Defense Date

2-29-2024

Graduation Date

Spring 5-18-2024

Availability

One-year Embargo

Submission Type

dissertation

Degree Name

PhD

Department

Pharmacology

School

School of Pharmacy

Committee Chair

Rehana K. Leak

Committee Member

Lauren A. O'Donnell

Committee Member

Aleem Gangjee

Committee Member

Paula A. Witt-Enderby

Committee Member

David A. Johnson

Keywords

Synuclein, Microtubules, Acetylation, Parkinson's Disease, Tyrosination, Detyrosination, Tubulin, Post-translational modification, Neurons, Lewy Body Disease, Dementia with Lewy bodies

Abstract

In Lewy body disorders, α-synuclein aggregates are believed to destabilize the microtubular framework. However, microtubules are a challenging therapeutic target, as excessive rigidity or flexibility afforded by microtubule modulators may be neurotoxic. Thus, the objective of the present study was to evaluate dual-acting microtubule- stabilizing/destabilizing agents in cellular and animal models of early-stage Lewy body disease.

In Aim 1 we tested the dual-acting tricylic pyrimido[4,5-b]indole analogs AG161-47 and AG161-41 in an in vitro preformed fibril induced model of Lewy body disease. We found that both compounds reduced preformed fibril-seeded α-synucleinopathy without toxicity in primary hippocampal cultures. AG161-47 subtly impacted microtubule stability markers in primary hippocampal cultures, and its mitigating effects on α-synucleinopathy and nuclear fragmentation were interrupted by nocodazole.

In aim 2 we tested two doses of AG161-47 (5, and 10 mg/kg/day) along with placebo tablets in CD-1 mice in an in vivo model of ⍺-synucleinopathies. We administered these doses in a new chewable tablet formulation for six weeks to mice infused with preformed fibrils in the bulbar anterior olfactory nucleus. Mice voluntarily consumed a tablet per day—without need for daily oral gavage or invasive injections. Neither 5 nor 10 mg/kg AG161-47 affected α-synuclein inclusion or neuron counts in histological assays, but the fraction of insoluble α-synuclein that was hyperphosphorylated was slightly lowered by 5 mg/kg AG161-47. AG161-47 did not affect acetylation, detyrosination, or tyrosination of α- tubulin in vivo. However, α-synucleinopathy was modestly correlated with α-tubulin acetylation—a marker of microtubule stability—and AG161-47 uncoupled this unexpected link.

In aim 3, we assessed the impact of Lewy body diseases on the microtubule stability markers in postmortem human brain samples. We report that biological sex has a significant impact on microtubule stability markers in human amygdala and olfactory bulb tissues. Women display significantly higher levels of nonstabilized and stabilized microtubule fractions, while displaying lower levels of highly stabilized microtubules. In contrast, Lewy body disease did not affect these markers in a statistically robust manner, suggesting that any impact of these neurodegenerative conditions on microtubule stability is subtler than the impact of biological sex.

In summary, the most important findings of our experimental work included the unexpectedly positive correlation between pathological α-synuclein aggregation and acetylated (highly stable) ⍺-tubulin, which warrants further study. A second important finding was the lack of a direct impact of Lewy body disorders on microtubular post-translational modifications in the human olfactory bulb or amygdala. Perhaps for these reasons, microtubule-modulating candidate drugs did not robustly alleviate pathological processes in mice infused with preformed fibrils. We conclude that microtubules remain a challenging therapeutic target. Excessive rigidity imparted by microtubule stabilizers might be toxic, and careful titration of stabilization vs. destabilization with dual-acting agents may be necessary.

Language

English

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