Date of Degree

9-2022

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Andreas H. Kottmann

Committee Members

Hysell Oviedo

Lice Ghilardi

Christoph Kellendonk

Stephan Rayport

Subject Categories

Molecular and Cellular Neuroscience

Keywords

Cholinergic Interneuron, Dopamine Neuron, GPR139, Parkinson's Disease, Sonic Hedgehog, Striatum

Abstract

Mesencephalic dopamine neurons (DAN) signal motivation and reward prediction errors. Their collective functions are considered critical for the translation of thought into context appropriate actions. Dopaminergic dysfunction has severe consequences: (1) DAN degeneration is a major determinate of Parkinson’s Disease (PD), (2) disturbed dopaminergic signaling in schizophrenia leads to unorganized learning, and (3) chemically induced dopaminergic signaling results in impulse control disorders (ICDs) and life-threatening addictions. The use of the dopamine (DA) precursor L-Dopa for DA replacement therapy in PD patients produces motor and behavioral side-effects such as L-Dopa induced dyskinesias (LID), ICDs, and a propensity for opioid addiction. Despite intense clinical and basic research efforts since the introduction of L-Dopa replacement therapy for PD management in the 1970’s, no therapeutic strategy has emerged that curtails the long-term side effects of L-Dopa therapy while preserving its anti-akinetic benefits. First pioneered by the laboratories of Steven Rayport and David Sulzer and subsequently involving many laboratories in the field of basal ganglia research, it has become increasingly clear during the last 20 years that DAN engage in neuronal co-transmission using multiple neurotransmitters and secreted peptides to communicate with their signaling targets. Specifically, the Kottmann Lab previously discovered that all DAN express the secreted cell signaling peptide sonic hedgehog (Shh) throughout life. DAN engagement in neuronal co-transmission has emerged as an explanation as to why there is poor success in treatment of DAN dysfunction by pharmacological manipulations of dopamine signaling alone. However, how the different modalities of dopaminergic co-transmission mechanistically work together to bring about the varied functions of DAN remains largely unknown. In my thesis work, I experimentally isolated the function of Shh signaling emanating from DAN (ShhDAN) from other dopaminergic signaling modalities through conditional genetic and pharmacological approaches and analyzed the circuit, cell-physiological, gene expression, and behavioral consequences of these manipulations. I first describe the establishment of a unilateral DAN-selective optogenetic stimulation paradigm that indicates that high frequency optogenetic stimulation of DAN releases ShhDAN and results in a graded exhaustion of releasable ShhDAN in a matter of minutes. This paradigm revealed that a progressive imbalance of ShhDAN causes a progressive loss of suppression of abnormal movements or dyskinesias. In collaborative studies based on this paradigm, I find that optogenetic stimulation released ShhDAN acts on cholinergic interneurons of the striatum (CIN) via the Shh signaling GPCR effector Smoothened. ShhDAN increases steady state cholinergic activity levels and decreases MAP kinase pathway activity in CIN. Together, these experiments reveal that ShhDAN impinges on the quality of locomotion via modulating CIN activity levels and opposing the effect of DA on CIN. Reduced ShhDAN signaling results in the expression of abnormal and involuntary movements that are reminiscent of L-Dopa induced dyskinesia. These results were published in Lauren Malave, Dustin Zuelke et al., 2021. Motivated by the dyskinesia experiments, I identify cell-autonomous mechanisms by which ShhDAN signaling counteracts DA signaling in CIN. Here I leverage mice with conditional ablation of ShhDAN to determine changes in gene expression in CIN using affinity purified polysomal RNA from CIN for comparative gene expression profiling. I discovered 44 CIN specific gene expression changes in CIN enriched RNA and 145 secondary gene expression changes in striatal RNA specifically depleted for CIN RNA. Employing a gene expression validation scheme based in part on previous knowledge of patterned Shh signaling strength in the striatum, I prioritized CIN RNA specific changes in gene expression and identified the orphan GPCR GPR139 as a potential target of ShhDAN signaling in CIN. I then begin to probe the physiological role of GPR139 function in CIN. GPR139 was previously recognized as a potent negative modulator of mu opioid receptor (MOR) signaling. MOR acts as a well-characterized negative modulator of CIN activity by inhibiting MOR’s activation of GIRK. Aberrant MOR signaling has been previously implicated in LID and MOR is the target of abused synthetic opioids. In preliminary results I find that GPR139 agonist treatment attenuates LID in mice with interrupted Shh signaling onto CIN. I also find that Shh pathways agonists that act on the Shh effector smoothened attenuate morphine induced place preference. Together with our results from Malave et al., in which we reveal that reduced Shh signaling onto CIN induces, my results begin to suggest that ShhDAN counteracts LID expression and opioid elicited habit formation in part by dampening opioid mediated inhibition of CIN (Zuelke et al., in prep.).

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