Steven Hou

Presenilin (PS)/γ-secretase plays a pivotal role in essential cellular events via proteolytic processing of transmembrane proteins that include APP and Notch receptors. However, how PS/γ-secretase activity is spatiotemporally regulated by other molecular and cellular factors and how the changes in PS/γ-secretase activity influence signaling pathways in live cells are poorly understood. These questions could be addressed by engineering a new tool that enables multiplexed imaging of PS/γ-secretase activity and additional cellular events in real-time. Here, we report the development of a near-infrared (NIR) FRET-based PS/γ-secretase biosensor, C99 720-670 probe, which incorporates an immediate PS/γ-secretase substrate APP C99 with miRFP670 and miRFP720 as the donor and acceptor fluorescent proteins, respectively. Extensive validation demonstrates that the C99 720-670 biosensor enables quantitative monitoring of endogenous PS/γ-secretase activity on a cell-by-cell basis in live cells (720/670 ratio: 2.47 ± 0.66 (vehicle) vs. 3.02 ± 1.17 (DAPT), ** p < 0.01). Importantly, the C99 720-670 and the previously developed APP C99 YPet-Turquoise-GL (C99 Y-T) biosensors simultaneously report PS/γ-secretase activity. This evidences the compatibility of the C99 720-670 biosensor with cyan (CFP)-yellow fluorescent protein (YFP)-based FRET biosensors for reporting other essential cellular events. Multiplexed imaging using the novel NIR biosensor C99 720-670 would open a new avenue to better understand the regulation and consequences of changes in PS/γ-secretase activity.

Amyloid-β oligomers (oAβ) are thought to mediate neurotoxicity in Alzheimer’s disease (AD), and previous studies in AD transgenic mice suggest that calcium dysregulation may contribute to these pathological effects. Even though AD mouse models remain a valuable resource to investigate amyloid neurotoxicity, the concomitant presence of soluble Aβ species, fibrillar Aβ, and fragments of amyloid precursor protein (APP) complicate the interpretation of the phenotypes. To explore the specific contribution of soluble oligomeric Aβ (oAβ) to calcium dyshomeostasis and synaptic morphological changes, we acutely exposed the healthy mouse brain, at 3 to 6 months of age, to naturally occurring soluble oligomers and investigated their effect on calcium levels using in vivo multiphoton imaging. We observed a dramatic increase in the levels of neuronal resting calcium, which was dependent upon extracellular calcium influx and activation of NMDA receptors. Ryanodine receptors, previously implicated in AD models, did not appear to be primarily involved using this experimental setting. We used the high resolution cortical volumes acquired in-vivo to measure the effect on synaptic densities and observed that, while spine density remained stable within the first hour of oAβ exposure, a significant decrease in the number of dendritic spines was observed 24 h post treatment, despite restoration of intraneuronal calcium levels at this time point. These observations demonstrate a specific effect of oAβ on NMDA-mediated calcium influx, which triggers synaptic collapse in vivo. Moreover, this work leverages a method to quantitatively measure calcium concentration at the level of neuronal processes, cell bodies and single synaptic elements repeatedly and thus can be applicable to testing putative drugs and/or other intervention methodologies.