The finding that neuronal age-dependent fate-instability and hypo-maturity are a key underlying feature of AD pathogenesis is a major new result that has a large impact on the AD research field. Our findings further relate to, and mechanistically adjusts, the established cell-cycle re-entry hypothesis for AD, which has previously only been accessible via post mortem histopathology. While PKM stood out as major regulator of the metabolic switch and cell fate in AD iNs, our multi-omics analysis also revealed several additional regulators (e.g. SIRT1, PGC1a and FOXO3) that might be harnessed as therapeutic targets in AD. We will thus further assess the total protein levels of these metabolic regulators and will study their subcellular localization and nucleo-cytoplasmic transport, and we have cloned and validated all necessary tagged with versions for live-cell imaging. We have performed a deep characterization of tagged PKM2 in healthy and AD iNs, and we will perform similar experiments for SIRT1, PGC1a and FOXO3. Further, to assess if AD iNs suffer from impaired nuclear shuttling, we will additionally perform FRAP experiments and compare the rate of shuttling of each metabolic regulator.
In the past project period, we have focused on PKM modulators and their effects on neuronal cell fate and the AD phenotype. We found that various PKM2-realted compounds can partially reverse AD phenotypes in patient-derived iNs. We will further test more compounds and combinations affecting also SIRT1 and PGC1a. Additionally, depending on the effects described in the nuclear shuttling experiments, we will treat AD iNs with nuclear export or import inhibitors and perform careful characterization and sequencing of the most promising approaches.
Our efforts on increasing the sample numbers for our MCI/AD cohort had been abruptly stopped with the Covid-19 pandemic, and are now ongoing again, however newly obtain biopsies from 78 donors already provide substantially increased power to our project, and only some anticipated disease sub-groups are still unrepresented. Our analysis of the AD disease trajectory and psychiatric risk factors has been so far only performed in cortical glutamatergic iNs, and while proving to be very informative, we also still plan to extend our efforts towards serotonergic iNs by the addition of FEV, GATA2, LMX1B and NKX2.2. We have already cloned the required plasmids and initial tests of their functionality have been performed.
