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.

Mutations in α-synuclein, TDP-43 and tau prolong protein half-life through diminished degradation by lysosomal proteases.

BACKGROUND: Autosomal dominant mutations in α-synuclein, TDP-43 and tau are thought to predispose to neurodegeneration by enhancing protein aggregation. While a subset of α-synuclein, TDP-43 and tau mutations has been shown to increase the structural propensity of these proteins toward self-association, rates of aggregation are also highly dependent on protein steady state concentrations, which are in large part regulated by their rates of lysosomal degradation. Previous studies have shown that lysosomal proteases operate precisely and not indiscriminately, cleaving their substrates at very specific linear amino acid sequences. With this knowledge, we hypothesized that certain coding mutations in α-synuclein, TDP-43 and tau may lead to increased protein steady state concentrations and eventual aggregation by an alternative mechanism, that is, through disrupting lysosomal protease cleavage recognition motifs and subsequently conferring protease resistance to these proteins. RESULTS: To test this possibility, we first generated comprehensive proteolysis maps containing all of the potential lysosomal protease cleavage sites for α-synuclein, TDP-43 and tau. In silico analyses of these maps indicated that certain mutations would diminish cathepsin cleavage, a prediction we confirmed utilizing in vitro protease assays. We then validated these findings in cell models and induced neurons, demonstrating that mutant forms of α-synuclein, TDP-43 and tau are degraded less efficiently than wild type despite being imported into lysosomes at similar rates. CONCLUSIONS: Together, this study provides evidence that pathogenic mutations in the N-terminal domain of α-synuclein (G51D, A53T), low complexity domain of TDP-43 (A315T, Q331K, M337V) and R1 and R2 domains of tau (K257T, N279K, S305N) directly impair their own lysosomal degradation, altering protein homeostasis and increasing cellular protein concentrations by extending the degradation half-lives of these proteins. These results also point to novel, shared, alternative mechanism by which different forms of neurodegeneration, including synucleinopathies, TDP-43 proteinopathies and tauopathies, may arise. Importantly, they also provide a roadmap for how the upregulation of particular lysosomal proteases could be targeted as potential therapeutics for human neurodegenerative disease.

Processing of progranulin into granulins involves multiple lysosomal proteases and is affected in frontotemporal lobar degeneration.

BACKGROUND: Progranulin loss-of-function mutations are linked to frontotemporal lobar degeneration with TDP-43 positive inclusions (FTLD-TDP-Pgrn). Progranulin (PGRN) is an intracellular and secreted pro-protein that is proteolytically cleaved into individual granulin peptides, which are increasingly thought to contribute to FTLD-TDP-Pgrn disease pathophysiology. Intracellular PGRN is processed into granulins in the endo-lysosomal compartments. Therefore, to better understand the conversion of intracellular PGRN into granulins, we systematically tested the ability of different classes of endo-lysosomal proteases to process PGRN at a range of pH setpoints. RESULTS: In vitro cleavage assays identified multiple enzymes that can process human PGRN into multi- and single-granulin fragments in a pH-dependent manner. We confirmed the role of cathepsin B and cathepsin L in PGRN processing and showed that these and several previously unidentified lysosomal proteases (cathepsins E, G, K, S and V) are able to process PGRN in distinctive, pH-dependent manners. In addition, we have demonstrated a new role for asparagine endopeptidase (AEP) in processing PGRN, with AEP having the unique ability to liberate granulin F from the pro-protein. Brain tissue from individuals with FTLD-TDP-Pgrn showed increased PGRN processing to granulin F and increased AEP activity in degenerating brain regions but not in regions unaffected by disease. CONCLUSIONS: This study demonstrates that multiple lysosomal proteases may work in concert to liberate multi-granulin fragments and granulins. It also implicates both AEP and granulin F in the neurobiology of FTLD-TDP-Pgrn. Modulating progranulin cleavage and granulin production may represent therapeutic strategies for FTLD-Pgrn and other progranulin-related diseases.

