Rothstein, Jeffrey D.

Abstract Apilimod dimesylate is a first-in-class phosphoinositide kinase, FYVE-type zinc finger-containing (PIKfyve) inhibitor with a favourable clinical safety profile and has demonstrated activity in preclinical C9orf72 and TDP-43 amyotrophic lateral sclerosis (ALS) models. In this ALS 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-end-point clinical trial was conducted in four US centres (ClinicalTrials.gov NCT05163886). Participants with C9orf72 repeat expansions were randomly assigned (2:1) to receive twice-daily oral treatment with 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 or serious adverse events attributable to the study drug and tolerability at trial completion or treatment over 12 weeks. Changes from baseline in plasma and CSF and concentrations of apilimod dimesylate 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)] were measured. Between 16 December 2021 and 7 July 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 [standard deviation (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 end-points in this phase 2a trial and demonstrated CNS penetrance and pharmacodynamic target engagement. Apilimod dimesylate was observed to result in the greatest reduction in CSF poly(GP) levels observed to date in C9orf72 clinical trials.

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.

AbstractObjectiveDual leucine zipper kinase (DLK), which regulates the c‐Jun N‐terminal kinase pathway involved in axon degeneration and apoptosis following neuronal injury, is a potential therapeutic target in amyotrophic lateral sclerosis (ALS). This first‐in‐human study investigated safety, tolerability, and pharmacokinetics (PK) of oral GDC‐0134, a small‐molecule DLK inhibitor. Plasma neurofilament light chain (NFL) levels were explored in GDC‐0134‐treated ALS patients and DLK conditional knockout (cKO) mice.MethodsThe study included placebo‐controlled, single and multiple ascending‐dose (SAD; MAD) stages, and an open‐label safety expansion (OLE) with adaptive dosing for up to 48 weeks.ResultsForty‐nine patients were enrolled. GDC‐0134 (up to 1200 mg daily) was well tolerated in the SAD and MAD stages, with no serious adverse events (SAEs). In the OLE, three study drug‐related SAEs occurred: thrombocytopenia, dysesthesia (both Grade 3), and optic ischemic neuropathy (Grade 4); Grade ≤2 sensory neurological AEs led to dose reductions/discontinuations. GDC‐0134 exposure was dose‐proportional (median half‐life = 84 h). Patients showed GDC‐0134 exposure‐dependent plasma NFL elevations; DLK cKO mice also exhibited plasma NFL compared to wild‐type littermates.InterpretationThis trial characterized GDC‐0134 safety and PK, but no adequately tolerated dose was identified. NFL elevations in GDC‐0134‐treated patients and DLK cKO mice raised questions about interpretation of biomarkers affected by both disease and on‐target drug effects. The safety profile of GDC‐0134 was considered unacceptable and led to discontinuation of further drug development for ALS. Further work is necessary to understand relationships between neuroprotective and potentially therapeutic effects of DLK knockout/inhibition and NFL changes in patients with ALS.

Small-molecule recruitment of an endogenous nuclease rescues pathologies associated with c9ALS/FTD in vivo by targeting an RNA repeat expansion.

Nuclear accumulation of CHMP7 leads to multiple cellular pathologies that can be mitigated via CHMP7 ASOs in familial and sporadic ALS iPSNs.

Disrupted nucleocytoplasmic transport (NCT) has been implicated in neurodegenerative disease pathogenesis; however, the mechanisms by which disrupted NCT causes neurodegeneration remain unclear. In a Drosophila screen, we identified ref(2)P/p62 , a key regulator of autophagy, as a potent suppressor of neurodegeneration caused by the GGGGCC hexanucleotide repeat expansion (G4C2 HRE) in C9orf72 that causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We found that p62 is increased and forms ubiquitinated aggregates due to decreased autophagic cargo degradation. Immunofluorescence and electron microscopy of Drosophila tissues demonstrate an accumulation of lysosome-like organelles that precedes neurodegeneration. These phenotypes are partially caused by cytoplasmic mislocalization of Mitf/TFEB, a key transcriptional regulator of autophagolysosomal function. Additionally, TFEB is mislocalized and downregulated in human cells expressing GGGGCC repeats and in C9-ALS patient motor cortex. Our data suggest that the C9orf72 -HRE impairs Mitf/TFEB nuclear import, thereby disrupting autophagy and exacerbating proteostasis defects in C9-ALS/FTD.

Poly(GP) proteins are a promising pharmacodynamic marker for developing and testing therapeutics for treating C9ORF72 -associated amyotrophic lateral sclerosis.

Mutations in the gene encoding superoxide dismutase 1 (SOD1) account for about 20% of the cases of familial amyotrophic lateral sclerosis (fALS). It is not known how the mutant protein causes disease, or why only a subset of cell types (motor neurons) are targeted. The aggregation and misfolding of mutant SOD1 are implicated in disease pathogenesis in both animal models and humans. We used a monoclonal antibody, C4F6, which specifically reacts with mutant and/or “misfolded” SOD1, to investigate the regional distribution of mutant SOD1 protein in rodent and human tissues. C4F6 reacted only with mutant SOD1 and showed remarkable selectivity for disease-affected tissues and cells. Tissue not affected by disease but containing high levels of mutant protein (sensory neurons) did not stain with C4F6. Additionally, C4F6 intensely stained some motor neurons while leaving adjacent motor neurons unstained. Although C4F6 was generated against the G93A SOD1 mutant, it also recognized other SOD1 mutants. In human autopsy tissues from patients carrying SOD1 mutations, C4F6 identified skein-like intracellular inclusions in motor neurons, similar to those seen in rodents, and again stained only a subset of motor neurons. In spinal cords from patients with sporadic ALS, other neurodegenerative diseases, and normal controls, C4F6-immunoreactive inclusions were not detected, but the antibody did reveal diffuse immunostaining of some spinal motor neurons. The ability of C4F6 to differentiate pathologically affected tissue in mutant SOD1 ALS rodent models and humans, specifically motor neuron populations, suggests that this antibody may recognize a “toxic” form of the mutant SOD1 protein.