Probing Alzheimer synaptopathy using genetically engineered human neurons derived from iPS cells

Stanford University, Stanford CA

Investigators

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Abstract

In the previous funding period of this R01, that we are seeking to renew, we have made the remarkable observation that the Swedish mutation in the Alzheimer Precursor Protein (APP), which causes familial Alzheimer's disease (AD), induces an increase in synapse formation in human iPS cell-derived neurons. Importantly, we established that this effect is caused by Amyloid beta (Ab) and not any other APP cleavage product altered due to the more efficient b-secretase cleavage. This finding was surprising since elevated Ab is considered one of the key drivers of neurodegeneration in AD. In the first aim we propose to elucidate the mechanism of how Ab induces synapses. We will test whether the synaptogenic effect is induced by Ab peptides more or less prone to aggregation and whether decreasing aggregated Ab affects synapse formation. After establishing the dynamics of Ab-induced synaptogenesis, we will map out the neuronal signaling pathways that lead to synapse formation in direct or indirect response to Ab. In our second aim, we will establish whether the Ab acts directly on neurons or indirectly via astrocytes or microglia. The latter hypothesis may be attractive since astrocytes promote synapse formation physiologically. Using a newly developed all-human iPS cell-derived neuron, astrocyte, and microglia tri-culture system we will further evaluate whether Ab may be presented differently in a human neuro-glial cell context as opposed to human neurons grown on mouse primary glia which is the context we have studied synaptic biology so far. Human neural organoids consisting of defined proportions of neurons, astrocytes, and microglia will further allow to address the question whether three-dimensional tissue and cell-produced extracellular matrix affects the synaptogenic effects of Ab. To investigate how Ab affects the three different cell types, we will perform single cell and nuclear transcriptional profiling of two-dimensional tri- cultures and organoids from APPSwe and control cells. In our third and final aim, we seek to further investigate the role of Tau pathology and ApoE on neuronal function. In the previous funding period we identified an ApoE- induced synapse formation by activation of ApoE receptors and downstream kinases DLK, MKK7, and ERK1 and phosphorylation of the transcription factor CREB. Intriguingly, ApoE4 stimulated this pathway and induced synapses better than ApoE3, and ApoE3 better than ApoE2. In addition, ApoE variants have been involved in many cell biological aspects. Tau aggregation and its prion-like spreading is another hallmark of AD pathology. We will apply recent breakthrough technologies to model Tau spreading in human neuronal cultures. Other than focusing on the mechanisms of Tau spreading, we here propose to assess its effect on synaptic function, consistent with the overall theme of this project. Here we propose to utilize our improved tri-culture and organoid models which allow more complex cell-cell interactions and our successful conditional gene editing approach that allows the derivation of truly isogenic controls in order to assess DLK/MKK7/ERK-dependent and independent effects of ApoE variants and perturbations on neuronal function.