Novel Optical Tools to Investigate Presenilin Control of Neurotransmission

Neurotransmission is a critical function for neurons that underlies the complex processes of the brain. Deficits in synaptic function often manifest in neurodegenerative diseases, motivating the study of control points of neurotransmission. The Endoplasmic Reticulum (ER) has recently been described as one such control point, as ER Ca2+ levels can regulate the magnitude of synaptic vesicle exocytosis. Here we investigate the controversial hypothesis that Presenilins are ER Ca2+ leak channels to determine if a role in ER Ca2+ leak can explain the previously described regulation of neurotransmission by Presenilins. While Presenilins are best known for their involvement in Alzheimer’s Disease, this potential secondary role of Presenilins may shed insight into Presenilin involvement in neurodegeneration. To effectively investigate Presenilin control of ER Ca2+, we created a ratiometric version of the low affinity ER-GCaMPs in which we fused the HaloTag protein to allow for direct reporting of ER Ca2+ levels. We coupled this new indicator with in-cell calibrations at physiological temperatures to create a highly robust readout of ER Ca2+ across various cell types. Our analysis revealed a large variation in ER Ca2+ levels in both fibroblasts and hippocampal neurons indicating that ER Ca2+ may be an important control point across cells. The newly designed ratiometric ER-GCaMP provides a tool to study ER Ca2+ biology across various cell types and conditions with increased confidence. The ER-GCaMPs were leveraged to investigate Presenilin control of ER Ca2+. Utilizing Presenilin double knockout mouse embryonic fibroblasts revealed a decrease in resting ER Ca2+ levels, unlike the increase in resting ER Ca2+ levels observed in Presenilin 1 KO Human Embryonic Kidney cells. This result suggests that ER Ca2+ regulation by Presenilins differs across various cell types. Looking in hippocampal neurons conversely showed no effect of Presenilin knockdown or Alzheimer’s mutations on ER Ca2+ levels or dynamics, thereby failing to find support for Presenilins as ER Ca2+ leak channels in neurons. The ratiometric ER-GCaMP was a powerful tool to investigate ER Ca2+ regulation by Presenilins across cell types. While Presenilins were not observed to control ER Ca2+ in neurons, they may still have a role in regulating neurotransmission. We utilized vGlut1-pHluorin, a pH sensitive analog of GFP tagged to a synaptic vesicle protein, that allows for reporting of synaptic vesicle cycling. These experiments found evidence that Presenilins do control neurotransmission and that they do so by impacting the fraction of responsive boutons. This result provided insight into the molecular mechanism of Presenilin control of neurotransmission. This thesis investigated Presenilin control of ER Ca2+ across cell types as well as regulation of neurotransmission in hippocampal neurons. Evidence for Presenilin control of ER Ca2+ was observed in non-neuronal cells, but not in hippocampal neurons. However, Presenilins were still found to control neurotransmission. The development of a robust ratiometric probe will also aid future investigations of ER Ca2+ biology and provides a framework for the creation of future ratiometric probes