Dopamine (DA) substitution therapy by L-DOPA effectively ameliorates many Parkinson's Disease (PD) symptoms caused by DA neuron (DAN) degeneration but leads to the formation of debilitating L-DOPA induced dyskinesia (LID) in most patients after several years of treatment. This treatment complication severely curtails the therapeutic window of L-DOPA and finding strategies that overcome LID formation remains a pressing clinical need. We recently found that DAN not only release dopamine but also the secreted cell signaling factor Sonic Hedgehog (Shh) which they use to signal selectively to cholinergic (CIN) and fast spiking (FS) interneurons among all dopaminergic targets. CIN have become implicated in LID independently via multiple lines of research and clinical observations. Thus our findings suggested that LID might emerge because CIN and FS are exposed to high DA but low Shh signaling during DA replacement therapy. Consistent with this hypothesis we found that stimulation of the Shh pathway through agonists of the Shh effector GPCR Smoothened (Smo) in addition to L-DOPA attenuates LID in mouse- and non-human primate-models of PD. While our primate studies suggest that augmenting Shh/Smo signaling together with L-DOPA might attenuate LID in PD it is unlikely that agonists of Smo are viable drugs in humans because of their severe oncogenic liability. Instead we aim to identify druggable events downstream of Shh/Smo signaling in CIN and FS in in vivo models of PD and LID. The hope is that emerging targets might be more selective and their pharmacological manipulation more tolerable than stimulating Smo signaling. To enable this goal we propose here to adapt and validate methods that allow (1) optic control over Shh/Smo signaling in the basal ganglia (BG) of LID expressing animals and (2) neuron subtype specific metabolic tagging and identification of proteins and their post-translational modifications in response to Smo activation in LID.
In aim1 we will produce chimeric Melanopsin:Smoothened proteins (Mel:Smo) and determine their light sensitivity in vitro. Light responsive Mel:Smo chimeras will then be expressed in vivo in a Cre dependent manner from Adeno-Associated Viruses (AAV) in CIN of the dorso-lateral striatum of the LID sensitive Aphakia mouse line. We will test whether light activation of Mel:Smo chimeras attenuates LID and will quantify that effect relative to Smo agonists that activate non recombinant Smo. These reagents will test the hypothesis that Smo signaling in CIN causes LID attenuation.
In aim2 we will identify proteins that become modified in response to Smo signaling selectively in either CIN or FS interneurons of mice that were acutely injected with either agonists or antagonists of Smo. This approach is enabled by mice expressing Cre in CIN or FS interneurons and that carry a conditional methionyl-tRNA synthetase L274G point mutation (MetRS) allele which causes the incorporation of azidonorleucine instead of methionine into de novo synthesized proteins. Tagged proteins and their modifications will be identified by mass spectrometry upon affinity purification. These tools will allow the mechanistic study of an emerging key signaling pathway at the molecular level in the healthy BG of behaving animals and might identify drug targets in LID and other BG diseases.
Dopamine neurons, those neurons that degenerate in Parkinson's Disease (PD), do not only communicate with their targets by dopamine but also several other factors among them Sonic Hedgehog (Shh). Complementing dopamine substitution therapy by the dopamine precursor L-Dopa with agonists for the Shh pathway in mouse and non human primate models of PD attenuates the development of utterly debilitating L-dopa induced dyskinesia that arise in the clinic after long term L-Dopa dosing. The current project aims to identify the molecular changes that occur in cholinergic and fast spiking target neurons of dopaminergic innervation in response to dopamine and Shh in order to find novel drug targets for PD therapy.
