Angela Ho, PhD - US grants

DESCRIPTION (Adapted from applicant's abstract): The goal of this proposal is to investigate whether Mint proteins are physiological regulators of synaptic vesicle and amyloid precursor protein (APP) trafficking targeted to the active zone of the presynaptic terminal of neurons. Mint1 and Mint2 are brain-specific proteins and bind with high affinity to Munc18-1, a protein required for synaptic vesicle exocytosis. The unique structure of Mint composed of an N- terminal Munc18-1 binding domain, a middle phosphotyrosine-binding (PTB) domain and two C-terminal PDZ domains, thus suggests that it may play an essential role in targeting and docking reactions of synaptic vesicles prior to exocytosis. Of particular interest, Mint proteins also bind to APP, the parent protein for amyloid protein beta4, a major component in the pathogenesis of Alzheimer's disease. Since it is not known how intracellular APP trafficking is regulated, investigating the role of Mint proteins is an important study to pursue. Therefore, the specific aims are: (1) to study the role of Mint domains by transfection of Mint deletion mutants in hippocampal neuronal cultures; (2) to study the function of Mints in mice by gene-inducible targeting using the Cre/lox recombination system.

[unreadable] DESCRIPTION (provided by applicant): Brain function requires the proper networking and communication between neurons. Improper development and maintenance of neuronal function leads to neurological abnormalities. As such, mechanisms underlying physiological to pathological processes in the brain are not clear. Currently, the laboratory of Dr. Thomas C. S[unreadable]dhof has allowed me to expand my knowledge in basic events of synaptic transmission to clinically associated problems in Alzheimer's disease. We have identified an essential family of adaptor proteins named Mints that have been implicated in coupling synaptic functions such as targeting of proteins to nerve terminals, and neurotransmission, to the regulation of amyloid precursor protein (APP) processing relating to Alzheimer's disease. To ascertain Mints function directly, we have generated mice lacking individual Mint proteins (isoforms 1-3), or all possible combination of Mint family members. We can now directly study: (1) membrane protein targeting by surface biotinylation of cell membranes, and morphologically examine the expression and cellular distribution of proteins that Mints interact with; (2) functionally examine synaptic transmission by using hippocampal slice electrophysiological recordings, and optical recording techniques of cultured neurons to look at kinetics of synaptic vesicles. We will characterize the structural dynamic of synaptic junctions by electron microscopy, and E-PTA staining to quantify morphological parameter of synapses. To explore the significance of Mint and APP processing, we have generated mice deficient of Mints which carry a transgene that coexpresses mutant APP, and presenilin 1. We will study the pathogenic events leading to disease state by examining age-dependent APP proteolysis and amyloid beta deposition by combining morphological and biochemical techniques. These studies will not only clarify the function of Mints in targeting, and synaptic transmission, but will broaden our understanding in the biology of Mints and. APP in Alzheimer's disease. My long-term goal is to pursue my understanding of molecular mechanisms underlying neuronal plasticity, and neurodegenerative diseases integrating the tools and conceptual approaches that I have learned and gained over my past graduate and postdoctoral training. This award will allow me to have a transition period during which I can expand my knowledge and technical foundations to become an independent principal investigator. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Amyloid plaques, which consist of fibrillar amyloid-ß (Aß) peptides, play a key role in Alzheimer's disease (AD) pathogenesis. It is well established that Aß is generated by sequential proteolysis of the amyloid precursor protein (APP) by ß- and ?-secretases, respectively. However, the cell biology and molecules controlling APP trafficking essential for Aß production in neurons are less defined. A key step in Aß generation is APP endocytosis that is mediated by the YENPTY sequence located in the cytoplasmic region of APP. Mints are adaptor proteins that are functionally important in regulating APP endocytosis and Aß production. We previously showed that the Mint adaptor proteins regulate APP endocytosis by directly binding to the YENPTY endocytic motif of APP, thereby influencing proteolytic processing of APP. The evidence that Mints are upregulated and found in Aß plaques in postmortem human AD brains supports a role for Mints in AD pathogenesis. Consistent with this finding, we showed that loss of any one of the three Mint proteins decreases Aß production in aging mice and mouse models of AD. These findings suggest that the APP-Mint interaction is a potential key therapeutic target to selectively reduce Aß production in AD. However, the mechanisms underlying the effects of Mints on APP binding and Aß production are unclear. Therefore, the overall goal of this research proposal is to understand Mint-dependent regulation of APP binding and processing. In Aim 1, we will determine the cell biology of APP trafficking and how Mints are essential for synaptic activity-induced APP endocytosis and Aß production. In Aim 2, we will investigate the effects of perturbing the APP-Mint1 interaction to decrease Aß production in both in vitro and in vivo mouse models. The identification of novel ways to modulate APP binding and Aß production will be an important tool that can lead to the development of alternative therapeutic strategies for treating AD. Through our structural studies, we found that autoinhibition of Mint1 regulates APP binding and processing; however, the molecular mechanism underlying Mint1 autoinhibition and the physiological relevance of this regulation in neurons are not known. In Aim 3, we will elucidate the biological mechanisms underlying Mint1 autoinhibition in regulating APP binding. A detailed delineation of the autoinhibitory mechanism regulating Mint1 binding to APP is an invaluable tool in exploring the critical routes to which it operates and a platform for future targeted therapeutics. The proposed research will provide new insights into understanding APP-Mint biology and the outcomes of this research are expected to have strong translational implications.

