Stress Granule Assembly Disrupts Nucleocytoplasmic Transport.

Defects in nucleocytoplasmic transport have been identified as a key pathogenic event in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) mediated by a GGGGCC hexanucleotide repeat expansion in C9ORF72, the most common genetic cause of ALS/FTD. Furthermore, nucleocytoplasmic transport disruption has also been implicated in other neurodegenerative diseases with protein aggregation, suggesting a shared mechanism by which protein stress disrupts nucleocytoplasmic transport. Here, we show that cellular stress disrupts nucleocytoplasmic transport by localizing critical nucleocytoplasmic transport factors into stress granules, RNA/protein complexes that play a crucial role in ALS pathogenesis. Importantly, inhibiting stress granule assembly, such as by knocking down Ataxin-2, suppresses nucleocytoplasmic transport defects as well as neurodegeneration in C9ORF72-mediated ALS/FTD. Our findings identify a link between stress granule assembly and nucleocytoplasmic transport, two fundamental cellular processes implicated in the pathogenesis of C9ORF72-mediated ALS/FTD and other neurodegenerative diseases.

SUN1 facilitates CHMP7 nuclear influx and injury cascades in sporadic amyotrophic lateral sclerosis.

We have recently identified the aberrant nuclear accumulation of the ESCRT-III protein CHMP7 as an initiating event that leads to a significant injury to the nuclear pore complex (NPC) characterized by the reduction of specific nucleoporins from the neuronal NPC in sporadic amyotrophic lateral sclerosis (sALS) and C9orf72 ALS/frontotemporal dementia (FTD)-induced pluripotent stem cell-derived neurons (iPSNs), a phenomenon also observed in post-mortem patient tissues. Importantly, this NPC injury is sufficient to contribute to TDP-43 dysfunction and mislocalization, a common pathological hallmark of neurodegenerative diseases. However, the molecular mechanisms and events that give rise to increased nuclear translocation and/or retention of CHMP7 to initiate this pathophysiological cascade remain largely unknown. Here, using an iPSN model of sALS, we demonstrate that impaired NPC permeability barrier integrity and interactions with the LINC complex protein SUN1 facilitate CHMP7 nuclear localization and the subsequent 'activation' of NPC injury cascades. Collectively, our data provide mechanistic insights in the pathophysiological underpinnings of ALS/FTD and highlight SUN1 as a potent contributor to and modifier of CHMP7-mediated toxicity in sALS pathogenesis.

G2C4 targeting antisense oligonucleotides potently mitigate TDP-43 dysfunction in human C9orf72 ALS/FTD induced pluripotent stem cell derived neurons.

The G4C2 repeat expansion in the C9orf72 gene is the most common genetic cause of Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Many studies suggest that dipeptide repeat proteins produced from this repeat are toxic, yet, the contribution of repeat RNA toxicity is under investigated and even less is known regarding the pathogenicity of antisense repeat RNA. Recently, two clinical trials targeting G4C2 (sense) repeat RNA via antisense oligonucleotide failed despite a robust decrease in sense-encoded dipeptide repeat proteins demonstrating target engagement. Here, in this brief report, we show that G2C4 antisense, but not G4C2 sense, repeat RNA is sufficient to induce TDP-43 dysfunction in induced pluripotent stem cell (iPSC) derived neurons (iPSNs). Unexpectedly, only G2C4, but not G4C2 sense strand targeting, ASOs mitigate deficits in TDP-43 function in authentic C9orf72 ALS/FTD patient iPSNs. Collectively, our data suggest that the G2C4 antisense repeat RNA may be an important therapeutic target and provide insights into a possible explanation for the recent G4C2 ASO clinical trial failure.

Fragile X protein mitigates TDP-43 toxicity by remodeling RNA granules and restoring translation.

