Date of Award

Spring 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Neuroscience

First Advisor

Arnsten, Amy

Abstract

Alzheimer’s disease (AD) is a devastating, progressive neurodegenerative disease, characterized by amyloid plaques and tau tangles, with synapse loss, calcium dysregulation, and accumulation of advanced glycation end products (AGEs). These pathologies develop in a region-specific manner, targeting regions responsible for higher-order integrative processing, such as the prefrontal cortex (PFC) and entorhinal cortex. Calcium signaling, through cAMP-PKA-calcium signaling, is critical to support the persistent firing needed for working memory. However, under various conditions, e.g., aging, inflammation, chronic stress, these circuits lose regulation of cAMP-PKA-calcium signaling, leading to calcium leak from the smooth endoplasmic reticulum (SER) and chronically elevated levels of cytosolic calcium. For instance, PKA phosphorylation of ryanodine receptors 2 (RyR2) at S2808 causes calcium to “leak” from the SER, which is observed in postmortem Alzheimer’s disease samples, chronic stress rodent models, and postmortem COVID-19 brain samples. However, the direct downstream consequences of such chronic calcium dysregulation remain elusive. The following chapters detail my investigation of an upstream regulator of this calcium pathway in the rodent medial PFC (mPFC) (Chapter 2) followed by a detailed characterization of the downstream consequences of chronic dysregulation of calcium in a genetic knock-in mouse model and aging macaques (Chapter 3), as well as mechanistic studies using in vitro models (Chapter 4). In Chapter 1, I comprehensively review previous literature on calcium dysregulation in the aging and AD brain as well as the glyoxalase system in the aging and AD brain. Building on prior macaque work, in Chapter 2, I report the localization of mGlu3 in the rodent mPFC. mGlu3 has been implicated in many cognitive disorders, including schizophrenia, and thus, has been of interest as a therapeutic target. However, few studies have carefully characterized the localization of mGlu3. In the rodent mPFC, we found mGlu3 to be localized not only on astrocytes, as expected, but also on dendritic spines. Unlike macaques where mGlu2 was the predominant isoform in macaques presynaptically, mGlu3 was robustly found presynaptically in rodent mPFC. I discuss the ramifications of the species differences for mGlu3 signaling. In Chapter 3, I examine the direct downstream consequences of chronic calcium leak through RyR2. Using the aging rhesus macaque model and a genetic mouse model, S2808D-RyR2, which causes constitutive calcium leak through a knock-in mutation, we performed mass spectrometry on synaptosomes harvested from the frontal cortex and hippocampus of young (3-month-old) mice. We identified one of the highest enriched proteins in S2808D-RyR2 mice to be glyoxalase I (GLO1). GLO1 was increased in expression and activity across the age span of the mouse. However, we also identified a bell-shaped pattern of GLO1 expression in the S2808D-RyR2 mouse with age. For the first time, we found this potential restorative mechanism also occurs in primates, as GLO1 increased with age in the PFC. Using multiple-label immunofluorescence and immuno-electron microscopy, we characterized in detail, the cellular and ultrastructural localization of GLO1 in macaque PFC and mouse PFC and dentate gyrus (DG). Altogether, our results reveal a novel link between RyR2-mediated calcium dysregulation and increased GLO1 expression in the brain. Next, in Chapter 4, I tested potential regulatory mechanisms of GLO1 using in vitro models. Using IPA, we identified upregulation of NRF2 pathways in the S2808D-RyR2 mice, a known mediator of GLO1. Using a neuro-epithelioma cell line, SK-N-MC, and a primary mixed glial culture system, we were able to increase NRF2 activation through a NRF2 activator, tert-Butylhydroquinone (TBHQ), and an activator of RyR2, 4-chloro-m-cresol (4CMC). However, neither led to increases in GLO1 expression nor activity. Instead, 4CMC led to a decrease in GLO1 activity in the primary mixed glial culture, suggesting potential limitations of the in vitro models. In summary, this work reveals new insights into an upstream mediator and a novel potential compensatory mechanism of chronic calcium dysregulation, including important species differences, with broad implications for cognitive disorders.

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