Investigating the Neuronal Signals Initiating Synapse Loss in Aging and Alzheimer's Disease

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Abstract: Late-onset Alzheimer’s disease (LOAD) targets the association cortices to cause profound dementia, and available treatments do not alter disease progression. The greatest risk factor for LOAD is advanced age, yet it is unknown why the aging association cortices are vulnerable to degeneration. Cognitive impairment tightly correlates with synapse loss in the dorsolateral prefrontal association cortex (dlPFC), which subserves higher-order cognition. Mouse models have shown that microglia can remove synapses by interacting with molecular “tags” on neurons, including complement (C1q) and phosphatidylserine (PS). However, it is unclear what upstream changes within neurons trigger the generation of these molecules, and whether these mechanisms are activated in the aging dlPFC. The proposed research will utilize an aged rhesus macaque model with mechanistic in vitro experiments, to identify the intraneuronal mechanisms that contribute to synapse loss in the aging association cortex. Aging rhesus macaques have an expanded dlPFC and naturally develop cognitive deficits, plaques and tangles, complement C1q expression, and region-specific synapse loss. Aged macaques also develop an abnormal mitochondrial phenotype termed “Mitochondria-on-a- string” (MOAS) that is seen in human LOAD. I hypothesize that MOAS may arise from chronic calcium (Ca2+) overload of mitochondria, generating molecules participating in synapse removal, including activated C1q, PS, and Caspase 3. Synapses in dlPFC are especially vulnerable to Ca2+ dysregulation as they express cAMP- protein kinase A (PKA) signaling to magnify internal Ca2+ release through ryanodine receptor 2 (RyR2) and inositol triphosphate receptor type 1 (IP3R1). This process is regulated by the phosphodiesterase PDE4D in young brain, which is lost with advancing age. I hypothesize that sustained elevations in cytosolic calcium leads to Ca2+ overload of mitochondria and the induction of MOAS with advancing age, leading to the expression of molecules (C1q, PS, and cleaved caspase 3) that mediate synapse removal by nearby microglia. Aims 1 and 2 will utilize high resolution immuno-Electron Microscopy (EM) and 3D EM reconstruction to elucidate the interactions between MOAS, and (Aim 1) molecules known to mediate synapse removal, and (Aim 2) markers of Ca2+ dysregulation in the dlPFC of young vs. aged macaques. Preliminary data indicate that MOAS preferentially associate with C1q and are more frequent under conditions when PDE4D expression is absent. Aim 3 will use primary murine cortical neuron cultures, immunofluorescence, super-resolution microscopy, and biochemistry to model Ca2+ dysregulation in vitro and test whether chronically elevated Ca2+ levels can induce the MOAS phenotype and generate the molecules mediating synapse removal. Relevant in vitro findings will be cross-validated in the macaque tissue. Identification of the intraneuronal events that lead to synapse loss in the vulnerable aging cortex will provide key insights into how advancing age contributes to LOAD pathogenesis, and help identify potential targets for early therapeutic interventions.