RNA dysregulation is a newly recognized disease mechanism in amyotrophic lateral sclerosis (ALS). Here we identify Drosophila fragile X mental retardation protein (dFMRP) as a robust genetic modifier of TDP-43-dependent toxicity in a Drosophila model of ALS. We find that dFMRP overexpression (dFMRP OE) mitigates TDP-43 dependent locomotor defects and reduced lifespan in Drosophila. TDP-43 and FMRP form a complex in flies and human cells. In motor neurons, TDP-43 expression increases the association of dFMRP with stress granules and colocalizes with polyA binding protein in a variant-dependent manner. Furthermore, dFMRP dosage modulates TDP-43 solubility and molecular mobility with overexpression of dFMRP resulting in a significant reduction of TDP-43 in the aggregate fraction. Polysome fractionation experiments indicate that dFMRP OE also relieves the translation inhibition of futsch mRNA, a TDP-43 target mRNA, which regulates neuromuscular synapse architecture. Restoration of futsch translation by dFMRP OE mitigates Futsch-dependent morphological phenotypes at the neuromuscular junction including synaptic size and presence of satellite boutons. Our data suggest a model whereby dFMRP is neuroprotective by remodeling TDP-43 containing RNA granules, reducing aggregation and restoring the translation of specific mRNAs in motor neurons.

Futsch/MAP1B mRNA is a translational target of TDP-43 and is neuroprotective in a Drosophila model of amyotrophic lateral sclerosis.

TDP-43 is an RNA-binding protein linked to amyotrophic lateral sclerosis (ALS) that is known to regulate the splicing, transport, and storage of specific mRNAs into stress granules. Although TDP-43 has been shown to interact with translation factors, its role in protein synthesis remains unclear, and no in vivo translation targets have been reported to date. Here we provide evidence that TDP-43 associates with futsch mRNA in a complex and regulates its expression at the neuromuscular junction (NMJ) in Drosophila. In the context of TDP-43-induced proteinopathy, there is a significant reduction of futsch mRNA at the NMJ compared with motor neuron cell bodies where we find higher levels of transcript compared with controls. TDP-43 also leads to a significant reduction in Futsch protein expression at the NMJ. Polysome fractionations coupled with quantitative PCR experiments indicate that TDP-43 leads to a futsch mRNA shift from actively translating polysomes to nontranslating ribonuclear protein particles, suggesting that in addition to its effect on localization, TDP-43 also regulates the translation of futsch mRNA. We also show that futsch overexpression is neuroprotective by extending life span, reducing TDP-43 aggregation, and suppressing ALS-like locomotor dysfunction as well as NMJ abnormalities linked to microtubule and synaptic stabilization. Furthermore, the localization of MAP1B, the mammalian homolog of Futsch, is altered in ALS spinal cords in a manner similar to our observations in Drosophila motor neurons. Together, our results suggest a microtubule-dependent mechanism in motor neuron disease caused by TDP-43-dependent alterations in futsch mRNA localization and translation in vivo.

Nuclear and degradative functions of the ESCRT-III pathway: implications for neurodegenerative disease.

The ESCRT machinery plays a pivotal role in membrane-remodeling events across multiple cellular processes including nuclear envelope repair and reformation, nuclear pore complex surveillance, endolysosomal trafficking, and neuronal pruning. Alterations in ESCRT-III functionality have been associated with neurodegenerative diseases including Frontotemporal Dementia (FTD), Amyotrophic Lateral Sclerosis (ALS), and Alzheimer's Disease (AD). In addition, mutations in specific ESCRT-III proteins have been identified in FTD/ALS. Thus, understanding how disruptions in the fundamental functions of this pathway and its individual protein components in the human central nervous system (CNS) may offer valuable insights into mechanisms underlying neurodegenerative disease pathogenesis and identification of potential therapeutic targets. In this review, we discuss ESCRT components, dynamics, and functions, with a focus on the ESCRT-III pathway. In addition, we explore the implications of altered ESCRT-III function for neurodegeneration with a primary emphasis on nuclear surveillance and endolysosomal trafficking within the CNS.

Visualizing nuclear pore complex plasticity with Pan-Expansion Microscopy.

