UCSF

Parkinson’s disease (PD), the second most common neurodgenerative disorder, affects millions of people worldwide. With movement-related disabilities and other cognitive impairment as well as psychiatric symptoms, PD not only greatly deteriorates the health and quality of life of patients, but also poses severe burdens on their families. The current drug therapies available for PD provide symptomatic relief but there is yet to be a therapy that can slow disease progression. Spanning across three chapters, the goal of this thesis project involves the notion of the bench-to-bedside approach of identifying promising drug candidates that could provide protective benefits in neurodegenerative disorders and understanding the mechanism behind the neuroprotective effects. Chapter 1 focuses on developing a whole organism based phenotypic screening assay using larval zebrafish and performing high throughput screening of 1403 bioactive compounds. While therapeutic drug discovery has traditionally focused on target-based drug discovery, the implementation of phenotypic drug discovery particularly for neurological diseases allows us to focus on the direct therapeutic impact and bypass the complex biological process of neurodegeneration and in many cases provide leads to novel targets. The transgenic model used in our assay expresses the E. coli nitroreductase (NTR) controlled by the promoter of tyrosine hydroxylase (th), a rate-limiting enzyme in DA synthesis. The NTR converts the pro-drug MTZ to the toxic nitroso radical form in vivo causing DA neuronal loss in the ventral forebrain region. 57 compounds passed the threshold for strictly standardized mean difference (SSMD) and brain health score (BHS) when compared to the MTZ treatment alone. Pathways implicated in the pathophysiology of PD were shown significant including deubiquination, cyclooxygenase (COX), respiratory electron transport, and mitochondrial biogenesis. Novel pathways were also identified including sleep cycle, cell development, insulin regulation, and blood pressure regulation. In particular, aliskiren, captopril, and olmesartan, which all target the renin angiotensin pathway, showed significant neuroprotection and was validated in a blinded manual counting of DA neurons. In chapter 2, the neuroprotective mechanism of the renin angiotensin pathway was extensively studied by incorporating genetic engineering with conditional CRISPR, RNA-seq, and examining human clinical data. Using conditional CRISPR to knock out the angiotensin receptor type 1 (agtr1) in DA neurons, we revealed a cell-autonomous mechanism of neuroprotection through agtr1 inhibition. DA neuron-specific RNA-seq further identified pathways including the mitochondrial electron transport chain that are significantly perturbed in DA neuron degeneration and is abated by RAAS inhibitor treatment. The neuroprotective effect of RAAS inhibitors was validated with brain imaging and functional analysis in a chemically induced zebrafish Gaucher’s disease model and in a Drosophila PD model of pink1 deficiency. Finally, examination of 308 clinical PD patient data revealed a significant effect of RAAS inhibitors in delaying the onset of levodopa therapy and increasing performance in symptom assessment scores. In the final chapter, the transcriptional modification of RAAS pathway genes were examined at the organ level upon the administration of RAAS inhibitors. This was in light during the recent COVID-19 pandemic in which we tried to address the knowledge gap of whether RAAS inhibitors might affect the expression levels of angiotensin-converting-enzyme-2 (ace2), which could impact patient susceptibility to SARS-CoV-2. Upon daily treatment with aliskiren, olmesartan, and captopril for 7 consecutive days, the qRT-PCR analysis of major RAAS pathway genes in the brain, gill, heart, intestine, kidney, and liver showed organ specific changes in ace2 expression and the discontinuation of compound treatments for 7 days did not return ace2 expression to baseline levels. In conclusion, this dissertation work demonstrates the pipeline of accelerated drug discovery in the field of neurodegeneration from the initial drug screening to elucidating the underlying molecular mechanisms and the evaluation of clinical data. The work provided in this thesis hopes to potentially further the development on the therapeutic potential of RAAS inhibitors through the local signaling cascade that could impact diverse physiological functions aside from the classical cardiovascular system.