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.

Edaravone: A new drug approved for ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive, adult onset neurodegenerative disease that is always fatal. The history of ALS drug discovery is fraught with many stops and starts. It took 22 years after the FDA approval of the anti-excitotoxic drug Riluzole before another drug was found to be effective in altering ALS progression: the anti-oxidant Edaravone.

Metabolic support of tumour-infiltrating regulatory T cells by lactic acid.

Regulatory T (Treg) cells, although vital for immune homeostasis, also represent a major barrier to anti-cancer immunity, as the tumour microenvironment (TME) promotes the recruitment, differentiation and activity of these cells1,2. Tumour cells show deregulated metabolism, leading to a metabolite-depleted, hypoxic and acidic TME3, which places infiltrating effector T cells in competition with the tumour for metabolites and impairs their function4-6. At the same time, Treg cells maintain a strong suppression of effector T cells within the TME7,8. As previous studies suggested that Treg cells possess a distinct metabolic profile from effector T cells9-11, we hypothesized that the altered metabolic landscape of the TME and increased activity of intratumoral Treg cells are linked. Here we show that Treg cells display broad heterogeneity in their metabolism of glucose within normal and transformed tissues, and can engage an alternative metabolic pathway to maintain suppressive function and proliferation. Glucose uptake correlates with poorer suppressive function and long-term instability, and high-glucose conditions impair the function and stability of Treg cells in vitro. Treg cells instead upregulate pathways involved in the metabolism of the glycolytic by-product lactic acid. Treg cells withstand high-lactate conditions, and treatment with lactate prevents the destabilizing effects of high-glucose conditions, generating intermediates necessary for proliferation. Deletion of MCT1-a lactate transporter-in Treg cells reveals that lactate uptake is dispensable for the function of peripheral Treg cells but required intratumorally, resulting in slowed tumour growth and an increased response to immunotherapy. Thus, Treg cells are metabolically flexible: they can use 'alternative' metabolites in the TME to maintain their suppressive identity. Further, our results suggest that tumours avoid destruction by not only depriving effector T cells of nutrients, but also metabolically supporting regulatory populations.

Apilimod dimesylate in C9orf72 amyotrophic lateral sclerosis: a randomized phase 2a clinical trial.

Apilimod dimesylate is a first-in-class phosphoinositide kinase, FYVE-type zinc finger containing (PIKfyve) inhibitor with favourable clinical safety profile and has demonstrated activity in preclinical C9orf72 and TDP-43 amyotrophic lateral sclerosis models. In this amyotrophic lateral sclerosis clinical trial, the safety, tolerability, CNS penetrance, and modulation of pharmacodynamic target engagement biomarkers were evaluated. This Phase 2a, randomized, double-blind, placebo-controlled, biomarker-endpoint clinical trial was conducted in four USA centres (ClinicalTrials.gov NCT05163886). Participants with C9orf72 repeat expansion were randomly assigned (2:1) to receive twice-daily oral treatment of 125 mg apilimod dimesylate capsules or matching placebo for 12 weeks, followed by a 12-week open-label extension. Safety was measured as the occurrence of treatment-emergent adverse or serious adverse events attributable to study drug, and tolerability as trial completion on treatment over 12 weeks. Changes from baseline in plasma and CSF and concentrations of apilimod and its active metabolites and of pharmacodynamic biomarkers of PIKfyve inhibition (soluble glycoprotein nonmetastatic melanoma protein B [sGPNMB] upregulation) and disease-specific CNS target engagement (poly[GP]). Between Dec 16, 2021, and Jul 7, 2022, 15 eligible participants were enrolled. There were no drug-related serious adverse events reported in the trial. Fourteen (93%) participants completed the double-blind period with 99% dose compliance (N=9 [90%] apilimod dimesylate; N=5 [100%] placebo). At Week 12, apilimod dimesylate was measurable in CSF at 1.63 ng/mL (SD: 0.937). At Week 12, apilimod dimesylate increased plasma sGPNMB by > 2.5-fold (p < 0.001) indicating PIKfyve inhibition and lowered CSF poly(GP) protein levels by 73% (p < 0.001) indicating CNS tissue-level proof of mechanism. Apilimod dimesylate met prespecified key safety and biomarker endpoints in this Phase 2a trial and demonstrated CNS penetrance and pharmacodynamic target engagement. Apilimod dimesylate was observed to have the greatest reduction in CSF poly(GP) levels observed to date in C9orf72 clinical trials.