PROJECT SUMMARY: Disruption in neuronal migration results in severe neurological and developmental impairments such as cognitive deficits and epilepsy that are recognized primarily in the pediatric population. A signaling pathway crucial for proper neuronal migration and brain development is initiated by the evolutionarily conserved glycoprotein Reelin. Human mutations in the Reelin pathway generate phenotypes that mimic those induced by mutations in cytoskeletal proteins that disrupt the function of microtubules and actin. The culmination of these genetic studies in children strongly suggests that several signaling pathways including the Reelin pathway converge on downstream cytoskeletal proteins to affect proper neuronal migration, brain development and cognition. We used a systems biology approach to identify the microtubule-stabilizing CLASP2 as a key cytoskeletal modifier of Reelin signaling. We previously found that CLASP2 regulates several important phenotypes during neuronal development in vitro including Golgi morphology, neuronal branching, axon specification and synaptic activity, phenotypes that are also regulated by Reelin signaling. However, little is known about the role of CLASP2 and its association with the Reelin signaling pathway in the developing brain. Therefore, our goal is to understand how Reelin signaling regulates CLASP2-mediated cytoskeletal function during neuronal and brain development. In the first aim, we will define the interaction of CLASP2 with Dab1, a downstream node in the Reelin pathway, and then determine the functional consequences of this interaction. In the second aim, we will define the in vivo function of CLASP2 during brain development. The proposed studies aim to advance the understanding of how Reelin controls neuronal migration through cytoskeleton reorganization, key elements of normal brain development.

? DESCRIPTION (provided by applicant): The Undiagnosed Disease Network (UDN) of the NIH has identified an individual presenting with severe neurological symptoms including microcephaly, progressive brain atrophy on MRI and global developmental delay. The UDN identified a specific mutation in the FOXR1 (forehead [FH] box protein R1) gene in this individual resulting in a non-synonymous protein alteration. FOXR1 is a member of the FH transcription factor family that in humans comprises at least 50 distinct human genes. The function of FOXR1 is currently unknown; however, several genes within the FOX family including FOXG1 and FOXP2 are critical for proper neuronal and brain development and mutations in FOXG1, FOXC2 and FOXL2 can also lead to microcephaly. Based on these observations, the central hypothesis of this application is that FOXR1 is a nuclear transcription factor that plays a central role in neuronal proliferation, differentiation and migration in the developing cortex. Therefore, our aims are to determine the role of FOXR1 in brain development and determine the molecular mechanism underlying FOXR1 function and how the FOXR1 mutation leads to disease pathogenesis. With innovative approaches such as molecular genetics including ChIP to identify transcriptional targets and in utero electroporation to obtain n vivo data, the proposed research will provide new insights into FOXR1 in brain development and disease pathogenesis. In addition, the results of this study may shed light on mechanisms relevant to the etiology of many neurological and psychiatric disorders related to cortical function.

Amyloid-? (A?) generation is a key pathological event in Alzheimer's disease (AD). A? is produced by the sequential proteolytic processing of the amyloid precursor protein (APP) by ?- and ?- secretases. APP endocytosis is an important step in A? generation. APP is internalized to endosomes where APP is cleaved by ?-secretase, initiating the amyloidogenic pathway. The sorting signal that regulates APP endocytic processing required for A? generation is the highly conserved endocytic YENPTY sequence located in the cytoplasmic region of APP. APP mice that lack the endocytic YENPTY motif have reduced APP internalization and lower brain A? levels. Through our studies, we found Mints (also known as APP binding family A, APBA) are a family of neuronal adaptor proteins that bind directly to the endocytic YENPTY motif of APP and are essential for regulating APP endocytosis and amyloidgenic processing. In addition, Mints interact with presenilin-1 (PS1), the catalytic core of the ?-secretase complex, facilitating APP-PS1 colocalization and promoting A? production. Further, we found that loss of any one of the three Mint proteins decreases A? production in aging mice and mouse models of AD. Together, we hypothesize that the APP-Mint interaction is a potential and novel therapeutic target to selectively reduce A? production in AD. We identified a novel cell-permeable APP mimetic peptide (TAT-APPMP) that interferes with the APP-Mint interaction. The TAT-APPMP is designed to outcompete endogenous APP binding to Mints to reduce A? production. Preliminary data reveals that treatment of primary neuronal cultures from an AD mouse model with TAT-APPMP reduced A? production with minimal toxicity. This provides compelling evidence that the APP-Mint interface is a viable therapeutic target for AD treatment and is expected to have strong translational implications. However, the biological characterization of the APPMP, examining its specificity, efficacy and its potential for in vivo AD treatment is lacking. The overall goal of this proposal is to determine the specificity of the cell-permeable APPMP to disrupt the APP-Mint interaction and reduce A? accumulation in AD mouse models.