Cell-type specific and environmentally-responsive plasticity in nuclear pore complex (NPC) composition and structure is an emerging area of investigation, but its molecular underpinnings remain ill defined. To understand the cause and consequence of NPC plasticity requires technologies to visualize differences within individual NPCs across the thousands in a given nucleus. We evaluate the utility of Pan Expansion Microscopy (Pan-ExM), which enables 16-20 fold isotropic cell enlargement while preserving the proteome, to reveal NPC plasticity. NPCs are robustly identified by deep learning-facilitated segmentation as tripartite structures corresponding to the nucleoplasmic ring, inner ring with central transport channel, and cytoplasmic ring, as confirmed by immunostaining. We demonstrate a range of NPC diameters with a bias for dilated NPCs at the basal nuclear surface, often in local clusters. These diameter biases are eliminated by disrupting linker of nucleoskeleton and cytoskeleton (LINC) complex-dependent connections between the nuclear envelope (NE) and the cytoskeleton, supporting that they reflect local variations in NE tension. Pan-ExM further reveals that the transmembrane nucleoporin/nup POM121 resides specifically at the nuclear ring in multiple model cell lines, surprising given the expectation that it would be a component of the inner ring like other transmembrane nups. Remarkably, however, POM121 shifts from the nuclear ring to the inner ring specifically in aged induced pluripotent stem cell derived neurons (iPSNs) from a patient with C9orf72 amyotrophic lateral sclerosis (ALS). Thus, Pan-ExM allows the visualization of changes in NPC architecture that may underlie early steps in an ALS pathomechanism. Taken together, Pan-ExM is a powerful and accessible tool to visualize NPC plasticity in physiological and pathological contexts at single NPC resolution.

PolyGR and polyPR knock-in mice reveal a conserved neuroprotective extracellular matrix signature in C9orf72 ALS/FTD neurons.

Dipeptide repeat proteins are a major pathogenic feature of C9orf72 amyotrophic lateral sclerosis (C9ALS)/frontotemporal dementia (FTD) pathology, but their physiological impact has yet to be fully determined. Here we generated C9orf72 dipeptide repeat knock-in mouse models characterized by expression of 400 codon-optimized polyGR or polyPR repeats, and heterozygous C9orf72 reduction. (GR)400 and (PR)400 knock-in mice recapitulate key features of C9ALS/FTD, including cortical neuronal hyperexcitability, age-dependent spinal motor neuron loss and progressive motor dysfunction. Quantitative proteomics revealed an increase in extracellular matrix (ECM) proteins in (GR)400 and (PR)400 spinal cord, with the collagen COL6A1 the most increased protein. TGF-ß1 was one of the top predicted regulators of this ECM signature and polyGR expression in human induced pluripotent stem cell neurons was sufficient to induce TGF-ß1 followed by COL6A1. Knockdown of TGF-ß1 or COL6A1 orthologues in polyGR model Drosophila exacerbated neurodegeneration, while expression of TGF-ß1 or COL6A1 in induced pluripotent stem cell-derived motor neurons of patients with C9ALS/FTD protected against glutamate-induced cell death. Altogether, our findings reveal a neuroprotective and conserved ECM signature in C9ALS/FTD.

Nuclear pore complex and nucleocytoplasmic transport disruption in neurodegeneration.

Nuclear pore complexes (NPCs) play a critical role in maintaining the equilibrium between the nucleus and cytoplasm, enabling bidirectional transport across the nuclear envelope, and are essential for proper nuclear organization and gene regulation. Perturbations in the regulatory mechanisms governing NPCs and nuclear envelope homeostasis have been implicated in the pathogenesis of several neurodegenerative diseases. The ESCRT-III pathway emerges as a critical player in the surveillance and preservation of well-assembled, functional NPCs, as well as nuclear envelope sealing. Recent studies have provided insights into the involvement of nuclear ESCRT-III in the selective reduction of specific nucleoporins associated with neurodegenerative pathologies. Thus, maintaining quality control of the nuclear envelope and NPCs represents a pivotal element in the pathological cascade leading to neurodegenerative diseases. This review describes the constituents of the nuclear-cytoplasmic transport machinery, encompassing the nuclear envelope, NPC, and ESCRT proteins, and how their structural and functional alterations contribute to the development of neurodegenerative diseases.

Highly variable molecular signatures of TDP-43 loss of function are associated with nuclear pore complex injury in a population study of sporadic ALS patient iPSNs.