Trial of Sodium Phenylbutyrate-Taurursodiol for Amyotrophic Lateral Sclerosis.

BACKGROUND: Sodium phenylbutyrate and taurursodiol have been found to reduce neuronal death in experimental models. The efficacy and safety of a combination of the two compounds in persons with amyotrophic lateral sclerosis (ALS) are not known. METHODS: In this multicenter, randomized, double-blind trial, we enrolled participants with definite ALS who had had an onset of symptoms within the previous 18 months. Participants were randomly assigned in a 2:1 ratio to receive sodium phenylbutyrate-taurursodiol (3 g of sodium phenylbutyrate and 1 g of taurursodiol, administered once a day for 3 weeks and then twice a day) or placebo. The primary outcome was the rate of decline in the total score on the Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R; range, 0 to 48, with higher scores indicating better function) through 24 weeks. Secondary outcomes were the rates of decline in isometric muscle strength, plasma phosphorylated axonal neurofilament H subunit levels, and the slow vital capacity; the time to death, tracheostomy, or permanent ventilation; and the time to death, tracheostomy, permanent ventilation, or hospitalization. RESULTS: A total of 177 persons with ALS were screened for eligibility, and 137 were randomly assigned to receive sodium phenylbutyrate-taurursodiol (89 participants) or placebo (48 participants). In a modified intention-to-treat analysis, the mean rate of change in the ALSFRS-R score was -1.24 points per month with the active drug and -1.66 points per month with placebo (difference, 0.42 points per month; 95% confidence interval, 0.03 to 0.81; P = 0.03). Secondary outcomes did not differ significantly between the two groups. Adverse events with the active drug were mainly gastrointestinal. CONCLUSIONS: Sodium phenylbutyrate-taurursodiol resulted in slower functional decline than placebo as measured by the ALSFRS-R score over a period of 24 weeks. Secondary outcomes were not significantly different between the two groups. Longer and larger trials are necessary to evaluate the efficacy and safety of sodium phenylbutyrate-taurursodiol in persons with ALS. (Funded by Amylyx Pharmaceuticals and others; CENTAUR ClinicalTrials.gov number, NCT03127514.).

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.

Effect of sodium phenylbutyrate/taurursodiol on tracheostomy/ventilation-free survival and hospitalisation in amyotrophic lateral sclerosis: long-term results from the CENTAUR trial.

BACKGROUND: Coformulated sodium phenylbutyrate/taurursodiol (PB/TURSO) was shown to prolong survival and slow functional decline in amyotrophic lateral sclerosis (ALS). OBJECTIVE: Determine whether PB/TURSO prolonged tracheostomy/ventilation-free survival and/or reduced first hospitalisation in participants with ALS in the CENTAUR trial. METHODS: Adults with El Escorial Definite ALS ≤18 months from symptom onset were randomised to PB/ TURSO or placebo for 6 months. Those completing randomised treatment could enrol in an open-label extension (OLE) phase and receive PB/TURSO for ≤30 months. Times to the following individual or combined key events were compared in the originally randomised treatment groups over a period spanning trial start through July 2020 (longest postrandomisation follow-up, 35 months): death, tracheostomy, permanent assisted ventilation (PAV) and first hospitalisation. RESULTS: Risk of any key event was 47% lower in those originally randomised to PB/TURSO (n=87) versus placebo (n=48, 71% of whom received delayed-start PB/TURSO in the OLE phase) (HR=0.53; 95% CI 0.35 to 0.81; p=0.003). Risks of death or tracheostomy/PAV (HR=0.51; 95% CI 0.32 to 0.84; p=0.007) and first hospitalisation (HR=0.56; 95% CI 0.34 to 0.95; p=0.03) were also decreased in those originally randomised to PB/TURSO. CONCLUSIONS: Early PB/TURSO prolonged tracheostomy/PAV-free survival and delayed first hospitalisation in ALS. TRIAL REGISTRATION NUMBER: NCT03127514; NCT03488524.

