Human SOD1 ALS Mutations in a Drosophila Knock-In Model Cause Severe Phenotypes and Reveal Dosage-Sensitive Gain- and Loss-of-Function Components

Abstract

Amyotrophic Lateral Sclerosis (ALS) is the most common adult-onset motor neuron disease and familial forms can be caused by numerous dominant mutations of the copper-zinc superoxide dismutase 1 (SOD1) gene. Substantial efforts have been invested in studying SOD1-ALS transgenic animal models; yet, the molecular mechanisms by which ALS-mutant SOD1 protein acquires toxicity are not well understood. ALS-like phenotypes in animal models are highly dependent on transgene dosage. Thus, issues of whether the ALS-like phenotypes of these models stem from overexpression of mutant alleles or from aspects of the SOD1 mutation itself are not easily deconvolved. To address concerns about levels of mutant SOD1 in disease pathogenesis, we have genetically engineered four human ALS-causing SOD1 point mutations (G37R, H48R, H71Y, and G85R) into the endogenous locus of Drosophila SOD1 (dsod) via ends-out homologous recombination and analyzed the resulting molecular, biochemical, and behavioral phenotypes. Contrary to previous transgenic models, we have recapitulated ALS-like phenotypes without overexpression of the mutant protein. Drosophila carrying homozygous mutations rendering SOD1 protein enzymatically inactive (G85R, H48R, and H71Y) exhibited neurodegeneration, locomotor deficits, and shortened life span. The mutation retaining enzymatic activity (G37R) was phenotypically indistinguishable from controls. While the observed mutant dsod phenotypes were recessive, a gain-of-function component was uncovered through dosage studies and comparisons with age-matched dsod null animals, which failed to show severe locomotor defects or nerve degeneration. We conclude that the Drosophila knock-in model captures important aspects of human SOD1-based ALS and provides a powerful and useful tool for further genetic studies.

Carbonic Anhydrase I modifies SOD1-induced motor neuron toxicity in Drosophila via ER stress pathway

“…Similar to what's discovered in mouse [20], dsod null lies had defect but did not readily exhibit ALS-like phenotype [25]. Interestingly, the Drosophila knock-in model of hSOD1 mutations was reported [25]. Flies homozygous for the knock-in dsod which contained the equivalent human ALS mutation were lethal, while lies heterozygous for the same knock-in mutant gene exhibited ALS-like pathology in a recessive manner [25].…”

“…Stress response of Hsp70 activation in glia was reported to be associated with the toxicity [24]. Similar to what's discovered in mouse [20], dsod null lies had defect but did not readily exhibit ALS-like phenotype [25]. Interestingly, the Drosophila knock-in model of hSOD1 mutations was reported [25].…”

Phenotypic Suppression of ALS/FTD-Associated Neurodegeneration Highlights Mechanisms of Dysfunction

“…The K.A.W. laboratory tested a subset of these genetic modifiers in a fourth ALS model, a knock-in of the SOD1-G85R lesion into the endogenous locus (dSod1 G85R ) (Sahin et al, 2017), for suppression of degenerative phenotypes. More than 30 genes were identified that could modify all four ALS models.…”

Genetic strategies to tackle neurological diseases in fruit flies

“…On the other hand, a large collection of nervous system-specific GAL4 lines [14,15] is available, permitting knockdown of genes in specific neuronal and glial cell (sub)populations. Finally, CRISPR based strategies can be employed to edit genes [16,17]. In all cases, it is highly recommended that the phenotypes associated with mutations that are being studied are rescued using either a UAS-cDNA approach or better, a genomic fragment that encodes the gene (see Sections C and D).…”

“…This requires that the human and fly proteins are likely orthologues and that the residues affected in humans are conserved in flies. Mutations or variants can be integrated into the fly homologue of disease-associated genes by homologous recombination (HR) [65] and this strategy was utilized to introduce ALS-associated SOD1 point mutations in the fly homologue of SOD1 [16]. This study provided valuable insights about the nature of the different mutations that affect SOD1 .…”