Izumi Maezawa, Ph.D. - US grants

DESCRIPTION (provided by applicant): Rett syndrome (RTT) is a devastating neurodevelopmental disorder caused by loss-of-function mutations in the X-linked MECP2 gene. MECP2 encodes methyl-CpG-binding protein 2 (MeCP2), an epigenetic modulator that binds the methyl CpG dinucleotide in target genes to regulate transcription. How MeCP2 deficiency causes neurological deficits remains poorly understood, but it is clearly related to dendritic and synaptic abnormalities. We previously reported that MeCP2-deficient microglia (MDM) cause excitotoxicity by constitutively releasing five times more glutamate than wild-type microglia, thus damaging dendrites and synapses. We subsequently found that MeCP2 is a potent transcriptional suppressor of the glutamine transporters SNAT1 and SNAT2. MDM consequently show over-expression of SNAT1 and SNAT2, resulting in increased glutamine uptake, disruption of microglial glutamine homeostasis, mitochondrial oxidative stress, and over-production of glutamate. This novel MeCP2-regulated pathway is highly significant for identification of therapeutic targets to block microglial neurotoxicity in RTT. Because our studies reveal that microglial glutamate production is regulated by a major epigenetic factor MeCP2, the research into this pathway will advance our knowledge about how neural activities regulate heterogeneous microglia response patterns and neuron-glia interactions through the epigenetic interface of DNA methylation. To consolidate the support for this mechanism, the purpose of this proposal, therefore, is (1) to firmly establish the link between MeCP2-regulated SNAT1/SNAT2 expression and MDM abnormalities and (2) to examine the relevance of this pathway to RTT. In Aim 1, we will determine the causal relationship between SNAT1/SNAT2 expression and the MDM phenotype, such as mitochondrial abnormalities and glutamate over- production. We will determine if over-expression of SNAT1 and/or SNAT2 in wild-type microglia induces the MDM phenotype. We will also determine if knock-down of SNAT1 and/or SNAT2 expression in MDM reduces the MDM phenotype. In Aim 2, we will determine the progression of microglial abnormalities (SNAT1/SNAT2 expression/activity and mitochondrial abnormalities) at different disease stages, using microglia freshly isolated from brains of MECP2 knockout mice and control wild-type mice. We recently have established a series of techniques to study microglia freshly isolated from juvenile and mature rodent brains, which would best represent microglia in vivo. We will employ qRT-PCR, flow cytometry, patch-clamp, electron microscopy, and fluorescent imaging to study the MDM phenotype in this preparation. The results of these studies are expected to be highly significant by 1) helping us to understand how MeCP2 deficiency causes dendritic and synaptic abnormalities in RTT through a microglia-mediated mechanism; 2) exploring the role of glutamine transporters in microglial pathology, which is presently poorly understood; and 3) providing one of the first few studies to examine epigenetic control of microglia function. PUBLIC HEALTH RELEVANCE: In Rett syndrome, abnormalities of microglia, the brain immune cells, may cause interruption of the neuronal network. We will investigate if abnormal levels of glutamine transporters SNAT1 and SNAT2 cause microglial abnormalities in Rett syndrome.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the most common cause of dementia in the elderly. To meet the public health challenge posed by AD, a goal set by the National Alzheimer's Project Act is to prevent or treat AD by 2025. Therefore currently there is an urgent need for new therapeutic target discovery and corresponding compound development. A protein deposited in AD brains called amyloid-beta (Abeta) has been hypothesized to play a critical role in AD pathogenesis. Abeta can activate microglia to clear Abeta but at the same time also stimulate microglia to release cytotoxic substances that cause neuronal damage. Using the small molecule TRAM-34, which was synthesized by our group, as a pharmacological tool we recently demonstrated that the calcium-activated potassium channel KCa3.1 plays an important role in microglia activation and microglial neurotoxicity. In vivo evidence indicates that blockade of KCa3.1 by TRAM-34 can inhibit microglia-mediated neuronal killing without affecting their migration and phagocytotic activities. Relevant to AD, our results suggest that TRAM-34 blocks the neurotoxicity induced by Abeta-activated microglia, but does not inhibit their beneficial function of phagocytosing Abeta. KCa3.1 blockade, therefore, is a potential new approach for the treatment of AD. With the help of this grant we wish to perform proof-of-principle studies to validate KCa3.1 as a novel therapeutic target for reducing microglia-mediated neurotoxicity and microglial dysfunction in Alzheimer's disease (AD), through the following three Specific Aims: Aim-1: Determine the effect of KCa3.1 blockade on Ab-induced microglial activation. We will treat cultured microglia with Abeta aggregates (oligomer and fibril) and evaluate the effect of KCa3.1 blockade by TRAM-34 treatment (Aim-1a) or genetic knockout (Aim-1b) on chemotactic and phagocytotic activities, signaling pathways, and the production of chemokines, cytokines, reactive oxygen species, and nitric oxide. Aim-2: Assess the contribution of KCa3.1 to AD-like pathology and cognitive deficits seen in the APPswe/PS1dE9 (APP-PS1) model using the genetic knockout approach. We will cross-breed APP-PS1 mice with KCa3.1-/- mice and evaluate whether KCa3.1 reduction affects neuropathological and behavioral abnormalities in APP-PS1 mice. We will further determine if KCa3.1 reduction can alleviate microglial dysfunction seen in APP-PS1 mice. Aim-3: Validate KCa3.1 as a therapeutic target for AD by performing preclinical studies with TRAM-34. We will determine if a 2-month course of TRAM-34, a selective inhibitor of KCa3.1, administered to APP-PS1 mice will reduce neuroinflammation, alleviate microglial dysfunction, and improve the neuropathological and behavioral outcomes of APP-PS1 mice. Because KCa3.1 blockade is relatively safe, our preclinical studies will have translational significance for future developmen of drugs for treating individuals with mild cognitive impairment or AD through inhibition of detrimental microglia functions.

