Description

sym-2 encodes an ortholog of the RNA-binding proteins hnRNPF and hnRNPH. A putative loss-of-function allele, sym-2(mn617) from (Davies et al., 1999) is a Y163N missense mutation, which lies N-terminal to the first RNA-recognition motif of SYM-2. Homozygous animals have no overt defects but animals heterozygous for sym-2(mn617) and homozygous for mec-8 mutations are embryonic lethal (Davies et al., 1999; Yochem et al., 2004). Additionally, sym-2(mn617) suppresses the exhaustion-induced locomotion defect of smn-1(cb131) animals (Walsh et al., 2020). We have previously shown that human hnRNPA2, an ortholog of the protein encoded by C. elegans hrpa-1, interacts with hnRNPF in vitro (Ryan et al., 2020). As the low complexity (LC) domains of hnRNPA2 and HRPA-1 are not well conserved, we replaced the third coding exon of the C. elegans hrpa-1 gene with the corresponding human protein sequence codon optimized for C. elegans expression, resulting in a chimeric HRPA-1 protein with the human LC domain (HRPA-1HsLC^WT) (Ryan et al., 2020). A mutation in hnRNPA2, D290V, is associated with multisystem proteinopathy, a disease that causes degeneration of muscles, bone, and neurons (Kim et al., 2013). Expression of the mutant chimeric version of hrpa-1, hrpa-1HsLC^D290V, causes glutamatergic neurodegeneration in phasmid neurons after oxidative stress in animals lacking endogenous hrpa-1 function (Ryan et al., 2020). To control for defects associated with array integration, we also created an empty array, hrpa-1HsLC^empty, which contains no hrpa-1. We hypothesized that sym-2(mn617) might rescue hrpa-1HsLC^D290V stress-induced neurodegeneration.

To test this hypothesis, we generated animals carrying sym-2(mn617) with hrpa-1(tm781) and hrpa-1HsLC variants. We first exposed all genotypes to 22 hours of paraquat-induced oxidative stress and counted intact neurons after dye filling. We found that sym-2(mn617) rescued hrpa-1HsLC^D290V stress-induced neurodegeneration but we also found that about 25% of the neurons in sym-2(mn617) animals failed to dye fill after 22 hours of stress. Virtually all sym-2(mn617) neurons dye filled after only 4 hours of stress; therefore, the dye filling defect is degeneration induced by stress. Interestingly, we found that sym-2(mn617) neurodegeneration is sensitive to hrpa-1 function. Animals expressing both sym-2(mn617) and hrpa-1HsLC^WT showed significant rescue of the sym-2(mn617) defect back to WT levels. hrpa-1(tm781), hrpa-1HsLC^D290V, hrpa-1HsLC^empty did not rescue the sym-2(mn617) defect.

The identification of neurodegeneration in sym-2(mn617) animals after longer exposure to oxidative stress was surprising, especially given the suppression of neuromuscular defects in smn-1(cb131) (Walsh et al., 2020) and improvement of hrpa-1HsLC^D290V defects. However, HNRNPF, an ortholog of sym-2, is associated with the neurodevelopmental disorder Rett syndrome (Newnham et al., 2010), suggesting that HNRNPF has an important role in preserving neuronal integrity. Interestingly, although hrpa-1HsLC^WT can rescue the sym-2(mn617) neurodegeneration, hrpa-1HsLC^D290V has similar levels of neurodegeneration as sym-2(mn617), with substantially less neurodegeneration than observed for hrpa-1HsLC^D290V alone. As such, sym-2 may be in a genetic pathway upstream of hrpa-1, although more work is needed to elucidate what this pathway is and how it leads to neurodegeneration.