Enrichment of SARM1 alleles encoding variants with constitutively hyperactive NADase in patients with ALS and other motor nerve disorders

Abstract

SARM1, a protein with critical NADase activity, is a central executioner in a conserved programme of axon degeneration. We describe eight rare missense or in-frame microdeletion human SARM1 variant alleles, in patients with amyotrophic lateral sclerosis (ALS), hereditary spastic paraparesis (HSP), or other motor nerve disorders, that alter the SARM1 auto-inhibitory ARM domain and constitutively hyperactivate SARM1 NADase activity. The constitutive NADase activities of six of the variants are at least as high as that of SARM1 lacking the entire ARM domain and greatly exceed the basal activity of wild-type SARM1, even in the presence of nicotinamide mononucleotide (NMN), its physiological activator. This rise in constitutive activity alone is enough to promote neuronal degeneration in response to otherwise non-harmful mild stress. Importantly, we provide evidence that these gain-of-function alleles are enriched in ALS and HSP patients compared to matched individuals without these conditions within ALS and other motor nerve disorder databases. Together, these data suggest these are risk alleles for ALS and other motor nerve disorders. The broad phenotypic heterogeneity and variable age-of-onset in patients with these alleles raises intriguing questions about the pathogenic mechanism of hyperactive SARM1 variants.

Programmed axon death executor SARM1 is a multi-functional NAD(P)ase with prominent base exchange activity, all regulated by physiological levels of NMN, NAD, NADP and other metabolites

“…Our data also have many implications for the development of therapies for axonopathies. Animal and cell culture data strongly implicate SARM1-dependent axon loss in some peripheral neuropathies, glaucoma, Parkinson's disease and other conditions [1,3,59] and human genetic studies support its involvement in rare axonopathies involving NMNAT2 mutation [7,8] and in ALS [5,6]. This, together with the complete rescue of axons by SARM1 removal when the pathway is very specifically activated [10,20] has led to considerable focus on inhibiting and knocking down SARM1 as a therapeutic strategy [3,28,32,60].…”

“…Wallerian degeneration occurs when axons are physically transected but it is now clear that the underlying programmed axon death mechanism is also activated by many toxins, gene mutations or metabolic disruption [3,4]. SARM1 is also constitutively hyperactivated by mutations associated with ALS [5,6]. The crucial step common to many of these is loss of functional NMNAT2 from axons, an enzyme synthesising the dinucleotide NAD from its precursor mononucleotide NMN.…”

“…HEK 293T cells were grown in DMEM with 4,500 mg/L glucose and 110 mg/L sodium pyruvate (PAA), supplemented with 2 mM glutamine, 1% penicillin/streptomycin (both Invitrogen), and 10% fetal bovine serum (PAA). Cells (50-70% confluence) in 10 cm dishes were transfected with 24 µg expression constructs for SARM1 or SAM-TIR[5,20] using Lipofectamine 2000 reagent (Invitrogen) as per manufacturer instructions. Media was supplemented with 2 mM NR at the time of transfection to boost expression.…”

“…[3][4][5][6][7][8][9][10][11] (167 mM Tris, 167 mM Bis-Tris, 167 mM sodium acetate). Stocks of 10X universal buffer were adjusted to each pH with 5 N HCl and diluted in the assay.…”

A Novel NAD Signaling Mechanism in Axon Degeneration and its Relationship to Innate Immunity

“…Furthermore, stillborn fetuses with biallelic NMNAT2 null mutations were also reported (Lukacs et al, 2019). Following earlier human studies implicating this pathway in ALS by GWAS association to the SARM1 chromosomal locus and loss of potential SARM1 regulator STMN2 in human induced pluripotent stem cell (hiPSC)derived motor neurons (Fogh et al, 2014;van Rheenen et al, 2016;Klim et al, 2019;Melamed et al, 2019), recent work has revealed that human SARM1 variant alleles that hyperactivate SARM1 NADase function and enhance neuronal vulnerability are enriched in patients with sporadic ALS, hereditary spastic paraplegia and other motor nerve disorders (Bloom et al, 2021;Gilley et al, 2021). Whether further mutations to NMNAT2, SARM1 or other proteins involved in the Wallerian degeneration pathway represent risk factors for other neurological diseases in living humans, is yet to be seen.…”