Tae-wan Kim, Phd - US grants

DESCRIPTION (Adapted from the application): Protein aggregates, or cellular "inclusions", are a prominent feature in a wide variety of neurodegenerative diseases, including Alzheimer's disease (AD). Proteasomal degradation appears to play a key role in removing aggregate prone molecules and submicroscopic aggregates, both of which can ultimately lead to visible inclusions. These investigators initially observed that cells stably expressing the N141I FAD mutant version of presenilin 2 (PS2) exhibited an increased tendency to form ubiquitin-positive aggregates consisting of misfolded PS2, as compared to cells expressing wild-type PS2. They have also found that capacitative Ca entry (CCE) was dramatically reduced by treatment with a proteasome inhibitor as well as by the N141I PS2 FAD mutation. CCE is a novel Ca entry mechanism that is induced by intracellular Ca pool depletion. These observations suggest a link between Ca dyshomeostasis and both proteasomal dysfunction and subsequent formation of protein aggregates. Consequently, they propose that conformational changes in the presenilins caused by FAD mutations promote misfolding and subsequent aggregation (e.g. submicroscopic aggregates), which would lead to Ca dyshomeostasis. Alternatively, mutant presenilins may first alter intracellular Ca homeostasis, which subsequently affects protein folding and proteasomal degradation. In either case, interplay between presenilin aggregates and perturbed Ca homeostasis (e.g. reduced CCE and depletion of the ER store) may contribute to other FAD-associated molecular phenotypes, such as increased AB42, by influencing protein trafficking or proteolytic processing. The overall goals will be to search for further evidence of proteasomal dysfunction in AD, to study the relationship between Ca signaling and protein aggregation, and to determine the underlying mechanism of how presenilin FAD mutations affect intracellular Ca homeostasis and proteasomal degradation.

DESCRIPTION (provided by applicant): Gamma-secretase is an unusual aspartyl protease that cleaves substrates within the transmembrane (TM) region. The presenilins (PS 1 and PS2) are essential for g-secretase-mediated cleavage of select TM proteins, including amyloid b-protein precursor (APP) to yield amyloid b-peptide (Ab). Familial Alzheimer's disease (FAD)-associated mutations in the presenilins give rise to an increased production of a longer and more amyloidogenic form of Ab (Ab42). In addition to their proteolytic activity, we reported that the functional presenilins are required to modulate the pathway for capacitative calcium entry (CCE), the refilling mechanism for depleted internal Ca2+ store. Furthermore, presenilin FAD mutations universally attenuate CCE. "Conformational coupling" mediated by direct physical interaction between plasma membrane CCE channels and ER constituents is known to be an underlying mechanism for CCE. Our studies suggest that Ca2+-mediated presenilin function and presenilin-associated g-secretase activity are functionally coupled and may be governed by a shared regulatory mechanism involving conformational coupling. We plan to study the underlying mechanism as to how the presenilins regulate Ca2+ entry and g-secretase-like proteolytic activity and what is the temporal and spatial relationship between these two separate presenilin functions (e.g. Ca2+-related vs. proteolytic). Additionally, we will identify relevant molecular effectors and substrates for these pathways. Our two central hypotheses are: i) the presenilin-associated g-secretase cleaves a substrate that is critical for calcium entry; ii) alternatively, the presenilins directly regulate "conformational coupling" between plasma membrane and ER, simultaneously serving as a regulatory mechanism for g-secretase and Ca2+ entry (e.g. CCE). To address these hypotheses, we will carry out three aims: 1) To define the precise relationship of Ca2+-related presenilin function with presenilin-associated g-secretase activity; 2) To determine the molecular nature and subcellular locus for the proteolytic and Ca2+-related functions of the presenilins; 3) To identify molecular substrates for presenilin-associated g-secretase. Our studies should provide information relevant in general to the integrated mechanism of Ca2+ mobilization and the proteolysis of TM proteins, and in particular to the underlying mechanism of FAD-associated neuropathogenesis.

