SUMMARY Neuronal hyperactivity is an early primary dysfunction in Alzheimer’s disease (AD) in humans and animal models, but effective neuronal hyperactivity-directed anti-AD therapeutic agents are lacking. Here we define a previously unknown mode of ryanodine receptor 2 (RyR2) control of neuronal hyperactivity and AD progression. We show that a single RyR2 point mutation, E4872Q, which reduces RyR2 open time, prevents hyperexcitability, hyperactivity, memory impairment, neuronal cell death, and dendritic spine loss in a severe early-onset AD mouse model (5xFAD). The RyR2-E4872Q mutation upregulates hippocampal CA1-pyramidal cell A-type K + current, a well-known neuronal excitability control that is downregulated in AD. Pharmacologically limiting RyR2 open time with the R -carvedilol enantiomer (but not racemic carvedilol) prevents and rescues neuronal hyperactivity, memory impairment, and neuron loss even in late stages of AD. These AD-related deficits are prevented even with continued β-amyloid accumulation. Thus, limiting RyR2 open time may be a hyperactivity-directed, non-β-amyloid-targeted anti-AD strategy.

Cardiac ryanodine receptors (RyR2s) are Ca 2þ release channels clustering in the sarcoplasmic reticulum membrane. These clusters are believed to be the elementary units of Ca 2þ release. The distribution of these Ca 2þ release units plays a critical role in determining the spatio-temporal profile and stability of sarcoplasmic reticulum Ca 2þ release. RyR2 clusters located in the interior of cardiomyocytes are arranged in highly ordered arrays. However, little is known about the distribution and function of RyR2 clusters in the periphery of cardiomyocytes. Here, we used a knock-in mouse model expressing a green fluorescence protein (GFP)-tagged RyR2 to localize RyR2 clusters in live ventricular myocytes by virtue of their GFP fluorescence. Confocal imaging and total internal reflection fluorescence microscopy was employed to determine and compare the distribution of GFP-RyR2 in the interior and periphery of isolated live ventricular myocytes and in intact hearts. We found tightly ordered arrays of GFP-RyR2 clusters in the interior, as previously described. In contrast, irregular distribution of GFP-RyR2 clusters was observed in the periphery. Time-lapse total internal reflection fluorescence imaging revealed dynamic movements of GFP-RyR2 clusters in the periphery, which were affected by external Ca 2þ and RyR2 activator (caffeine) and inhibitor (tetracaine), but little detectable movement of GFP-RyR2 clusters in the interior. Furthermore, simultaneous Ca 2þ -and GFP-imaging demonstrated that peripheral RyR2 clusters with an irregular distribution pattern are functional with a Ca 2þ release profile similar to that in the interior. These results indicate that the distribution of RyR2 clusters in the periphery of live ventricular myocytes is irregular and dynamic, which is different from that of RyR2 clusters in the interior.

Background:Little is known about the distribution of cardiac ryanodine receptor (RyR2) and its functional correlation in living cells. Results: Imaging live GFP-tagged RyR2 cardiomyocytes revealed Ca 2ϩ sparks originated exclusively from RyR2 clusters distributed along z-lines and transverse tubules. Conclusion:The distribution of RyR2 clusters determines the spatial profile of Ca 2ϩ release. Significance: RyR2 cluster distribution is an important determinant of Ca 2ϩ release in the heart.