A Helicase Unwinds Hexanucleotide Repeat RNA G-Quadruplexes and Facilitates Repeat-Associated Non-AUG Translation.

The expansion of a hexanucleotide repeat GGGGCC (G4C2) in the C9orf72 gene is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The G4C2 expansion leads to repeat-associated non-AUG (RAN) translation and the production of toxic dipeptide repeat (DPR) proteins, but the mechanisms of RAN translation remain enigmatic. Here, we report that the RNA helicase DHX36 is a robust positive regulator of C9orf72 RAN translation. DHX36 has a high affinity for the G4C2 repeat RNA, preferentially binds to the repeat RNA's G-quadruplex conformation, and efficiently unwinds the G4C2 G-quadruplex structures. Native DHX36 interacts with the G4C2 repeat RNA and is essential for effective RAN translation in the cell. In induced pluripotent stem cells and differentiated motor neurons derived from C9orf72-linked ALS patients, reducing DHX36 significantly decreased the levels of endogenous DPR proteins. DHX36 is also aberrantly upregulated in tissues of C9orf72-linked ALS patients. These results indicate that DHX36 facilitates C9orf72 RAN translation by resolving repeat RNA G-quadruplex structures and may be a potential target for therapeutic intervention.

MC-100093, a Novel ß-Lactam Glutamate Transporter-1 Enhancer Devoid of Antimicrobial Properties, Attenuates Cocaine Relapse in Rats.

Cocaine use disorder currently lacks Food and Drug Administration-approved treatments. In rodents, the glutamate transporter-1 (GLT-1) is downregulated in the nucleus accumbens after cocaine self-administration, and increasing the expression and function of GLT-1 reduces the reinstatement of cocaine seeking. The ß-lactam antibiotic ceftriaxone upregulates GLT-1 and attenuates cue- and cocaine-induced cocaine seeking without affecting motivation for natural rewards. Although ceftriaxone shows promise for treating cocaine use disorder, it possesses characteristics that limit successful translation from bench to bedside, including poor brain penetration, a lack of oral bioavailability, and a risk of bacterial resistance when used chronically. Thus, we aimed to develop novel molecules that retained the GLT-1-enhancing effects of ceftriaxone but displayed superior drug-like properties. Here, we describe a new monocyclic ß-lactam, MC-100093, as a potent upregulator of GLT-1 that is orally bioavailable and devoid of antimicrobial properties. MC-100093 was synthesized and tested in vitro and in vivo to determine physiochemical, pharmacokinetic, and pharmacodynamic properties. Next, adult male rats underwent cocaine self-administration and extinction training. During extinction training, rats received one of four doses of MC-100093 for 6-8 days prior to a single cue-primed reinstatement test. Separate cohorts of rats were used to assess nucleus accumbens GLT-1 expression and MC-100093 effects on sucrose self-administration. We found that 50 mg/kg MC-100093 attenuated cue-primed reinstatement of cocaine seeking while upregulating GLT-1 expression in the nucleus accumbens core. This dose did not produce sedation, nor did it decrease sucrose consumption or body weight. Thus, MC-100093 represents a potential treatment to reduce cocaine relapse. SIGNIFICANCE STATEMENT: Increasing GLT-1 activity reliably reduces drug-seeking across classes of drugs; however, existing GLT1-enhancers have side effects and lack oral bioavailability. To address this issue, novel GLT-1 enhancers were synthesized, and the compound with the most favorable pharmacokinetic and pharmacodynamic properties, MC-100093, was selected for further testing. MC-100093 attenuated cued cocaine seeking without reducing food seeking or locomotion and upregulated GLT-1 expression in the nucleus accumbens.

