Pharmacologic Activation of Integrated Stress Response Kinases Inhibits Pathologic Mitochondrial Fragmentation.

Excessive mitochondrial fragmentation is associated with the pathologic mitochondrial dysfunction implicated in the pathogenesis of etiologically-diverse diseases, including many neurodegenerative disorders. The integrated stress response (ISR) - comprising the four eIF2α kinases PERK, GCN2, PKR, and HRI - is a prominent stress-responsive signaling pathway that regulates mitochondrial morphology and function in response to diverse types of pathologic insult. This suggests that pharmacologic, stress-independent activation of the ISR represents a potential strategy to mitigate pathologic mitochondrial fragmentation associated with human disease. Here, we show that pharmacologic, stress-independent activation of the ISR kinases HRI or GCN2 promotes adaptive mitochondrial elongation and prevents mitochondrial fragmentation induced by the calcium ionophore ionomycin. Further, we show that stress-independent activation of these ISR kinases reduces mitochondrial fragmentation and restores basal mitochondrial morphology in patient fibroblasts expressing the pathogenic D414V variant of the pro-fusion mitochondrial GTPase MFN2 associated with neurological dysfunctions including ataxia, optic atrophy, and sensorineural hearing loss. These results identify pharmacologic, stress-independent activation of ISR kinases as a potential strategy to prevent pathologic mitochondrial fragmentation induced by disease-relevant chemical and genetic insults, further motivating the pursuit of highly selective ISR kinase-activating compounds as a therapeutic strategy to mitigate mitochondrial dysfunction implicated in diverse human diseases.

Uncompetitive, adduct-forming SARM1 inhibitors are neuroprotective in preclinical models of nerve injury and disease.

Axon degeneration is an early pathological event in many neurological diseases. The identification of the nicotinamide adenine dinucleotide (NAD) hydrolase SARM1 as a central metabolic sensor and axon executioner presents an exciting opportunity to develop novel neuroprotective therapies that can prevent or halt the degenerative process, yet limited progress has been made on advancing efficacious inhibitors. We describe a class of NAD-dependent active-site SARM1 inhibitors that function by intercepting NAD hydrolysis and undergoing covalent conjugation with the reaction product adenosine diphosphate ribose (ADPR). The resulting small-molecule ADPR adducts are highly potent and confer compelling neuroprotection in preclinical models of neurological injury and disease, validating this mode of inhibition as a viable therapeutic strategy. Additionally, we show that the most potent inhibitor of CD38, a related NAD hydrolase, also functions by the same mechanism, further underscoring the broader applicability of this mechanism in developing therapies against this class of enzymes.

Structural and Mechanistic Regulation of the Pro-degenerative NAD Hydrolase SARM1.

The NADase SARM1 is a central switch in injury-activated axon degeneration, an early hallmark of many neurological diseases. Here, we present cryo-electron microscopy (cryo-EM) structures of autoinhibited (3.3 Å) and active SARM1 (6.8 Å) and provide mechanistic insight into the tight regulation of SARM1's function by the local metabolic environment. Although both states retain an octameric core, the defining feature of the autoinhibited state is a lock between the autoinhibitory Armadillo/HEAT motif (ARM) and catalytic Toll/interleukin-1 receptor (TIR) domains, which traps SARM1 in an inactive state. Mutations that break this lock activate SARM1, resulting in catastrophic neuronal death. Notably, the mutants cannot be further activated by the endogenous activator nicotinamide mononucleotide (NMN), and active SARM1 is product inhibited by Nicotinamide (NAM), highlighting SARM1's functional dependence on key metabolites in the NAD salvage pathway. Our studies provide a molecular understanding of SARM1's transition from an autoinhibited to an injury-activated state and lay the foundation for future SARM1-based therapies to treat axonopathies.