Riqiang Yan - US grants

Alzheimer's disease is the most common age-related neurodegenerative disorder. It has been widely accepted that abnormal production or accumulation of amyloidogenic peptides (Abeta), derived from sequential processing of amyloid precursor protein (APP) by beta-secretase and gamma-secretase, is tightly linked to AD pathogenesis. Our main research objectives will be focused on the regulated APP processing and production of (Abeta) in cells. A type I transmembrane aspartyl protease have been recently identified as beta-secretase, or called BACE1. We believe that all physiological functions are well balanced by various negative and positive modifiers. It is conceivable that BACE1 activity in cells is also modulated by natural factors and a shift of the modulating balance will clearly affect Abeta production. We hypothesize that abnormal fluctuations of BACE1 modifiers in human brains contribute to the amyloid depositions and the subsequent AD pathogenesis. Recently, we found that reticulon 3 (RTN3) clearly modulates BACE1 activity. RTN3 is one of the members of RTN family that comprises four members and all the members share highly conserved C-terminal Reticulon Homology Domain (RHD). Our specific objectives in this proposal are to delineate the molecular mechanism in which RTN3, and probably its family members, negatively modulate BACE1 activity and to explore potential applications of reticulon proteins in Alzheimer's therapeutics. Vigorous biochemical, molecular cell biology approaches combined with genetic methods will be used to address the following Specific Aims. Aim 1 -To investigate interactions of BACE1 with RTN family members. Aim 2 - To explore the mechanism by which BACE1 activity is modified by RTN3. Aim 3 To validate RTN3 as a negative BACE1 modifier in vivo. We believe that results from this study will provide valuable insights into the molecular mechanisms of RTN3 and its family members in negatively modulating BACE1 activity.

DESCRIPTION (provided by applicant): BACE1 is a type I transmembrane aspartyl protease which is essential for cleaving amyloid precursor protein (APP) at the -site. Only after this initial cleavage does - secretase further process the released APP C-terminal fragment to excise -amyloid peptide (A). While inhibiting BACE1 activity will reduce BACE1 processing of APP and will thus reduce the release of A, this inhibition will also lead to decrease cleavage of another BACE1 substrate, neuregulin-1 (Nrg1), and will reduce its signaling activity, which regulates various central nervous system functions such as myelination, synaptic plasticity and astrogenesis. We have demonstrated that genetic deletion of BACE1 causes hypomyelination, perhaps via reduced Nrg1 signaling. BACE1-null mice also exhibit schizophrenia-like behaviors, epileptic seizures and neurodegeneration, indicating that BACE1 plays a critical role in many brain functions. In this proposal, we will specifically investigate the contribution of Nrg1 signaling to the observed phenotypes because many of these neurological dysfunctions exhibited in BACE1-null mice are potentially related to alterations in the Nrg1/ErbB signaling pathway. We will test our central hypothesis that reduced BACE1-dependent Nrg1 signaling activity contributes to the observed multiple neurological dysfunctions in BACE1-null mice. Specifically, we will answer important questions as to whether elevated Nrg1 signaling activity will ameliorate or exacerbate BACE1-null phenotypes through examining BACE1-null mice engineered to express BACE1-cleaved Nrg1 N-terminal fragment and whether knock-in mice with disrupted cleavage in Nrg1 will produce phenotypes mimicking BACE1-null mice. Results from this study will not only resolve many ambiguous questions related to BACE1 and Nrg1 functions, but will also provide important guidance as to whether enhancing Nrg1 signaling activity will reverse or ameliorate potential side effects associated with long-term significant inhibition of BACE1 in AD patients.

