CONCLUSIONS: The medium from hypoxic-preconditioned BMSCs robustly restored retinal function and prevented cell loss after ischemia when injected 24 hours after ischemia. The protective effect was even more pronounced than in our previous studies of normoxic conditioned medium. Prosurvival signals triggered by the secretome may play a role in this neuroprotective effect.