The expanding biology of the C9orf72 nucleotide repeat expansion in neurodegenerative disease.

A nucleotide repeat expansion (NRE) within the chromosome 9 open reading frame 72 (C9orf72) gene was the first of this type of mutation to be linked to multiple neurological conditions, including amyotrophic lateral sclerosis and frontotemporal dementia. The pathogenic mechanisms through which the C9orf72 NRE contributes to these disorders include loss of C9orf72 function and gain-of-function mechanisms of C9orf72 driven by toxic RNA and protein species encoded by the NRE. These mechanisms have been linked to several cellular defects - including nucleocytoplasmic trafficking deficits and nuclear stress - that have been observed in both patients and animal models.

Long-term survival of participants in the CENTAUR trial of sodium phenylbutyrate-taurursodiol in amyotrophic lateral sclerosis.

An orally administered, fixed-dose coformulation of sodium phenylbutyrate-taurursodiol (PB-TURSO) significantly slowed functional decline in a randomized, placebo-controlled, phase 2 trial in ALS (CENTAUR). Herein we report results of a long-term survival analysis of participants in CENTAUR. In CENTAUR, adults with ALS were randomized 2:1 to PB-TURSO or placebo. Participants completing the 6-month (24-week) randomized phase were eligible to receive PB-TURSO in the open-label extension. An all-cause mortality analysis (35-month maximum follow-up post-randomization) incorporated all randomized participants. Participants and site investigators were blinded to treatment assignments through the duration of follow-up of this analysis. Vital status was obtained for 135 of 137 participants originally randomized in CENTAUR. Median overall survival was 25.0 months among participants originally randomized to PB-TURSO and 18.5 months among those originally randomized to placebo (hazard ratio, 0.56; 95% confidence interval, 0.34-0.92; P = .023). Initiation of PB-TURSO treatment at baseline resulted in a 6.5-month longer median survival as compared with placebo. Combined with results from CENTAUR, these results suggest that PB-TURSO has both functional and survival benefits in ALS.

The C9orf72 repeat expansion disrupts nucleocytoplasmic transport.

The hexanucleotide repeat expansion (HRE) GGGGCC (G4C2) in C9orf72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent studies support an HRE RNA gain-of-function mechanism of neurotoxicity, and we previously identified protein interactors for the G4C2 RNA including RanGAP1. A candidate-based genetic screen in Drosophila expressing 30 G4C2 repeats identified RanGAP (Drosophila orthologue of human RanGAP1), a key regulator of nucleocytoplasmic transport, as a potent suppressor of neurodegeneration. Enhancing nuclear import or suppressing nuclear export of proteins also suppresses neurodegeneration. RanGAP physically interacts with HRE RNA and is mislocalized in HRE-expressing flies, neurons from C9orf72 ALS patient-derived induced pluripotent stem cells (iPSC-derived neurons), and in C9orf72 ALS patient brain tissue. Nuclear import is impaired as a result of HRE expression in the fly model and in C9orf72 iPSC-derived neurons, and these deficits are rescued by small molecules and antisense oligonucleotides targeting the HRE G-quadruplexes. Nucleocytoplasmic transport defects may be a fundamental pathway for ALS and FTD that is amenable to pharmacotherapeutic intervention.

Monocarboxylate transporter 1 in Schwann cells contributes to maintenance of sensory nerve myelination during aging.

