The calm before dSTORM: neuronal RyR2 and its role in Alzheimer’s disease and comorbid epilepsy

Alzheimer’s disease (AD) is an age-related, multifactorial neurodegenerative disease that makes up 50-75% of New Zealand and Australia’s dementia patients. An emerging hypothesis of AD pathogenesis is the calcium (Ca²⁺) hypothesis, which postulates that Ca²⁺ homeostasis is essential for neuronal function, and becomes disrupted in AD. One of the Ca²⁺ handling proteins involved in maintaining Ca²⁺ homeostasis is the ryanodine receptor type II (RyR2), a Ca²⁺ release channel highly expressed in CA1 hippocampal neurons. In AD, RyR2 dysfunction causes excessive RyR2-mediated Ca²⁺ leak, which impairs synaptic transmission and neuronal excitability, resulting in the development of AD-like symptoms. One of the mechanisms by which RyR2 can alter its Ca²⁺ leak is the formation of clusters and Ca²⁺ release units (CRUs). This thesis aimed to investigate RyR2’s role in early-onset AD (EOAD) using APP_swe/PS1dE9 mice.

Meta-analyses of a large APP_swe/PS1dE9 mouse colony and their WT littermates over an 80-week-period revealed APP_swe/PS1dE9 animals had a lower survival. Median survival for APP_swe/PS1dE9 animals was 33 weeks, whereas almost all WT survived the study period. When stratified by sex, females had a lower survival probability compared to male APP_swe/PS1dE9 animals. Congo Red staining revealed mortality preceded Ab plaque formation, suggesting other mechanisms drive hyperexcitability.

To measure the incidence of unprovoked, spontaneous seizures, a wireless EEG telemetric implant was developed and implanted in 7-9-week-old APP_swe/PS1dE9 animals for continuous in vivo EEG recordings with video recordings in parallel over a 19-week-period. Seizures and interictal epileptiform discharges were detected in APP_swe/PS1dE9 animals but absent in WT controls. Quantitative analysis of seizure burden was not conducted, representing a limitation and a focus for future studies.

Using direct stochastic optical reconstruction microscopy (dSTORM), our findings revealed that RyR2 remodeling is distinct between WT and APP_swe/PS1dE9, and they remodel differently with age. In young APP_swe/PS1dE9 animals, there are fewer clusters and fewer, smaller CRUs compared to their WT littermates. As age increases, CRUs undergo fragmentation, similar to what is seen in cardiac disease. These changes in RyR2 nanoscale arrangement highlights that young APP_swe/PS1dE9 animals exhibit reduced Ca²⁺ leak compared to their WT littermates, and this increases as they age.

As it was evident that RyR2 remodels in EOAD, our aim was to target RyR2 pharmacologically using RyR2 antagonist, R-carvedilol, to see if we could reduce APP_swe/PS1dE9 mortality. Our findings revealed while there was no difference between WT and APP_swe/PS1dE9 mice, when stratified by sex, improved survival was. a general trend seen in female APP_swe/PS1dE9 mice. This was the opposite in male APP_swe/PS1dE9 animals.

In summary, this thesis demonstrates APP_swe/PS1dE9 animals exhibit early mortality likely linked to seizures, with females being more susceptible than males. Mortality in APP_swe/PS1dE9 preceded Ab plaque formation, but coincided with RyR2 remodeling, implicating RyR2 dysfunction in AD pathogenesis. R-carvedilol improved survival in female but not male APP_swe/PS1dE9 mice, emphasizing the importance of sex-specific considerations in AD therapeutic interventions. These findings establish a link between RyR2 remodeling, seizures in EOAD, and support RyR2 as a potential therapeutic target.