Elaine Roby

A common genetic form of Parkinson's disease (PD) is caused by mutations in LRRK2. We identify WSB1 as a LRRK2 interacting protein. WSB1 ubiquitinates LRRK2 through K27 and K29 linkage chains, leading to LRRK2 aggregation and neuronal protection in primary neurons and a Drosophila model of G2019S LRRK2. Knocking down endogenous WSB1 exacerbates mutant LRRK2 neuronal toxicity in neurons and the Drosophila model, indicating a role for endogenous WSB1 in modulating LRRK2 cell toxicity. WSB1 is in Lewy bodies in human PD post-mortem tissue. These data demonstrate a role for WSB1 in mutant LRRK2 pathogenesis, and suggest involvement in Lewy body pathology in sporadic PD. Our data indicate a role in PD for ubiquitin K27 and K29 linkages, and suggest that ubiquitination may be a signal for aggregation and neuronal protection in PD, which may be relevant for other neurodegenerative disorders. Finally, our study identifies a novel therapeutic target for PD.

Post-translational modifications (PTMs) of proteins regulate various cellular processes. PTMs of polyglutamine-expanded huntingtin (Htt) protein, which causes Huntington's disease (HD), are likely modulators of HD pathogenesis. Previous studies have identified and characterized several PTMs on exogenously expressed Htt fragments, but none of them were designed to systematically characterize PTMs on the endogenous full-length Htt protein. We found that full-length endogenous Htt, which was immunoprecipitated from HD knock-in mouse and human post-mortem brain, is suitable for detection of PTMs by mass spectrometry. Using label-free and mass tag labeling-based approaches, we identified near 40 PTMs, of which half are novel (data are available via ProteomeXchange with identifier PXD005753). Most PTMs were located in clusters within predicted unstructured domains rather than within the predicted α-helical structured HEAT repeats. Using quantitative mass spectrometry, we detected significant differences in the stoichiometry of several PTMs between HD and WT mouse brain. The mass-spectrometry identification and quantitation were verified using phospho-specific antibodies for selected PTMs. To further validate our findings, we introduced individual PTM alterations within full-length Htt and identified several PTMs that can modulate its subcellular localization in striatal cells. These findings will be instrumental in further assembling the Htt PTM framework and highlight several PTMs as potential therapeutic targets for HD.

Huntington's disease (HD) is caused in large part by a polyglutamine expansion within the huntingtin (Htt) protein. Post-translational modifications (PTMs) control and regulate many protein functions and cellular pathways, and PTMs of mutant Htt are likely important modulators of HD pathogenesis. Alterations of selected numbers of PTMs of Htt fragments have been shown to modulate Htt cellular localization and toxicity. In this study, we systematically introduced site-directed alterations in individual phosphorylation and acetylation sites in full-length Htt constructs. The effects of each of these PTM alteration constructs were tested on cell toxicity using our nuclear condensation assay and on mitochondrial viability by measuring mitochondrial potential and size. Using these functional assays in primary neurons, we identified several PTMs whose alteration can block neuronal toxicity and prevent potential loss and swelling of the mitochondria caused by mutant Htt. These PTMs included previously described sites such as serine 116 and newly found sites such as serine 2652 throughout the protein. We found that these functionally relevant sites are clustered in protease-sensitive domains throughout full-length Htt. These findings advance our understanding of the Htt PTM code and its role in HD pathogenesis. Because PTMs are catalyzed by enzymes, the toxicity-modulating Htt PTMs identified here may be promising therapeutic targets for managing HD.

BACKGROUND: The potential benefit of cysteamine for Huntington's disease has been demonstrated in HD animal models. Cysteamine and its derivate cystamine were shown to reduce neuropathology and prolong lifespan. Human studies have demonstrated safety, and suggestive results regarding efficacy. Despite all the studies available in vivo, there are only few in vitro studies, and the mechanism of action of cysteamine remains unclear. OBJECTIVE: The objective of this study is to assess the capacity of cysteamine for neuroprotection against mutant Huntingtin in vitro using cellular models of HD, and to provide initial data regarding mechanism of action. METHODS: We tested the neuroprotective properties of cysteamine in vitro in our primary neuron and iPSC models of HD. RESULTS: Cysteamine showed a strong neuroprotective effect (EC50 = 7.1 nM) against mutant Htt-(aa-1-586 82Q) toxicity, in a nuclear condensation cell toxicity assay. Cysteamine also rescued mitochondrial changes induced by mutant Htt. Modulation of the levels of cysteine or glutathione failed to protect neurons, suggesting that cysteamine neuroprotection is not mediated through cysteine metabolism. Taurine and Hypotaurine, which are metabolites of cysteamine can protect neurons against Htt toxicity, but the inhibition of the enzyme converting cysteamine to hypotaurine does not block either protective activity, suggesting independent protective pathways. Cysteamine has been suggested to activate BDNF secretion; however, cysteamine protection was not blocked by BDNF pathway antagonists. CONCLUSIONS: Cysteamine was strongly neuroprotective with relatively high potency. We demonstrated that the main neuroprotective pathways that have been proposed to be the mechanism of protection by cysteamine can all be blocked and still not prevent the neuroprotective effect. The results suggest the involvement of other yet-to-be-determined neuroprotective pathways.