Schwann cell (SC)-specific monocarboxylate transporter 1 (MCT1) knockout mice were generated by mating MCT1 f/f mice with myelin protein zero (P0)-Cre mice. P0-Cre+/- , MCT1 f/f mice have no detectable early developmental defects, but develop hypomyelination and reduced conduction velocity in sensory, but not motor, peripheral nerves during maturation and aging. Furthermore, reduced mechanical sensitivity is evident in aged P0-Cre+/- , MCT1 f/f mice. MCT1 deletion in SCs impairs both their glycolytic and mitochondrial functions, leading to altered lipid metabolism of triacylglycerides, diacylglycerides, and sphingomyelin, decreased expression of myelin-associated glycoprotein, and increased expression of c-Jun and p75-neurotrophin receptor, suggesting a regression of SCs to a less mature developmental state. Taken together, our results define the contribution of SC MCT1 to both SC metabolism and peripheral nerve maturation and aging.

Loss of cerebellar glutamate transporters EAAT4 and GLAST differentially affects the spontaneous firing pattern and survival of Purkinje cells.

Loss of excitatory amino acid transporters (EAATs) has been implicated in a number of human diseases including spinocerebellar ataxias, Alzhiemer's disease and motor neuron disease. EAAT4 and GLAST/EAAT1 are the two predominant EAATs responsible for maintaining low extracellular glutamate levels and preventing neurotoxicity in the cerebellum, the brain region essential for motor control. Here using genetically modified mice we identify new critical roles for EAAT4 and GLAST/EAAT1 as modulators of Purkinje cell (PC) spontaneous firing patterns. We show high EAAT4 levels, by limiting mGluR1 signalling, are essential in constraining inherently heterogeneous firing of zebrin-positive PCs. Moreover mGluR1 antagonists were found to restore regular spontaneous PC activity and motor behaviour in EAAT4 knockout mice. In contrast, GLAST/EAAT1 expression is required to sustain normal spontaneous simple spike activity in low EAAT4 expressing (zebrin-negative) PCs by restricting NMDA receptor activation. Blockade of NMDA receptor activity restores spontaneous activity in zebrin-negative PCs of GLAST knockout mice and furthermore alleviates motor deficits. In addition both transporters have differential effects on PC survival, with zebrin-negative PCs more vulnerable to loss of GLAST/EAAT1 and zebrin-positive PCs more vulnerable to loss of EAAT4. These findings reveal that glutamate transporter dysfunction through elevated extracellular glutamate and the aberrant activation of extrasynaptic receptors can disrupt cerebellar output by altering spontaneous PC firing. This expands our understanding of disease mechanisms in cerebellar ataxias and establishes EAATs as targets for restoring homeostasis in a variety of neurological diseases where altered cerebellar output is now thought to play a key role in pathogenesis.

Astrocyte Diversity: Current Insights and Future Directions.

Astrocytes make up 20-40% of glial cells within the central nervous system (CNS) and provide several crucial functions, ranging from metabolic and structural support to regulation of synaptogenesis and synaptic transmission. Although these cells are morphologically and functionally complex, astrocytes have been historically regarded as homogenous cell populations and studied as one population of cells. Fortunately, recent evidence in RNA profiling and imaging data has begun to refute this view. These studies suggest heterogeneity of astrocytes across brain regions, differing in many aspects such as morphology, function, physiological properties, developmental origins, and response to disease. Increased understanding of astrocyte heterogeneity is critical for investigations into the function of astrocytes in the brain and neuro-glia interactions. Furthermore, insights into astrocyte heterogeneity can help better understand their role in neurological disorders and potentially produce novel approaches to treating these diseases.

Amyotrophic lateral sclerosis care and research in the United States during the COVID-19 pandemic: Challenges and opportunities.

Coronavirus disease 2019 has created unprecedented challenges for amyotrophic lateral sclerosis (ALS) clinical care and research in the United States. Traditional evaluations for making an ALS diagnosis, measuring progression, and planning interventions rely on in-person visits that may now be unsafe or impossible. Evidence- and experience-based treatment options, such as multidisciplinary team care, feeding tubes, wheelchairs, home health, and hospice, have become more difficult to obtain and in some places are unavailable. In addition, the pandemic has impacted ALS clinical trials by impairing the ability to obtain measurements for trial eligibility, to monitor safety and efficacy outcomes, and to dispense study drug, as these also often rely on in-person visits. We review opportunities for overcoming some of these challenges through telemedicine and novel measurements. These can reoptimize ALS care and research in the current setting and during future events that may limit travel and face-to-face interactions.