DESCRIPTION (PROVIDED BY APPLICANT): Genetic, biochemical and neuropathological studies support the idea that cerebral elevation and accumulation of the amyloid beta-peptide (Ab) are early and necessary steps in the pathogenesis of Alzheimer's disease (AD). Abeta is produced by sequential proteolytic cleavages of the amyloid precursor protein (APP) by a set of membrane-bound proteases termed beta-and gamma-secretases. Heterogeneous gamma-secretase cleavage at the C-terminal end of Abeta produces two major isoforms of Abeta, Abeta40 and Abeta42. While Abeta40 is the predominant cleavage product, the less abundant, highly amyloidogenic Abeta42 is believed to be one of the key pathogenic agents in AD and increased cerebrocorical Abeta42 is closely related to synaptic/neuronal dysfunction associated with AD. Furthermore, mutations in the APP and presenilin genes that cause rare early-onset forms of familial Alzheimer's disease (FAD), universally lead to an increased production of Abeta42. Thus, agents which are able to selectively reduce Abeta42 production are attractive and promising as therapeutic reagents for treating AD. Our preliminary studies showed that several triterpene natural products (known as "ginsenosides") derived from heat-processed ginseng (e.g. red ginseng), selectively lower the production of Abeta42. Based on this key preliminary data, the major goal of this proposal is to investigate the mechanistic basis of anti-amyloid (e.g. Abeta42-reducing) activity of these ginsenosides and other related natural products, and to evaluate their therapeutic potential using in vitro and in vivo models of (AD). To address these issues, we will carry out the following Specific Aims: (1) To investigate the mechanism of the anti-amyloid activity of selected ginsenosides; (2) To investigate the effects of Abeta42-lowering ginsenosides on Alzheimer-like pathology in a mouse model of AD; (3) To test the effects of Abeta42-lowering ginsenosides on neuronal dysfunction in a mouse model of AD: parallel analyses using fMRI, electrophysiology and behavioral approaches. Since these Abeta42-reducing ginsenosides can directly antagonize the key pathological event in AD, successful completion of our studies will help determine the therapeutic benefit of ginsenosides in AD.

Genetic, biochemical and neuropathological studies support the idea that cerebral elevation and accumulation of amyloid beta-peptide (Abeta) is an early and necessary step in AD pathogenesis. Abeta is generated from the amyloid beta-protein precursor (APP) by two proteolytic cleavages mediated by beta- and gamma-secretases. Mutations in the APP and presenilin genes cause rare early-onset forms of familial Alzheimer's disease (FAD), which shares many pathological features with the more common sporadic AD. Although FAD-associated genes (e.g. PS and APP) are expressed throughout the brain, early and most severe AD-associated neurodegenerative changes manifest in selective populations of neurons, e.g. neurons in entorhinal cortex and basal forebrain. Thus, additional cellular factors may contribute to selective neuronal vulnerability in AD. The hippocampus, a vital brain region for memory function, consists of multiple sub-regions each containing a unique population of neurons. The various subregions of hippocampus seem to be differentially affected in AD: for instance, entorhinal cortex is the most severely affected area, while dentate gyrus is relatively spared. To examine the underlying molecular mechanisms of selective vulnerability associated with common sporadic AD, we have performed carefully controlled microarray analyses to identify genes that are selectively up- or down-regulated in AD entorhinal cortex relative to dentate gyrus (in the same patient). Our microarray analyses revealed that VPS35p, a putative endosome/TGN trafficking molecule, is selectively upregulated in AD entorhinal cortex. Parallel analyses revealed that suppression of endogenous VPS35p using RNA interference (RNAi) in APP-expressing cells selectively lowers AP generation, suggesting that VPS35p is a modulatory factor in Abeta biogenesis. In light of our findings, the overall goal of this proposal is to perform molecular and functional characterization of VPS35p to delineate the role of this protein in APP processing and selective cellular dysfunction of entorhinal cortex in sporadic AD. The successful completion of this project will help identify cellular pathways that are directly involved in Abeta biogenesis and selective neuronal degeneration in AD entorhinal cortex, and may lead to development of a clinically valuable assay system for novel therapeutic reagents.

