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

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. The authors would like to thank the technical and research support officers and the use of facilities within the Imaging Facility, Molecular Horizons, and Building 32, University of Wollongong. The authors also thank the donors and their families for which this work is possible.

This research was funded by US Department of Defense Therapeutic Ideas award AL210047 and a FightMND Impact grant (07) and was undertaken with the assistance of resources from the National Computational Infrastructure (NCI Australia), an NCRIS-enabled capability supported by the Australian Government. Parts of this research were funded by an Australian Research Council Centre of Excellence in Quantum Biotechnology CE230100021. Aspects of this research were conducted using instrumentation from the National NeuroProteomics Facility (NNPF), funded by a FightMND (ROR: https://ror.org/02htmqw41; Crossref Funder ID: 100014012) Infrastructure Grant (INF-202408-01396).

The Australian Research Council (ARC) supported NMR and MS equipment (grants LE0668477, LE140100119, and LE0237908) that were essential for the full characterisation and identification of all compounds that constitute the Davis Open Access Compound Library. Compounds Australia (https://www.griffith.edu.au/griffith-sciences/compounds-australia) is acknowledged for curating, plating, and shipping the Davis Open Access Compound Library from which the bioactive compounds described in this paper were initially sourced. We acknowledge the Benefit Sharing Agreement between the Queensland Government and Griffith University under the Biodiscovery Act 2004 (QLD), enabling equitable benefit sharing from biodiscovery using Queensland-sourced Native Biological Material.