Single copy/knock-in models of ALS SOD1 in C. elegans suggest loss and gain of function have different contributions to cholinergic and glutamatergic neurodegeneration

Abstract

Mutations in Cu/Zn superoxide dismutase 1 (SOD1) lead to Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disease that disproportionately affects glutamatergic and cholinergic motor neurons. Previous work with SOD1 overexpression models supports a role for SOD1 toxic gain of function in ALS pathogenesis. However, the impact of SOD1 loss of function in ALS cannot be directly examined in overexpression models. In addition, overexpression may obscure the contribution of SOD1 loss of function in the degeneration of different neuronal populations. Here, we report the first single-copy, ALS knock-in models in C. elegans generated by transposon- or CRISPR/Cas9- mediated genome editing of the endogenous sod-1 gene. Introduction of ALS patient amino acid changes A4V, H71Y, L84V, G85R or G93A into the C. elegans sod-1 gene yielded single-copy/knock-in ALS SOD1 models. These differ from previously reported overexpression models in multiple assays. In single-copy/knock-in models, we observed differential impact of sod-1 ALS alleles on glutamatergic and cholinergic neurodegeneration. A4V, H71Y, G85R, and G93A animals showed increased SOD1 protein accumulation and oxidative stress induced degeneration, consistent with a toxic gain of function in cholinergic motor neurons. By contrast, H71Y, L84V, and G85R lead to glutamatergic neuron degeneration due to sod-1 loss of function after oxidative stress. However, dopaminergic and serotonergic neuronal populations were spared in single-copy ALS models, suggesting a neuronal-subtype specificity previously not reported in invertebrate ALS SOD1 models. Combined, these results suggest that knock-in models may reproduce the neurotransmitter-type specificity of ALS and that both SOD1 loss and gain of toxic function differentially contribute to ALS pathogenesis in different neuronal populations.

Age-dependent degeneration of an identified adult leg motor neuron in a Drosophila SOD1 model of ALS

“…Thus, our data indicate a combination of gain- and loss-of-function phenotypes in sod H71Y /sod H71Y flies. Other groups have suggested that ALS caused by sod1 mutations is due to both gain and loss-of-function and our data generally support this model ( Baskoylu et al, 2018 ; Saccon et al, 2013 ; Sau et al, 2007 ).…”

Modeling neurodegeneration in Caenorhabditis elegans

“…Notably, these same two mutations (G85R and G93R) were subsequently modeled as single-copy insertions, along with three other common SOD-1 mutations (A4V, H71Y, L84) in C. elegans . The impact on both glutamatergic and cholinergic neuron degeneration was examined for all five of these mutations ( Baskoylu et al, 2018 ). G93R displayed phenotypes consistent with a toxic gain-of-function phenotype in cholinergic neurons, while G85R caused glutamatergic neurodegeneration following exposure to the neurotoxin paraquat ( Baskoylu et al, 2018 ).…”

“…As another example, animals treated with quinolinic acid exhibited neurodegeneration due to glutamatergic neurotransmission defects ( da Silveira et al, 2018 ). Similarly, in single-copy knock-in SOD1 models of ALS, loss of sod-1 function produced defects in light touch response indicative of a disruption in glutamate signaling ( Baskoylu et al, 2018 ).…”

Tyrosine phosphorylation regulates hnRNPA2 granule protein partitioning & reduces neurodegeneration

“…No overt defects were observed in any lines. To assess neurodegeneration, we used dye uptake assays, which provide a sensitive readout of glutamatergic neurodegeneration in other disease models (Baskoylu et al, 2018;Faber et al, 1999). In dye uptake assays, the intact sensory endings of specific glutamatergic sensory neurons take up fluorescent dyes from the environment and backfill cell bodies.…”