DESCRIPTION (provided by applicant): The elevation and accumulation of soluble amyloid [unreadable]-peptide (A[unreadable]) oligomers closely correlate with cognitive decline and/or disease progression in Alzheimer's disease (AD). Furthermore, at the cellular level, A[unreadable] oligomers induce synaptic dysfunction, including the impairment of long-term potentiation (LTP), an electrophysiological correlate of learning and memory in the mammalian hippocampus. Our preliminary studies showed that A[unreadable] oligomers, the A[unreadable] species known to trigger synaptic dysfunction, caused a profound decrease in the levels of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], a phospholipid that regulates key aspects of neural function. The destabilizing effect of A[unreadable] oligomers on PI(4,5)P2 metabolism was not observed in neurons derived from mice containing higher brain levels of PI(4,5)P2 levels owing to the hemizygous deletion of synaptojanin 1 (Synj1 +/-), the main PI(4,5)P2 phosphatase [PI(4,5)P2 degrading enzyme] in the brain and synapses. Furthermore, the characterized inhibitory effect of A[unreadable] on LTP was strongly suppressed in brain slices from the Synj1 +/- mice. Thus, based our robust preliminary studies, we hypothesize that inhibition of Synj1, which is mainly responsible for PI(4,5)P2 turnover at the synapse, and corresponding increases in a specific pool of PI(4,5)P2 in neurons may ameliorate A[unreadable]-induced synaptic dysfunctions. Synj1 is expressed and concentrated at the synapse in the brain regions known to be vulnerable in AD. Additionally, it is reasonable to postulate that Synj1 is a druggable target, given the fact that SHIP2, an enzyme in the same lipid phosphatase family, is a validated therapeutic target in diabetes. These facts validate Synj1 as a promising druggable target in AD and we intend to determine if small molecule inhibitors of Synj1 can be potential therapeutic agents in AD. To this end, we will attempt to identify novel small molecule Synj1 inhibitors and determine if these compounds can prevent or reverse A[unreadable]-associated synaptic abnormalities. Our specific goals will be to identify novel small molecule inhibitors of Synj1 by i) developing a mid to high throughput enzyme assay, ii) screening small molecule libraries with the assay, iii) performing preliminary medicinal chemistry on hit compounds and iv) testing the biological activity of the resulting compounds and derivatives in secondary cell-based assays. Successful completion of this work will yield active lead compounds that can be further developed as effective therapeutic agents in AD. PUBLIC HEALTH RELEVANCE: Synaptic dysfunction caused by amyloid [unreadable]-peptide (A[unreadable]) has been linked to cognitive deficits in AD. Synaptojanin 1 (Synj1) is a lipid phosphatase that is highly expressed in the brain and the reduced Synj1 levels ameliorate A[unreadable]-induced synaptic dysfunction, suggesting Synj1 as a therapeutic target in AD. Our proposed study will identify novel, small molecule inhibitors of Synj1. Successful completion of this work will yield active lead compounds that can be further developed as an effective therapeutic agent in AD.

DESCRIPTION (provided by applicant): The amyloid ¿-peptide (A¿), which originates from the proteolytic cleavage of amyloid precursor protein (APP), plays a central role in the pathogenesis of Alzheimer's disease (AD). Mounting evidence indicates that different species of A¿, such as A¿ oligomers and fibrils, may contribute to AD pathogenesis via distinct mechanisms at different stages of the disease. Importantly, elevated levels of A¿ oligomers closely correlate with cognitive decline and disease progression in animal models of AD. At the cellular level, A¿ disrupts synaptic plasticity, including the impairment of long-term potentiation (LTP), an electrophysiological correlate of learning and memory in the mammalian hippocampus. Our recently published work demonstrated that A¿ oligomers caused a decrease in the levels of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], a phospholipid that regulates key aspects of neural function. The destabilizing effect of A¿ on PI(4,5)P2 metabolism was not observed in neurons derived from mice containing higher brain levels of PI(4,5)P2 levels owing to the hemizygous deletion of synaptojanin 1 (Synj1 +/-). Synj1 is the main PI(4,5)P2 phosphatase [PI(4,5)P2 degrading enzyme] in the brain and synapses. Furthermore, the well characterized inhibitory effect of A¿ on LTP was strongly suppressed in brain slices from the Synj1 +/- mice. Furthermore, our preliminary results showed that A¿-induced suppression of phosphorylation of cAMP response element-binding protein (CREB), a critical transcription factor associated with synaptic plasticity and memory, was absent in primary neurons derived from Synj1+/- mice. Thus, based on these results, we hypothesize that inhibition of Synj1 may ameliorate A¿-induced synaptic dysfunction and memory impairment in AD. To this end, the main goals of this proposal are to employ biochemical and mouse genetic approaches to investigate the role of Synj1 in A¿-induced disruption of neuronal signaling and further validate Synj1 as a therapeutic target using cultured neurons and in vivo mouse models of AD. Specifically, we will investigate the role of Synj1 in A¿2-induced alterations in neuronal signaling, and will also determine if hemizygous deletion of Synj1 can ameliorate learning and memory impairments in an animal model of AD. Thus, our study will establish Synj1 as a validated target based on a molecular and system level target characterization study. Successful completion of this work will establish a solid rationale for the development of small molecule inhibitors that could selectively target Synj1, as potential therapeutic agents in AD.

