Phenotypic screening in ALS motor neurons identifies SIRT1 activation as a genotype-independent neuroprotective strategy

Abstract Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, characterised by motor neuron degeneration and intrinsic neuronal hyperexcitability, with a lack of disease modifying treatments. One of the major challenges in therapeutic development for ALS is the genetic heterogeneity. Thus, there is a pressing need for human models of sporadic and familial ALS that enable identification of convergent, genotype-independent neuroprotective mechanisms that correct multiple ALS-associated phenotypes. This study screened spinal motor neurons generated from sporadic and familial ALS donor induced pluripotent stem cells (iPSCs) with 512 natural products isolated from fungal, plant and marine invertebrate sources. We utilised live cell imaging, whole cell patch clamp and proteomics to identify compounds that could protect against neuronal degeneration, neurite damage and intrinsic hyperexcitability in ALS motor neurons. Two resveratrol tetramers isolated from a Papua New Guinean rainforest canopy tree, vaticanol B and (-)-hopeaphenol, protected against neuronal degeneration and neurite damage across genotypes. Both tetramers were more potent than resveratrol, a well-characterised SIRT1 activator, with (-)-hopeaphenol exhibiting the greatest effects. (-)-hopeaphenol conferred broad neuroprotection in an expanded cohort of sporadic and familial ALS motor neurons spanning TARDBP, SOD1 , or C9ORF72 genotypes ( n = 14 iPSC lines). Additionally, (-)-hopeaphenol treatment protected against intrinsic hyperexcitability, reversing aberrant increases in Na + currents and alterations in repetitive firing. As a pathway to clinical translation, we showed SRT2104, a second generation SIRT1 activator with blood-brain barrier permeability and tolerability in humans, protected against neurite degeneration, ALS-related hyperexcitability changes and upregulated neuroprotective pathways at threefold lower concentration than (-)-hopeaphenol. These results provide proof-of-concept that SIRT1 activation in motor neurons offers a convergent, genotype-independent neuroprotective strategy for ALS.