C9orf72 nucleotide repeat structures initiate molecular cascades of disease.

A hexanucleotide repeat expansion (HRE), (GGGGCC)n, in C9orf72 is the most common genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we identify a molecular mechanism by which structural polymorphism of the HRE leads to ALS/FTD pathology and defects. The HRE forms DNA and RNA G-quadruplexes with distinct structures and promotes RNA•DNA hybrids (R-loops). The structural polymorphism causes a repeat-length-dependent accumulation of transcripts aborted in the HRE region. These transcribed repeats bind to ribonucleoproteins in a conformation-dependent manner. Specifically, nucleolin, an essential nucleolar protein, preferentially binds the HRE G-quadruplex, and patient cells show evidence of nucleolar stress. Our results demonstrate that distinct C9orf72 HRE structural polymorphism at both DNA and RNA levels initiates molecular cascades leading to ALS/FTD pathologies, and provide the basis for a mechanistic model for repeat-associated neurodegenerative diseases.

Motor neuron-derived microRNAs cause astrocyte dysfunction in amyotrophic lateral sclerosis.

We recently demonstrated that microRNA-218 (miR-218) is greatly enriched in motor neurons and is released extracellularly in amyotrophic lateral sclerosis model rats. To determine if the released, motor neuron-derived miR-218 may have a functional role in amyotrophic lateral sclerosis, we examined the effect of miR-218 on neighbouring astrocytes. Surprisingly, we found that extracellular, motor neuron-derived miR-218 can be taken up by astrocytes and is sufficient to downregulate an important glutamate transporter in astrocytes [excitatory amino acid transporter 2 (EAAT2)]. The effect of miR-218 on astrocytes extends beyond EAAT2 since miR-218 binding sites are enriched in mRNAs translationally downregulated in amyotrophic lateral sclerosis astrocytes. Inhibiting miR-218 with antisense oligonucleotides in amyotrophic lateral sclerosis model mice mitigates the loss of EAAT2 and other miR-218-mediated changes, providing an important in vivo demonstration of the relevance of microRNA-mediated communication between neurons and astrocytes. These data define a novel mechanism in neurodegeneration whereby microRNAs derived from dying neurons can directly modify the glial phenotype and cause astrocyte dysfunction.

Motor neuron disease, TDP-43 pathology, and memory deficits in mice expressing ALS-FTD-linked UBQLN2 mutations.

Missense mutations in ubiquilin 2 (UBQLN2) cause ALS with frontotemporal dementia (ALS-FTD). Animal models of ALS are useful for understanding the mechanisms of pathogenesis and for preclinical investigations. However, previous rodent models carrying UBQLN2 mutations failed to manifest any sign of motor neuron disease. Here, we show that lines of mice expressing either the ALS-FTD-linked P497S or P506T UBQLN2 mutations have cognitive deficits, shortened lifespans, and develop motor neuron disease, mimicking the human disease. Neuropathologic analysis of the mice with end-stage disease revealed the accumulation of ubiquitinated inclusions in the brain and spinal cord, astrocytosis, a reduction in the number of hippocampal neurons, and reduced staining of TAR-DNA binding protein 43 in the nucleus, with concomitant formation of ubiquitin+ inclusions in the cytoplasm of spinal motor neurons. Moreover, both lines displayed denervation muscle atrophy and age-dependent loss of motor neurons that correlated with a reduction in the number of large-caliber axons. By contrast, two mouse lines expressing WT UBQLN2 were mostly devoid of clinical and pathological signs of disease. These UBQLN2 mouse models provide valuable tools for identifying the mechanisms underlying ALS-FTD pathogenesis and for investigating therapeutic strategies to halt disease.