Project Summary/Abstract Rett syndrome (RTT), a devastating neurodevelopmental disorder, is largely caused by loss-of-function mutations in the X-linked gene encoding the epigenetic regulator MeCP2. Our group was the first to show a detrimental role of microglial abnormalities in a MECP2 knockout (MECP2-KO) mouse model of RTT. Recent in vivo data suggest that correcting microglial abnormalities is sufficient to rectify major RTT-like symptoms in this model. Therefore, how MeCP2 deficiency causes microglial abnormalities and how the functional states of MeCP2-deficient microglia (RTT-MG) influence RTT pathology is a critical question. Recently we found that MeCP2 deficiency in RTT-MG resulted in enhanced mitochondrial reactive oxygen species (mtROS) production, which we hypothesize would lead to metabolic derangements and abnormal microglial functions. Interestingly, in vitro studies showed that several key RTT-MG abnormalities were rescued by mitochondria- targeted transgene mCAT (the antioxidant enzyme catalase being expressed only in mitochondria, which usually do not harbor catalase) and two FDA-approved mitoactive Nrf2 activators, opening a possibility for intervention. Our preliminary data further showed that global expression of mCAT (GL-mCAT) prolonged the lifespan and improved motor and respiratory functions of the MECP2-KO mice, supporting the promise of our approach in a whole animal setting. Here we propose to extend this line of research to gain a deeper understanding of how MeCP2 is related to functional and pathological states of microglia, and to generate preclinical proof of principle that is instrumental for developing novel therapies for RTT: Aim 1: Determine the pathological role of microglial mtROS in vivo: Several lines of evidence support that microglia abnormalities drive the progression of RTT. We have shown that global expression of mCAT ameliorates the RTT-like phenotype in MECP2-KO mice. Based on this encouraging result, we further hypothesize that quenching mtROS selectively in microglia to rectify microglial abnormalities will also ameliorate RTT-like deficits in MECP2-KO mice. For this aim, we will generate and analyze the phenotype and microglial pathology of MECP2-KO/MG-mCAT mice with microglia-targeted expression of mCAT. The result would support the role of microglial mtROS in the pathogenesis of RTT. Aim 2: Determine the role of mtROS in pathological characteristics and functional responsiveness of RTT microglia: Our previous data, mostly in vitro work, support the hypothesis that mtROS-related metabolic/molecular derangements lead to RTT-MG abnormalities including the loss of the ?metabolic flexibility? required to drive their functional responsiveness. Now in this aim we will test this hypothesis in vivo, mainly by analyzing microglia acutely isolated from MECP2-KO and MECP2-KO/mCAT mice. We will determine if mCAT, expressed in vivo, is able to (a) rectify the metabolic/molecular derangements of RTT-MG, and (b) recover the ability of RTT-MG to respond to M1/M2-inducing signals with proper functional differentiation. Key microglial cellular features such as phagocytic function, mitochondrial integrity, aerobic glycolysis, functional polarization, and Nrf2 antioxidation pathway will be analyzed to determine the beneficial effects of mtROS suppression by mCAT. Aim 3: Test the therapeutic effects of two FDA-approved mitoactive/Nrf2 activating drugs in vivo: The success of the mCAT approach provides a rationale for anti-mtROS therapy. To translate this finding to potential therapies, we screened 1,600 FDA-approved drugs to identify mitoactive anti-mtROS compounds, and subsequently screened the hits on a RTT-MG culture platform. We now have two leads able to correct RTT- MG abnormalities in vitro. Interestingly, both are known activators of the Nrf2 antioxidation pathway, supporting our hypothesized pivotal role of Nrf2 in RTT-MG. Because of their known favorable pharmacokinetics/safety profiles, now we will test them in vivo; efficacy shown in this aim would enable rapid translation by re-purposing these FDA-approved drugs for RTT. In summary, these studies will clarify mechanisms of MeCP2-regulated microglial function, the role of microglial mtROS in RTT, and advance novel therapies for RTT, for which no effective treatment is available. We hope that findings in this proposal would set the foundation to explore several new areas, such as how mtROS and metabolic modules such as aerobic glycolysis mediate or modulate microglial function including M1/M2 differentiation and phagocytosis, and the role of Nrf2 in microglial function. Moreover, RTT is one of few Autism Spectrum Disorders (ASDs) whose cause is identified as a single gene mutation and shares important pathogenic pathways with autism. Our studies, therefore, may implicate novel mechanisms and therapeutic approaches as well for autism where mitochondrial and microglia dysfunction could also play a role.