Summary There are no therapies that reverse the proteotoxic misfolding events that underpin fatal neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and Parkinson disease (PD). Hsp104, a conserved hexameric AAA+ protein from yeast, solubilizes disordered aggregates and amyloid, but has no metazoan homologue and only limited activity against human neurodegenerative disease proteins. Here, we reprogram Hsp104 to rescue TDP-43, FUS, and α-synuclein proteotoxicity by mutating single residues in helix 1, 2, or 3 of the middle domain or the small domain of nucleotide-binding domain 1. Potentiated Hsp104 variants enhance aggregate dissolution, restore proper protein localization, suppress proteotoxicity, and in a C. elegans PD model attenuate dopaminergic neurodegeneration. Potentiating mutations reconfigure how Hsp 104 subunits collaborate, desensitize Hsp104 to inhibition, obviate any requirement for Hsp70, and enhance ATPase, translocation, and unfoldase activity. Our work establishes that disease-associated aggregates and amyloid are tractable targets and that enhanced disaggregases can restore proteostasis and mitigate neurodegeneration.

Genetic and idiopathic forms of Parkinson's disease (PD) are characterized by loss of dopamine (DA) neurons and typically the formation of protein inclusions containing the alpha-synuclein (α-syn) protein. Environmental contributors to PD remain largely unresolved but toxins, such as paraquat or rotenone, represent well-studied enhancers of susceptibility. Previously, we reported that a bacterial metabolite produced by Streptomyces venezuelae caused age- and dose-dependent DA neurodegeneration in Caenorhabditis elegans and human SH-SY5Y neurons. We hypothesized that this metabolite from a common soil bacterium could enhance neurodegeneration in combination with PD susceptibility gene mutations or toxicants. Here, we report that exposure to the metabolite in C. elegans DA neurons expressing human α-syn or LRRK2 G2019S exacerbates neurodegeneration. Using the PD toxin models 6-hydroxydopamine and rotenone, we demonstrate that exposure to more than one environmental risk factor has an additive effect in eliciting DA neurodegeneration. Evidence suggests that PD-related toxicants cause mitochondrial dysfunction, thus we examined the impact of the metabolite on mitochondrial activity and oxidative stress. An ex vivo assay of C. elegans extracts revealed that this metabolite causes excessive production of reactive oxygen species. Likewise, enhanced expression of a superoxide dismutase reporter was observed in vivo. The anti-oxidant probucol fully rescued metabolite-induced DA neurodegeneration, as well. Interestingly, the stress-responsive FOXO transcription factor DAF-16 was activated following exposure to the metabolite. Through further mechanistic analysis, we discerned the mitochondrial defects associated with metabolite exposure included adenosine triphosphate impairment and upregulation of the mitochondrial unfolded protein response. Metabolite-induced toxicity in DA neurons was rescued by complex I activators. RNA interference (RNAi) knockdown of mitochondrial complex I subunits resulted in rescue of metabolite-induced toxicity in DA neurons. Taken together, our characterization of cellular responses to the S. venezuelae metabolite indicates that this putative environmental trigger of neurotoxicity may cause cell death, in part, through mitochondrial dysfunction and oxidative stress.

Due to environmental insult or innate genetic deficiency, protein folding environments of the mitochondrial matrix are prone to dysregulation, prompting the activation of a specific organellar stress-response mechanism, the mitochondrial unfolded protein response (UPR). In , mitochondrial damage leads to nuclear translocation of the ATFS-1 transcription factor to activate the UPR After short-term acute stress has been mitigated, the UPR is eventually suppressed to restore homeostasis to hermaphrodites. In contrast, and reflective of the more chronic nature of progressive neurodegenerative disorders such as Parkinson's disease (PD), here, we report the consequences of prolonged, cell-autonomous activation of the UPR in dopaminergic neurons. We reveal that neuronal function and integrity decline rapidly with age, culminating in activity-dependent, non-apoptotic cell death. In a PD-like context wherein transgenic nematodes express the Lewy body constituent protein α-synuclein (αS), we not only find that this protein and its PD-associated disease variants have the capacity to induce the UPR, but also that coexpression of αS and ATFS-1-associated dysregulation of the UPR synergistically potentiate dopaminergic neurotoxicity. This genetic interaction is in parallel to mitophagic pathways dependent on the homolog, which is necessary for cellular resistance to chronic malfunction of the UPR Given the increasingly recognized role of mitochondrial quality control in neurodegenerative diseases, these studies illustrate, for the first time, an insidious aspect of mitochondrial signaling in which the UPR pathway, under disease-associated, context-specific dysregulation, exacerbates disruption of dopaminergic neurons , resulting in the neurodegeneration characteristic of PD. Disruptions or alterations in the activation of pathways that regulate mitochondrial quality control have been linked to neurodegenerative diseases due in part to the central role of mitochondria in metabolism, ROS regulation, and proteostasis. The extent to which these pathways, including the mitochondrial unfolded protein response (UPR) and mitophagy, are active may predict severity and progression of these disorders, as well as sensitivity to compounding stressors. Furthermore, therapeutic strategies that aim to induce these pathways may benefit from increased study into cellular responses that arise from long-term or ectopic stimulation, especially in neuronal compartments. By demonstrating the detrimental consequences of prolonged cellular activation of the UPR, we provide evidence that this pathway is not a universally beneficial mechanism because dysregulation has neurotoxic consequences.

In the nematode C. elegans, insulin signaling regulates development and aging in response to the secretion of numerous insulin peptides. Here, we describe a novel, non-signaling isoform of the nematode insulin receptor (IR), DAF-2B, that modulates insulin signaling by sequestration of insulin peptides. DAF-2B arises via alternative splicing and retains the extracellular ligand binding domain but lacks the intracellular signaling domain. A daf-2b splicing reporter revealed active regulation of this transcript through development, particularly in the dauer larva, a diapause stage associated with longevity. CRISPR knock-in of mScarlet into the daf-2b genomic locus confirmed that DAF-2B is expressed in vivo and is likely secreted. Genetic studies indicate that DAF-2B influences dauer entry, dauer recovery and adult lifespan by altering insulin sensitivity according to the prevailing insulin milieu. Thus, in C. elegans alternative splicing at the daf-2 locus generates a truncated IR that fine-tunes insulin signaling in response to the environment.

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