The rising global prevalence of Alzheimer disease (AD) has heightened the urgency to develop effective AD therapeutics. Despite extraordinary efforts, we have been less than successful to curb either the progression or initiation of AD through drug therapy. To do so, we need a stronger understanding of the fundamental elements of AD pathobiology. The multifactorial nature of AD pathogenesis is becoming increasing clear and thus we can benefit from a broad approach to understanding disease mechanisms for effective therapeutic targeting. Recent advances have revealed factors such as partial loss of presenilin function and non-cell autonomous interactions which may contribute to AD pathogenesis. For example, murine models in which both presenilin genes are absent in forebrain neurons develop AD-like neuropathological and clinical features including neurodegeneration. We have demonstrated that presenilin 2 (PSEN2) deficiency is associated with an exaggerated pro-inflammatory response in microglia and that the fAD associated PSEN2 N141I mutation leads to decreased gamma- secretase activity in microglia. We have also reported a novel AD associated PSEN2 mutation that leads to decreased c-terminus Presenilin 2 (PS2) protein, further supporting the hypothesis that PSEN2 loss of function contributes to AD. These findings in conjunction with the lack of success thus far of gamma secretase inhibitors in clinical trials and recent reports on partial loss of PSEN1 function associated with AD raise a critical question regarding the pathogenesis of AD. In addition to neuronal A?42 production what additional mechanisms are involved in AD pathogenesis? The dynamics and significance of A?42 production are being investigated;however, decreased CNS A? clearance itself has been implicated in AD. Microglia, are key mediators of A? clearance. Therefore altered microglia behavior, as we observed with PS2 deficiency, may play a critical non-cell autonomous role in AD pathogenesis. Taking all recently available data into consideration, we hypothesize that AD pathogenesis involves combinatorial dysfunction in multiple cell types and that PSEN2 fAD mutations contribute to disease through toxic-loss-of-function in addition to the previously described toxic-gain-of-function. The goal of our laboratory is to study cell autonomous and non-cell autonomous mechanisms of neuronal injury in AD. We are pursuing an R01 funded project examining the impact of PSEN2 mutations on microglia and neuroinflammation as it relates to non-cell autonomous neurodegeneration in AD. To bolster the significance and human disease relevance of the R01 project, we are developing additional techniques in our research program with exciting potential to address these hypotheses. The use of patient derived induced pluripotent stem cells (iPSCs) is an expedient approach to examine the molecular phenotype of specific mutations as well as their cell type specific effects. At the University of Washington (UW), we are uniquely positioned to address the questions posed above by employing several key resources. First, the UW Alzheimer Disease Research Center (ADRC) has banked fibroblasts from well-characterized fAD cohorts. Second, we have access to established facilities for the derivation and characterization of induced pluripotent stem cell (iPSC) lines. We have created multiple iPSC lines which are being fully characterized molecularly, epigenetically and for capacity for teratoma formation among other crucial iPSC requirements. The impact of fAD mutations on iPSC derived glial cells and the effect of specific PSEN2 mutations on the biology of any neural cell type has not been reported. In this K02 proposal, I aim to collaborate with iPSC pioneers in the field with dual purpose to 1) develop a new skill set for my career development and 2) contribute unique information about PSEN2 fAD mutations and identify potential pathways where neuronal and glial cell processes may interact, leading to neurodegeneration. Thus, we propose the following experimental plan. We will investigate the cell type specific effects of two different PSEN2 mutations that cause fAD. We hypothesize that AD associated PSEN2 mutations lead to partial loss of PSEN2 function that will alter the behavior of neurons and microglia. To address this hypothesis we will: A) Generate, characterize and assess APP processing activity in iPSC lines from patient fibroblasts containing PS2 mutations. B) Differentiate iPSCs containing PSEN2 N141I or PSEN2 deletion mutation (PS2del) into neurons. Determine the effects of PS2 deletion on intrinsic electrophysiological properties, synaptic physiology and gamma secretase activity of these neurons. C) Differentiate the iPSC lines used in 1B into microglia and evaluate for pro-inflammatory cytokine release, phagocytosis and inflammatory pathway signaling. Next, we will study the non-cell autonomous impact of fAD PS2 mutations on the interaction between neurons and glia. We hypothesize that these two PS2 mutations contribute to AD through consequences of glial dysfunction leading to neuronal injury. By employing neuronal-glial co-cultures we will study neuronal processes in the presence of PS2 fAD mutation carrying microglia. We will: A) Measure wildtype neuronal synaptic physiology in the absence and presence of A?42 when cocultured with wildtype or PS2 fAD microglia. B) Assess neuronal susceptibility to neurotoxicity in the presence of wildtype or PS2 fAD microglia.

Public Health Relevance

Alzheimer Disease (AD) is a growing global health crisis. Development of effective therapeutics will benefit from a broad approach to understanding AD pathogenic mechanisms. In this proposal we will employ AD patient derived induced pluripotent stem cells to study the function of Presenilin 2 as it relates to AD, and to investigate the consequences of neuron/glial interactions on neuronal health.

National Institute of Health (NIH)
National Institute on Aging (NIA)
Research Scientist Development Award - Research (K02)
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National Institute on Aging Initial Review Group (NIA)
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Wise, Bradley C
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University of Washington
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