Mutations in nucleotide metabolism genes bypass proteasome defects in png-1/NGLY1-deficient Caenorhabditis elegans.

The conserved SKN-1A/Nrf1 transcription factor regulates the expression of proteasome subunit genes and is essential for maintenance of adequate proteasome function in animal development, aging, and stress responses. Unusual among transcription factors, SKN-1A/Nrf1 is a glycoprotein synthesized in the endoplasmic reticulum (ER). N-glycosylated SKN-1A/Nrf1 exits the ER and is deglycosylated in the cytosol by the PNG-1/NGLY1 peptide:N-glycanase. Deglycosylation edits the protein sequence of SKN-1A/Nrf1 by converting N-glycosylated asparagine residues to aspartate, which is necessary for SKN-1A/Nrf1 transcriptional activation of proteasome subunit genes. Homozygous loss-of-function mutations in the peptide:N-glycanase (NGLY1) gene cause NGLY1 deficiency, a congenital disorder of deglycosylation. There are no effective treatments for NGLY1 deficiency. Since SKN-1A/Nrf1 is a major client of NGLY1, the resulting proteasome deficit contributes to NGLY1 disease. We sought to identify targets for mitigation of proteasome dysfunction in NGLY1 deficiency that might indicate new avenues for treatment. We isolated mutations that suppress the sensitivity to proteasome inhibitors caused by inactivation of the NGLY1 ortholog PNG-1 in Caenorhabditis elegans. We identified multiple suppressor mutations affecting 3 conserved genes: rsks-1, tald-1, and ent-4. We show that the suppressors act through a SKN-1/Nrf-independent mechanism and confer proteostasis benefits consistent with amelioration of proteasome dysfunction. ent-4 encodes an intestinal nucleoside/nucleotide transporter, and we show that restriction of nucleotide availability is beneficial, whereas a nucleotide-rich diet exacerbates proteasome dysfunction in PNG-1/NGLY1-deficient C. elegans. Our findings suggest that dietary or pharmacological interventions altering nucleotide availability have the potential to mitigate proteasome insufficiency in NGLY1 deficiency and other diseases associated with proteasome dysfunction.

Streamlined single shot safe harbor transgene integration in C. elegans using unc-119 rescue.

Transgenic animals are an invaluable tool in model organism genetics. The ease of modifying the C. elegans genome through high-copy integration of transgenes facilitates the investigation of diverse and fundamental biological processes. However, generation of new multicopy integrated transgenes is limited by the time and labor cost. Further, many transgenes are integrated using non-specific DNA damaging agents. These DNA damaging agents cause unwanted mutations during the integration process and may have deleterious effects. A recently described method for CRISPR/Cas9-based integration of multicopy transgenes at safe harbor loci using Fluorescent Landmark Interference (FLInt) greatly increases the efficiency of multicopy transgene integration and mitigates issues related to off-target mutagenesis during integration. unc-119 rescue is a simple and widely used phenotypic marker in C. elegans transgenesis and genome engineering. To streamline generation of multicopy transgenes via FLInt, we have generated a set of strains suitable for FLInt-mediated integration of transgenes using rescue of the unc-119 mutant phenotype to select transgenic animals. We demonstrate the utility of this approach and outline a protocol that uses unc-119 rescue as a selection marker for streamlined integration of multicopy transgenes at safe harbor loci.

Meta-analysis of Genetic Modifiers Reveals Candidate Dysregulated Pathways in Amyotrophic Lateral Sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease that has significant overlap with frontotemporal dementia (FTD). Mutations in specific genes have been identified that can cause and/or predispose patients to ALS. However, the clinical variability seen in ALS patients suggests that additional genes impact pathology, susceptibility, severity, and/or progression of the disease. To identify molecular pathways involved in ALS, we undertook a meta-analysis of published genetic modifiers both in patients and in model organisms, and undertook bioinformatic pathway analysis. From 72 published studies, we generated a list of 946 genes whose perturbation (1) impacted ALS in patient populations, (2) altered defects in laboratory models, or (3) modified defects caused by ALS gene ortholog loss of function. Herein, these are all called modifier genes. We found 727 modifier genes that encode proteins with human orthologs. Of these, 43 modifier genes were identified as modifiers of more than one ALS gene/model, consistent with the hypothesis that shared genes and pathways may underlie ALS. Further, we used a gene ontology-based bioinformatic analysis to identify pathways and associated genes that may be important in ALS. To our knowledge this is the first comprehensive survey of ALS modifier genes. This work suggests that shared molecular mechanisms may underlie pathology caused by different ALS disease genes. Surprisingly, few ALS modifier genes have been tested in more than one disease model. Understanding genes that modify ALS-associated defects will help to elucidate the molecular pathways that underlie ALS and provide additional targets for therapeutic intervention.

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.

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.