Doctor of Philosophy
Prado, Vania F.
Prado, Marco AM.
Cholinergic dysfunction is a major contributor to Alzheimer’s Disease (AD), a neurodegenerative disorder characterized by cognitive impairment and loss of functional abilities. Prior studies from our group have shown that mice with a forebrain-specific deletion of the vesicular acetylcholine transporter (VAChT), a key protein for acetylcholine (ACh) release, exhibit pathological features associated with AD. However, whether the modulation of cholinergic signaling or its downstream targets could affect AD-like pathology remains unclear. In this dissertation, we assessed the effects of both upregulating and downregulating cholinergic tone on AD-like pathology in male and female mice.
To study the effects of enhancing cholinergic neurotransmission, we treated forebrain VAChT-knock-out (VAChTKO) mice with BQCA, a highly selective positive allosteric modulator, to specifically activate the M1 muscarinic ACh receptor. We quantified the levels of AD-associated proteins including amyloid β (Aβ), the splicing regulator hnRNPA2/B1 and the enzyme initiating Aβ synthesis, BACE1. Our results show that M1 activation modulates the abnormal levels of BACE1 and hnRNPA2/B1 in VAChT-deficient males compared with vehicle-treated mice, while no difference was found in Tris-soluble Aβ. Interestingly, BQCA-treated females did not show any difference in these proteins when compared to controls. Next, to specifically investigate the role of BACE1 in AD-like pathology, we generated a new mouse model by crossing VAChTKO mice with BACE1KO mice. Our results suggest that BACE1 signaling pathway modulates the levels of hnRNPA2/B1 and the synaptic marker PSD95 independently of the cholinergic tone. Moreover, we found that the absence of BACE1 does not rescue the deficiency of the non-amyloidogenic pathway identified in VAChTKO mice. Lastly, to study whether VAChT levels are causally associated with Aβ deposition, as well as the role of biological sex in this relationship, we generated new mouse lines by crossing mice expressing humanized amyloid precursor protein with mice either lacking or overexpressing VAChT. We found that VAChT levels have a bidirectional causal effect on amyloid pathology in male mice and ovariectomized female mice, but not in ovary-intact or estradiol-treated ovariectomized female mice. Together, these results show that cholinergic signaling modulates AD-like pathology in specific hormonal enviroments, highlighting a sexual dimorphism in this regulation.
Summary for Lay Audience
The cholinergic system is an important chemical pathway in the body that plays a key role in the development of Alzheimer's Disease (AD). This system uses acetylcholine (ACh), a chemical messenger essential for learning and memory, both greatly affected in AD. In this disease, brain cells that produce ACh start to deteriorate. This leads to increased amyloid-beta (Aβ), a harmful protein that forms toxic deposits in the brain and causes cell death. However, the full details on how ACh impacts AD are still unclear. In this thesis, we investigated the effects of changing the levels of ACh (either increasing or decreasing them) in male and female mouse models of AD.
Our study shows that increasing cholinergic communication restores the balance of important proteins affected by AD. One of these proteins is BACE1, which is involved in Aβ production. Interestingly, this effect was only seen in male mice, while females did not respond to the boost in cholinergic signaling. We also studied the specific role of BACE1 in AD by creating mice missing both ACh and BACE1 in the brain. We discovered that BACE1 regulates several proteins, independently of the levels of ACh. However, even when the lack of BACE1 completely blocked the production of Aβ, it was not enough to rescue all the abnormalities caused by ACh loss. Finally, to investigate whether ACh levels are associated with the formation of the harmful Aβ buildup, we used mouse models that develop Aβ plaques at different ages. Our results showed that elevating cholinergic signalling decreases the formation of Aβ deposits, while reducing ACh levels increased toxic Aβ aggregation. Importantly, these results were only seen in male mice. To better understand this modulation, we also operated on female mice to remove their ovaries, essentially stopping the production of sex hormones. We found that female mice without ovaries had the same response to changes in cholinergic signaling as the males, indicating that sex-specific hormones play an important role in this regulation.
This thesis demonstrates that cholinergic signaling modulates AD-like abnormalities and calls attention to the sex differences in this regulation.
German-Castelan, Liliana, "Cholinergic Regulation of Alzheimer's-Like Pathology" (2023). Electronic Thesis and Dissertation Repository. 9745.
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