The nuclear depletion and cytoplasmic aggregation of the RNA binding protein TDP-43 is widely considered a pathological hallmark of Amyotrophic Lateral Sclerosis (ALS) and related neurodegenerative diseases. Recent studies have artificially reduced TDP-43 in wildtype human neurons to replicate loss of function associated events. Although this prior work has defined a number of gene expression and mRNA splicing changes that occur in a TDP-43 dependent manner, it is unclear how these alterations relate to authentic ALS where TDP-43 is not depleted from the cell but miscompartmentalized to variable extents. Here, in this population study, we generate ~30,000 qRT-PCR data points spanning 20 genes in induced pluripotent stem cell (iPSC) derived neurons (iPSNs) from >150 control, C9orf72 ALS/FTD, and sALS patients to examine molecular signatures of TDP-43 dysfunction. This data set defines a time dependent and variable profile of individual molecular hallmarks of TDP-43 loss of function within and amongst individual patient lines. Importantly, nearly identical changes are observed in postmortem CNS tissues obtained from a subset of patients whose iPSNs were examined. Notably, these studies provide evidence that induction of nuclear pore complex (NPC) injury via reduction of the transmembrane Nup POM121 in wildtype iPSNs is sufficient to phenocopy disease associated signatured of TDP-43 loss of function thereby directly linking NPC integrity to TDP-43 loss of function. Therapeutically, we demonstrate that the expression of all mRNA species associated with TDP-43 loss of function can be restored in sALS iPSNs via two independent methods to repair NPC injury. Collectively, this data 1) represents a substantial resource for the community to examine TDP-43 loss of function events in authentic sALS patient iPSNs, 2) demonstrates that patient derived iPSNs can accurately reflect actual TDP-43 associated alterations in patient brain, and 3) that targeting NPC injury events can be preclinically and reliably accomplished in an iPSN based platform of a sporadic disease.

Nuclear pore complexes - a doorway to neural injury in neurodegeneration.

The genetic underpinnings and end-stage pathological hallmarks of neurodegenerative diseases are increasingly well defined, but the cellular pathophysiology of disease initiation and propagation remains poorly understood, especially in sporadic forms of these diseases. Altered nucleocytoplasmic transport is emerging as a prominent pathomechanism of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis, Alzheimer disease, frontotemporal dementia and Huntington disease. The nuclear pore complex (NPC) and interactions between its individual nucleoporin components and nuclear transport receptors regulate nucleocytoplasmic transport, as well as genome organization and gene expression. Specific nucleoporin abnormalities have been identified in sporadic and familial forms of neurodegenerative disease, and these alterations are thought to contribute to disrupted nucleocytoplasmic transport. The specific nucleoporins and nucleocytoplasmic transport proteins that have been linked to different neurodegenerative diseases are partially distinct, suggesting that NPC injury contributes to the cellular specificity of neurodegenerative disease and could be an early initiator of the pathophysiological cascades that underlie neurodegenerative disease. This concept is consistent with the fact that rare genetic mutations in some nucleoporins cause cell-type-specific neurological disease. In this Review, we discuss nucleoporin and NPC disruptions and consider their impact on cellular function and the pathophysiology of neurodegenerative disease.

Poly(ADP-ribose) promotes toxicity of C9ORF72 arginine-rich dipeptide repeat proteins.

Arginine-rich dipeptide repeat proteins (R-DPRs), abnormal translational products of a GGGGCC hexanucleotide repeat expansion in C9ORF72, play a critical role in C9ORF72-related amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), the most common genetic form of the disorders (c9ALS/FTD). R-DPRs form liquid condensates in vitro, induce stress granule formation in cultured cells, aggregate, and sometimes coaggregate with TDP-43 in postmortem tissue from patients with c9ALS/FTD. However, how these processes are regulated is unclear. Here, we show that loss of poly(ADP-ribose) (PAR) suppresses neurodegeneration in c9ALS/FTD fly models and neurons differentiated from patient-derived induced pluripotent stem cells. Mechanistically, PAR induces R-DPR condensation and promotes R-DPR-induced stress granule formation and TDP-43 aggregation. Moreover, PAR associates with insoluble R-DPR and TDP-43 in postmortem tissue from patients. These findings identified PAR as a promoter of R-DPR toxicity and thus a potential target for treating c9ALS/FTD.

Answer ALS, a large-scale resource for sporadic and familial ALS combining clinical and multi-omics data from induced pluripotent cell lines.

