Periodic Reporting for period 3 - AGEMEC (Age-dependent mechanisms of sporadic Alzheimer’s Disease in patient-derived neurons)
Período documentado: 2023-02-01 hasta 2024-07-31
Aging is not merely a major risk factor, but a pre-requisites for several diseases such as cancer or neurodegeneration to develop. With growing life expectancy in Europe and worldwide, the incidence of age-related diseases is skyrocketing. Sporadic Alzheimer’s disease (AD) is a prime example of an age-associated disorder, with an onset above 65 years. AD already ranks as the sixth leading cause of death in the US, which is expected to increase exponentially in the next decades. This fatal neurodegenerative disease is characterized by the loss of neuronal function and cell death. The triggers for this sudden neuronal loss and severe dementia are however still unclear, hindering the development of effective therapeutics that are able to halt or reverse disease progression and preventing early diagnosis. In this project we aim at deciphering the molecular mechanisms driving AD onset and progression. This work is based on cohorts of skin punch biopsies that we obtained from healthy old donors and AD patients, which we directly converted into induced neurons (iNs). Patient-specific cultures of iNs resemble functional adult-like human neurons and preserve signatures of aging, and they thus represent a useful tool to study age-associated diseases in human neurons. We previously showed that iNs generated from AD patients regress toward a hypo-mature state, re-gaining features typical for immature neurons and losing functionality. In this project we aim to uncover the molecular features behind this hypo-mature neuronal identity and pinpoint specific molecular regulators that might be harnessed as a therapeutic target. One focus of the project is on metabolic changes in AD iNs, as metabolism is a known driver of de-differentiation in cancer cells. Here, we deeply characterize metabolic alterations in AD iNs and their role in neuronal de-differentiation to identify key metabolic regulators in the disease. Chemical modulators will be tested on their ability to revert the metabolic switch and the de-differentiated phenotype in AD iNs. Further, a major goal in this project is to identify early disease mechanisms that might be involved in disease onset, and to pinpoint how severe psychiatric disorders accelerate biological aging and increase the risk for developing AD. Therefore, we establish and iN cohort comprising healthy donors, donors that suffer from mild cognitive impairment (MCI), AD patients, and donors that suffer from psychiatric disorders.
In this project we advanced the characterization of the molecular mechanisms underlying the de-differentiation of AD iNs. Briefly, we assessed transcriptomic and epigenetic changes of a cohort of healthy control iNs and AD iNs that revealed that AD iNs are in a hypo-mature neuronal state. Interestingly, our data indicate that mechanisms that play a role in AD pathogenesis are similar to known mechanisms driving malignant transformation in cancer cells. Unbiased bioinformatic analyses revealed that a dysregulation in carbon metabolism is a major hallmark of AD iNs. We thus continued to perform mass-spectroscopy-based metabolomics and glucose tracing experiments on our cohort and found that indeed AD iNs undergo a metabolic switch to aerobic glycolysis, similar to what has been described previously in cancer cells. Using an integrative analysis, the iN model revealed that the glycolytic bottleneck enzyme pyruvate kinase (PKM) undergoes pathological isoform switching towards the cancer-associated PKM2 isoform. This checked and confirmed this surprising result in human post-mortem brain tissue transcriptome data banks, and in neuropathological brain sections. Importantly, PKM2 modulators have been developed in the cancer field for several years, and some of these compounds were capable of restoring a healthy neuronal phenotype in iNs from AD patients. This included a reversal of the metabolic switch back to low glycolytic activity, resilience towards cell death, and the re-gain of synaptic connections. To identify early mechanisms that are important for the onset of dementia, and the progression of mild cognitive impairment towards AD, we established a cohort of healthy iNs, MCI and AD patient iNs, and iNs from psychiatric patients, to explore disease trajectory and molecular crosstalk of the co-morbidities. To identify sequential molecular changes that orchestrate AD onset and progression, and to probe for overlapping cellular and molecular features between psychiatric disease and MCI/AD, we have generated cortical iNs from a large cohort of new donors. We have started to profile their genome-wide epigenetic DNA methylation landscape, gene expression profiles, and functional neuronal network activity, and perform integrative multi-omics analyses.
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.
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.