DESCRIPTION (provided by applicant): Schizophrenia is a devastating neuropsychiatric disorder, affecting about 1.1% of the population over the age of 18. Human and mouse genetic studies have identified several schizophrenia susceptibility genes. Among them, neuregulin-1 (Nrg1), a pleiotropic signaling molecule, is confirmed as a risk gene for schizophrenia (Stefansson et al., 2002;Hall et al., 2006;Law et al., 2006). How functional changes in Nrg1 signaling lead to schizophrenia is an important research topic. Proteolytic cleavage of Nrg1 is required to release a functional fragment that will interact with its cognate receptors of the ErbB family to exert cell-cell signaling (Falls, 2003;Mei and Xiong, 2008). In our studies of BACE1, which was initially discovered as the -secretase for cleaving amyloid precursor protein to release A (Vassar et al., 1999;Yan et al., 1999;Hussain et al., 1999;Sinha et al., 1999;Lin et al., 2000), we have shown that BACE1 cleaves transmembrane Nrg1 to release a secreted EGF-domain- containing N-terminal fragment and to exert a signaling function (Hu et al., 2008;Fleck et al., 2013). Mice with deficiency in BACE1 exhibit altered Nrg1 signaling function and develop schizophrenia-like phenotypes (Savonenko et al., 2008). One intriguing question is whether enhancing Nrg1 activity in BACE1-null mice will ameliorate behaviors. Related to this question, we have recently generated a mouse model which expressed BACE1-cleaved Nrg1 N-terminal fragment (termed as Nrg1-ntf ) under the control of tetracycline (Tet) responsible element (Tet-Off promoter). We found that overexpression of Nrg1-ntf in transgenic mice (Tg- N1 /T mice) enhances Nrg1 signaling activity, as its downstream signaling molecules Akt and Erk are activated. However, contrary to our expectations, our functional study shows that Tg-N1 /T mice develop schizophrenia- like behaviors, which can be reversed if transgene expression is switched off. Results from our lab and others imply that abnormally hypo- or hyper-functional Nrg1 can lead to schizophrenia. Since the dys-regulated expression of schizophrenia susceptibility genes may affect normal brain development and lead to the gradual appearance of different symptoms at different ages (Piper et al., 2012;Powell, 2010;Rapoport et al., 2005), we aim to test our hypothesis that increased Nrg1 activity during early development, but not during adulthood, contributes to subsequent schizophrenia-like behaviors in the adult. To test our hypothesis in this study, we propose two specific aims. Aim 1: To determine whether increased Nrg1 activity in the adult has an impact on schizophrenia-like behaviors. Aim 2: To explore molecular mechanisms associated with schizophrenia-like behaviors in N1 /T transgenic mice.

ABSTRACT Alzheimer's disease (AD) is the most common age-dependent neurodegenerative disease. How neurons are lost in AD brains remains contested, although many studies have postulated that toxic ?-amyloid peptide (A?) in various forms (such as soluble multimers or oligomers) as well as tau aggregates contribute to neuronal loss in aging AD brains and synaptic dysfunction in AD patients. AD mouse models such as PS19 and 5XFAD do develop age-dependent neurogeneration, supporting the above assertion. Currently, AD therapy is centered on developing drugs to block or remove amyloid deposition or tau aggregation. In this proposal, we aim to investigate how to revert neuronal loss in AD brains as an alternative therapeutic strategy by reversing degenerative processes. We have recently discovered that mice overexpressing either full-length CX3CL1 (Tg-CX3CL1) or the C-terminal domain of CX3CL1 (Tg-CX3CL1-ct) show enhanced neurogenesis. CX3CL1, which is also known as fractalkine, is a type I transmembrane chemokine (Bazan et al., 1997;Pan et al., 1997) and is cleaved by ADAM10 (Hurst et al., 2012;Hundhausen et al., 2003) to release its N-terminal fragment containing the C-XXX-C motif, which mediates binding to the G protein-coupled CX3CR1 receptor (Imai et al., 1997). Since the discovery of CX3CL1, its biological functions have exclusively been shown to occur through CX3CL1/CX3CR1 interactions, which activate signal transduction to regulate inflammatory responses, leukocyte capture and infiltration, as well as other immune functions. However, we have discovered that the C- terminal domain has a back-signaling function, which regulates the expression of genes important for cell growth or differentiation. We aim to test the hypothesis that neuronal expression of CX3CL1 enhances neurogenesis through its C-terminal domain, which replenishes neuronal loss and fosters recovery of synaptic functions in AD mouse models. Three specific aims are proposed to test this hypothesis: Aim 1: To determine the role of CX3CL1 C-terminal domain (CX3CL1-ct) in the control of neurogenesis; Aim 2: To enhance neurogenesis to reverse impaired synaptic functions in AD mouse models; and Aim 3: To explore potential therapeutic use of CX3CL1-ct in age-dependent neurogenesis for AD therapy. Accomplishing the experiments as proposed will provide novel answers as to the translational potential of CX3CL1 in AD treatment. Knowledge gained from this study will guide future development of molecules targeted as an AD combinatorial therapy that will not only reducing amyloid deposition or tau aggregation, but will also replenish neurons.