Answer ALS is a biological and clinical resource of patient-derived, induced pluripotent stem (iPS) cell lines, multi-omic data derived from iPS neurons and longitudinal clinical and smartphone data from over 1,000 patients with ALS. This resource provides population-level biological and clinical data that may be employed to identify clinical-molecular-biochemical subtypes of amyotrophic lateral sclerosis (ALS). A unique smartphone-based system was employed to collect deep clinical data, including fine motor activity, speech, breathing and linguistics/cognition. The iPS spinal neurons were blood derived from each patient and these cells underwent multi-omic analytics including whole-genome sequencing, RNA transcriptomics, ATAC-sequencing and proteomics. The intent of these data is for the generation of integrated clinical and biological signatures using bioinformatics, statistics and computational biology to establish patterns that may lead to a better understanding of the underlying mechanisms of disease, including subgroup identification. A web portal for open-source sharing of all data was developed for widespread community-based data analytics.

Nuclear lamina invaginations are not a pathological feature of C9orf72 ALS/FTD.

The most common genetic cause of familial and sporadic amyotrophic lateral sclerosis (ALS) is a GGGGCC hexanucleotide repeat expansion (HRE) in the C9orf72 gene. While direct molecular hallmarks of the C9orf72 HRE (repeat RNA foci, dipeptide repeat protein pathology) are well characterized, the mechanisms by which the C9orf72 HRE causes ALS and the related neurodegenerative disease frontotemporal dementia (FTD) remain poorly understood. Recently, alterations to the nuclear pore complex and nucleocytoplasmic transport have been accepted as a prominent pathomechanism underlying C9orf72 ALS/FTD. However, global disruptions to nuclear morphology and the nuclear lamina itself remain controversial. Here, we use a large number of induced pluripotent stem cell derived spinal neurons and postmortem human motor cortex sections to thoroughly examine nuclear morphology and nuclear lamina disruptions with light microscopy. In contrast to previous studies in artificial overexpression model systems, endogenous levels of the C9orf72 HRE do not increase the frequency of nuclear lamina invaginations. In addition, the C9orf72 HRE has no impact on overall nuclear shape and size. Notably, the frequency of nuclear Lamin B1 invaginations increases with cellular aging, independent of the C9orf72 HRE. Together, our data suggest that nuclear morphology is unaltered in C9orf72 ALS/FTD.

The ESCRT-III protein VPS4, but not CHMP4B or CHMP2B, is pathologically increased in familial and sporadic ALS neuronal nuclei.

Nuclear pore complex injury has recently emerged as an early and significant contributor to familial and sporadic ALS disease pathogenesis. However, the molecular events leading to this pathological phenomenon characterized by the reduction of specific nucleoporins from neuronal nuclear pore complexes remain largely unknown. This is due in part to a lack of knowledge regarding the biological pathways and proteins underlying nuclear pore complex homeostasis specifically in human neurons. We have recently uncovered that aberrant nuclear accumulation of the ESCRT-III protein CHMP7 initiates nuclear pore complex in familial and sporadic ALS neurons. In yeast and non-neuronal mammalian cells, nuclear relocalization of CHMP7 has been shown to recruit the ESCRT-III proteins CHMP4B, CHMP2B, and VPS4 to facilitate nuclear pore complex and nuclear envelope repair and homeostasis. Here, using super resolution structured illumination microscopy, we find that neither CHMP4B nor CHMP2B are increased in ALS neuronal nuclei. In contrast, VPS4 expression is significantly increased in ALS neuronal nuclei prior to the emergence of nuclear pore injury in a CHMP7 dependent manner. However, unlike our prior CHMP7 knockdown studies, impaired VPS4 function does not mitigate alterations to the NPC and the integral transmembrane nucleoporin POM121. Collectively our data suggest that while alterations in VPS4 subcellular localization appear to be coincident with nuclear pore complex injury, therapeutic efforts to mitigate this pathogenic cascade should be targeted towards upstream events such as the nuclear accumulation of CHMP7 as we have previously described.

Nuclei Isolation and Super-Resolution Structured Illumination Microscopy for Examining Nucleoporin Alterations in Human Neurodegeneration.

