Funded Studies
Objective/Rationale:
This project will evaluate the functional consequences of the interaction between 14-3-3 proteins and LRRK2. 14-3-3 proteins play a crucial role in inhibiting cell death pathways, and evidence is growing that 14-3-3 expression and function is disrupted in PD. Our work has shown that overexpression of 14-3-3s reduces toxicity in neurotoxin and alpha-synuclein-based models of PD, while disruption of 14-3-3s promotes toxicity. 14-3-3s have been recently demonstrated to bind LRRK2 and affect its cellular localization. We hypothesize that 14-3-3 dysfunction in PD produces toxic effects through dysregulation of LRRK2 activity.
Project Description:
We will examine the effect of 14-3-3s on LRRK2 toxicity in cell culture. One established measure of LRRK2 toxicity is the induction of neurite retraction by LRRK2 mutants in cell culture. Primary neuronal cultures from BAC transgenic models overexpressing G2019S LRRK2 show retraction of neurites, whereas neuronal cultures from LRRK2 pre-clinical models exhibit neurite extension. We will examine whether overexpression of different 14-3-3 isoforms can reduce or prevent neurite retraction in primary neuronal cultures from BAC G2019S LRRK2 pre-clinical models. Conversely, we will test whether the 14-3-3 inhibitor difopein promotes neurite retraction induced by G2019S in this system. We will also test whether difopein induces neurite retraction in neuronal cultures from BAC wildtype LRRK2 models that do not show neurite retraction. As a control for nonspecific actions, the effects of 14-3-3s and difopein will be tested in cells in which LRRK2 expression is absent.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
While the binding of 14-3-3s to several proteins implicated in the pathogenesis of PD, including alpha-synuclein and LRRK2, has been demonstrated, whether these interactions with 14-3-3s play a role in disease mechanisms is not known. If we find that 14-3-3 proteins can alter LRRK2 toxicity in this neurite retraction assay, these findings would support the hypothesis that 14-3-3 dysfunction observed in PD could produce toxic effects through dysregulation of LRRK2 function. 14-3-3 proteins could serve as a target for PD therapeutics.
Anticipated Outcome:
This project will determine whether the 14-3-3 proteins can regulate LRRK2 function and toxicity in primary neuronal cultures. If we find that 14-3-3 proteins can alter LRRK2 toxicity in this neurite retraction assay, such findings would support further evaluation of this interaction in pre-clinical models.