PROJECT SUMMARY Alzheimer disease (AD) is the most common cause of dementia and one of the leading sources of morbidity and mortality in the aging population. Despite enormous social and economic costs associated with AD, current drugs are directed towards symptomatic relief and none are curative. In this project titled ?An integrated reverse engineering approach toward rapid drug repositioning for Alzheimer?s disease,? we propose to develop an innovative integrated drug repositioning strategy that combines computation-based drug prediction, computation-based human brain-blood-barrier (BBB) permeability prediction, retrospective large-scale clinical corroboration, and prospective experimental testing to rapidly identify anti-AD drug candidates. First, we will develop novel computational approaches to identify repositioning anti-AD candidates from all (>2,600) FDA-approved drugs. Second, we will develop novel multifaceted biology-based computational methods to predict which repositioned drug candidates can cross BBB in humans. Third, we will perform large-scale retrospective case-control studies to corroborate the clinical efficacy of repositioned drug candidates using patient electronic health record (EHR) data of >50 million patients. Finally, we will evaluate the therapeutic potential of promising repositioned candidates in experimental models. Our study will generate a large amount of data/knowledge/hypotheses that could serve as a starting point for us and others to conduct hypothesis-driven drug repositioning studies in other animal models of AD and in AD patients. We will build a comprehensive Alzheimer Drug Repositioning Knowledge Base (ADRKB) and develop interactive web applications to make ADRKB publicly available. The unique and powerful strength of our project is our ability to seamlessly combine novel computational predictions, retrospective clinical corroboration using patient EHRs, and experimental testing in animal models of AD to rapidly identify innovative drug candidates that may work in real-world AD patients. The repositioned drug candidates will have interpretable mechanisms of action, are highly likely to cross BBB in humans, have clinical effectiveness evidence gathered from ?real-world? AD patients, and have demonstrated efficacy in mouse models of AD. We anticipate that these findings can be expeditiously translated into clinical trials and benefit 5.4 million AD patients in United States and 47 million AD patients worldwide.

Project Summary In Alzheimer?s disease (AD), the first signs of cognitive impairment are observed many years before a clinical AD diagnosis is established, and the loss of synaptic function in AD is evident long before any substantial loss of neurons. The excessive production or accumulation of ?-amyloid peptide (A?) has been documented to have deleterious effects on synaptic activity by various mechanisms. Understanding the cellular and molecular mechanisms of the early AD-associated synaptic dysfunction (before the behavioral manifestations of severe learning and memory deficits) may be critical for the development of new therapies for slowing down the progression of AD. However, detection of the AD-associated changes in synaptic function among cortical circuits is technically challenging, especially so if it is needed in the earliest stages of the AD process, before the formation of plaques and tangles, when changes are small and difficult to spot. Where exactly, at which cortical layer, or which synapse, one should investigate? The current assays for detecting neural circuit deficiencies in AD model animals are based on traditional electrode electrophysiology and have several practical limitations including: poor spatial resolution, blindness for cell-types, and a labor intensive nature of experiments. New technologies bring an improved temporal and spatial resolution for monitoring activity in many neurons simultaneously, thus facilitating studies on brain circuitry disruptions in neurological disorders. We propose to use GEVI imaging (multi-cell optical imaging of the membrane potential changes using genetically-encoded voltage indicators). Our hypothesis is that ?synaptic and neuronal dysfunctions emerge before significant A? deposition and pathological tau aggregation, and can be routinely detected by affordable imaging methods?. A simple and sensitive physiological assay for detecting changes in network physiology, prior to the substantial accumulation of the amyloid plaques or reproducible behavioral deficits in learning and memory, would accelerate the investigations of the earliest cellular and molecular changes mediated by the AD pathological process. Understanding the cellular and molecular mechanisms of the early AD-associated synaptic dysfunction (before the behavioral manifestations of the learning and memory deficits) may help the development of the new therapies for slowing down the progression of the AD.