PROJECT SUMMARY Apolipoprotein E (apoE), a cholesterol-transporting apolipoprotein, is critically involved in the pathophysiology of a number of human disorders, including cardiovascular diseases, ischemia and neurodegenerative diseases. Most notably, among the common allele variants of the APOE gene (APOE2, APOE3 and APOE4), the APOE4 allele (encoding apoE4 isoform) underlies the single strongest risk factor for late-onset Alzheimer?s disease (AD). ApoE regulates the clearance, aggregation, and deposition of amyloid-? (A?) in an isoform-dependent manner and also regulates other AD-relevant brain functions such as neuroinflammation and synaptic plasticity. ApoE is mainly produced and secreted from astrocytes in the brain. It has been postulated that an increase in the levels of apoE (especially the apoE3 isoform present in the majority of the human population) leads to decreased amyloid levels. Therefore, it is conceivable that an increase of apoE may furnish therapeutic benefits for AD. The idea of pharmacological enhancement of apoE has been tested using several nuclear receptor agonists, such as bexarotene and T0901317(retinoid X receptor (RXR) and liver X receptor (LXR) agonist, respectively). Since these compounds induce a number of genes other than apoE (such as ABCA1) which have widespread physiological effects, it is difficult to pin-point the exact contribution of apoE elevation in AD-associated phenotypic changes in the brain. To this end, we have conducted high throughput screening (HTS) in order to identify novel small molecules that can enhance apoE production in human primary astrocytes. We have identified a number of small molecule hits that can increase apoE levels via previously unknown mechanisms, including ones promoting apoE secretion without co-inducing ABCA1. Using these compounds as chemical tools, we will first confirm pharmacological activities of the identified apoE modulators in vivo and further test to discover compound(s) that can affect AD-like phenotypes in mouse models of AD. Thus, by using physiologically relevant brain cells for HTS, our proposed studies will help not only to establish translational significance of pharmacological modulation of apoE levels in the brain, but also to understand regulatory mechanisms of brain apoE levels which will provide broad translational significance on other apoE-linked human disease pathophysiology. Successful completion of our proposed studies will also lead to the identification of new tool compounds that modulate apoE secretion through previously unknown mechanisms of action in vivo, or that are ideal for further drug discovery efforts.

Triggering receptor expressed on myeloid cells 2 (TREM2) is an immune modulatory receptor expressed in microglial cells in the brain. Coding variants in TREM2 have been identified as risk factors for Alzheimer's disease (AD). Previous studies have extensively demonstrated that TREM2 plays a central role in microglia activation/survival and amyloid pathology in various cell-based and animal models of AD. Despite evidence demonstrating the functional importance of TREM2 in microglial biology relevant to AD, we currently do not have a complete mechanistic understanding of microglial TREM2 signaling, due in part to the lack of a comprehensive knowledge of TREM2-bearing molecular complex(es) in microglial cells. Most of the focus has been on the interaction of TREM2 with DNAX-activating protein 12 (DAP12), which appears to serve as a communal platform for downstream signaling. Given the complexity and high disease relevance of TREM2-associated biological processes, it is conceivable that additional molecular components may interact with TREM2 and contribute substantially to the regulation of relevant microglial functions. Therefore, we are proposing an unbiased proteomic approach to identify novel components of TREM2-harboring protein complex(es), not yet implicated in TREM2 signaling. Moreover, certain protein-protein-interactions involving TREM2 may be regulated in response to TREM2 receptor stimulation and/or affected by AD-associated TREM2 risk variants. Since conventional protein complex isolation methods are disruptive and often severely affect the stability of the complex, our experimental approach will use an enzyme-catalyzed proximity labeling technique to investigate the protein interactions in living cells. Specifically, the ascorbate peroxidase (APEX2)-based proximity-tagging method combined with mass spectrometry will identify proximal endogenously interacting proteins of TREM2 in the intact microglial cell. Successful completion of the proposed research will fill a critical gap in our understanding of the complex biological regulation of TREM2. Our proposal will establish a protein-protein interaction map of TREM2 in microglial cells and may discover novel TREM2-interacting proteins that are critical for TREM2-mediated microglial functions relevant to AD.