The nuclear pore complex (NPC) is a complex macromolecular structure comprised of multiple copies of ~30 different nucleoporin proteins (Nups). Collectively, these Nups function to regulate genome organization, gene expression, and nucleocytoplasmic transport (NCT). Recently, defects in NCT and alterations to specific Nups have been identified as early and prominent pathologies in multiple neurodegenerative diseases, including Amyotrophic Lateral Sclerosis (ALS), Alzheimer's Disease (AD)/Frontotemporal Dementia (FTD), and Huntington's Disease (HD). Advances in both light and electron microscopy allow for a thorough examination of sub-cellular structures, including the NPC and its Nup constituents, with increased precision and resolution. Of the commonly used techniques, super-resolution structured illumination microscopy (SIM) affords the unparalleled opportunity to study the localization and expression of individual Nups using conventional antibody-based labeling strategies. Isolation of nuclei prior to SIM enables the visualization of individual Nup proteins within the NPC and nucleoplasm in fully and accurately reconstructed 3D space. This protocol describes a procedure for nuclei isolation and SIM to evaluate Nup expression and distribution in human iPSC-derived CNS cells and postmortem tissues.

Nuclear accumulation of CHMP7 initiates nuclear pore complex injury and subsequent TDP-43 dysfunction in sporadic and familial ALS.

Alterations in the components [nucleoporins (Nups)] and function of the nuclear pore complex (NPC) have been implicated as contributors to the pathogenesis of genetic forms of neurodegeneration including C9orf72 amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD). We hypothesized that Nup alterations and the consequential loss of NPC function may lie upstream of TDP-43 dysfunction and mislocalization widely observed in ALS, FTD, and related neurodegenerative diseases. Here, we provide evidence that CHMP7, a critical mediator of NPC quality control, is increased in nuclei of C9orf72 and sporadic ALS induced pluripotent stem cell (iPSC)-derived spinal neurons (iPSNs) and postmortem human motor cortex before the emergence of Nup alterations. Inhibiting the nuclear export of CHMP7 triggered Nup reduction and TDP-43 dysfunction and pathology in human neurons. Knockdown of CHMP7 alleviated disease-associated Nup alterations, deficits in Ran GTPase localization, defects in TDP-43-associated mRNA expression, and downstream glutamate-induced neuronal death. Thus, our data support a role for altered CHMP7-mediated Nup homeostasis as a prominent initiating pathological mechanism for familial and sporadic ALS and highlight the potential for CHMP7 as therapeutic target.

UPF1 reduces C9orf72 HRE-induced neurotoxicity in the absence of nonsense-mediated decay dysfunction.

Multiple cellular pathways have been suggested to be altered by the C9orf72 GGGGCC (G4C2) hexanucleotide repeat expansion (HRE), including aspects of RNA regulation such as nonsense-mediated decay (NMD). Here, we investigate the role that overexpression of UPF1, a protein involved in NMD, plays in mitigating neurotoxicity in multiple models of C9orf72 ALS/FTD. First, we show that NMD is not altered in our endogenous induced pluripotent stem cell (iPSC)-derived spinal neuron (iPSN) model of C9orf72 ALS (C9-ALS) or postmortem motor cortex tissue from C9-ALS patients. Unexpectedly, we find that UPF1 overexpression significantly reduces the severity of known neurodegenerative phenotypes without altering NMD function itself. UPF1 overexpression reduces poly(GP) abundance without altering the amount of repeat RNA, providing a potential mechanism by which UPF1 reduces dipeptide repeat (DPR) protein-mediated toxicity. Together, these findings indicate that UPF1 is neuroprotective in the context of C9-ALS, albeit independent of known UPF1-mediated NMD pathways.

Ribonuclease recruitment using a small molecule reduced c9ALS/FTD r(G4C2) repeat expansion in vitro and in vivo ALS models.

The most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD) is an expanded G4C2 RNA repeat [r(G4C2)exp] in chromosome 9 open reading frame 72 (C9orf72), which elicits pathology through several mechanisms. Here, we developed and characterized a small molecule for targeted degradation of r(G4C2)exp. The compound was able to selectively bind r(G4C2)exp's structure and to assemble an endogenous nuclease onto the target, provoking removal of the transcript by native RNA quality control mechanisms. In c9ALS patient­derived spinal neurons, the compound selectively degraded the mutant C9orf72 allele with limited off-targets and reduced quantities of toxic dipeptide repeat proteins (DPRs) translated from r(G4C2)exp. In vivo work in a rodent model showed that abundance of both the mutant allele harboring the repeat expansion and DPRs were selectively reduced by this compound. These results demonstrate that targeted small-molecule degradation of r(G4C2)exp is a strategy for mitigating c9ALS/FTD-associated pathologies and studying disease-associated pathways in preclinical models.