Progranulin Adsorbs to Polypropylene Tubes and Disrupts Functional Assays: Implications for Research, Biomarker Studies, and Therapeutics.

Progranulin (PGRN) is a tightly regulated, secreted glycoprotein involved in a wide range of biological processes that is of tremendous interest to the scientific community due to its involvement in neoplastic, neurodevelopmental, and neurodegenerative diseases. In particular, progranulin haploinsufficiency leads to frontotemporal dementia. While performing experiments with a HIS-tagged recombinant human (rh) PGRN protein, we observed a measurable depletion of protein from solution due to its adsorption onto polypropylene (PPE) microcentrifuge tubes. In this study, we have quantified the extent of rhPGRN adsorption to PPE tubes while varying experimental conditions, including incubation time and temperature. We found that ∼25-35% of rhPGRN becomes adsorbed to the surface of PPE tubes even after a short incubation period. We then directly showed the deleterious impact of PGRN adsorption in functional assays and have recommended alternative labware to minimize these effects. Although the risk of adsorption of some purified proteins and peptides to polymer plastics has been characterized previously, this is the first report of rhPGRN adsorption. Moreover, since PGRN is currently being studied and utilized in both basic science laboratories to perform in vitro studies and translational laboratories to survey PGRN as a quantitative dementia biomarker and potential replacement therapy, the reported observations here are broadly impactful and will likely significantly affect the design and interpretation of future experiments centered on progranulin biology.

EphA7 signaling guides cortical dendritic development and spine maturation.

The process by which excitatory neurons are generated and mature during the development of the cerebral cortex occurs in a stereotyped manner; coordinated neuronal birth, migration, and differentiation during embryonic and early postnatal life are prerequisites for selective synaptic connections that mediate meaningful neurotransmission in maturity. Normal cortical function depends upon the proper elaboration of neurons, including the initial extension of cellular processes that lead to the formation of axons and dendrites and the subsequent maturation of synapses. Here, we examine the role of cell-based signaling via the receptor tyrosine kinase EphA7 in guiding the extension and maturation of cortical dendrites. EphA7, localized to dendritic shafts and spines of pyramidal cells, is uniquely expressed during cortical neuronal development. On patterned substrates, EphA7 signaling restricts dendritic extent, with Src and Tsc1 serving as downstream mediators. Perturbation of EphA7 signaling in vitro and in vivo alters dendritic elaboration: Dendrites are longer and more complex when EphA7 is absent and are shorter and simpler when EphA7 is ectopically expressed. Later in neuronal maturation, EphA7 influences protrusions from dendritic shafts and the assembling of synaptic components. Indeed, synaptic function relies on EphA7; the electrophysiological maturation of pyramidal neurons is delayed in cultures lacking EphA7, indicating that EphA7 enhances synaptic function. These results provide evidence of roles for Eph signaling, first in limiting the elaboration of cortical neuronal dendrites and then in coordinating the maturation and function of synapses.

Histone deacetylase inhibition suppresses myogenin-dependent atrogene activation in spinal muscular atrophy mice.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease caused by mutations in the survival of motor neuron 1 (SMN1) gene and deficient expression of the ubiquitously expressed SMN protein. Pathologically, SMA is characterized by motor neuron loss and severe muscle atrophy. During muscle atrophy, the E3 ligase atrogenes, atrogin-1 and muscle ring finger 1 (MuRF1), mediate muscle protein breakdown through the ubiquitin proteasome system. Atrogene expression can be induced by various upstream regulators. During acute denervation, they are activated by myogenin, which is in turn regulated by histone deacetylases 4 and 5. Here we show that atrogenes are induced in SMA model mice and in SMA patient muscle in association with increased myogenin and histone deacetylase-4 (HDAC4) expression. This activation during both acute denervation and SMA disease progression is suppressed by treatment with a histone deacetylase inhibitor; however, this treatment has no effect when atrogene induction occurs independently of myogenin. These results indicate that myogenin-dependent atrogene induction is amenable to pharmacological intervention with histone deacetylase inhibitors and help to explain the beneficial effects of these agents on SMA and other denervating diseases.