Xiongwei Zhu, PhD, Case Western Reserve University - US grants

[unreadable] DESCRIPTION (provided by applicant): Since Abeta (AB) appears to play a key role in the onset and progression of Alzheimer's disease (AD), understanding the pathway by which AB injures and kills neurons has the potential to identify molecular targets for therapies. We and others have demonstrated that JNK is activated in degenerating neurons in AD. That JNK is involved in neuronal degeneration in AD is supported by the fact that JNK is activated by AB in vjtro and AB-induced JNK activation mediates AB toxicity. However, given that JNK is not activated in all neurons containing increased AB in vivo, the mechanism of AB-induced JNK activation is still not fully understood and other factor(s) are clearly involved. The proposed studies will focus on understanding these additional factor(s) which will not only gain an appreciation for an important basic biological process, but also provide novel therapeutic target(s). Previously, we identified that iron is associated with the pathological hallmark lesions of AD, which is similar to the distribution pattern of activated JNK in AD case. Further, we demonstrated that AB toxicity is significantly attenuated when AB is pretreated with the iron chelator deferoxamine, suggesting that iron augments AB toxicity. Most importantly, we found that JNK is activated in Tg2576 ABPP transgenic mice which accumulate iron but not in Van Leuvan's ABPP transgenic mice which do not accumulate iron. Therefore, we hypothesize that iron is critical for AB-induced JNK activation. Additionally, since lesion-associated iron is able to participate in in situ oxidation and readily catalyzes an H2O2-dependent oxidation that mediates AB toxicity, we further hypothesize that H2O2 mediates metal-augmented AB-induced JNK activation. The specific goals are as follows: Aim 1 and 2: Determine the relationship between JNK activation, Ab deposition and iron accumulation in susceptible neurons in AD patients and ABPP mice. Aim 3: Determine the effect of metal ion chelation on Ab-induced JNK activation. Aim 4: Determine whether H2O2 mediates metal-augmented AB-induced JNK activation. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Early changes in AD brain include loss of synapses. AB, considered to have a central role in the pathogenesis of AD, bind to dendritic spines and cause synaptic dysfunction. However, the mechanisms responsible for AB-induced synaptic dysfunction and spine loss are not firmly established. Notably, synaptic terminals have abundant mitochondria which play an indispensable role at these sites. Along this line, mitochondrial dysfunction is an early prominent feature of AD neurons. Mitochondria are dynamic organelles that undergo continual fission and fusion events which are regulated by a machinery involving large dynamin-related GTPase that exert opposing effects, e.g., dynamin-like protein 1 (DLP1) and Fis1 for fission, and mitofusins (Mfn1 and Mfn2C) and OPA1 for fusion. These mitochondria fission and fusion proteins control not only mitochondrial number and morphology but also mitochondrial distribution and function. Indeed, defects in the mitochondrial fission/fusion balance and thus, the morphology and distribution have the potential to cause localized energy and calcium imbalance, which is especially damaging to polarized cells such as neurons, resulting in cellular dysfunction and death. Our preliminary studies suggest that the normally strict regulation of mitochondria morphology and distribution is impaired in AD neurons and fibroblasts which may be caused by differential expression of mitochondrial fission/fusion proteins induced by AB. Our central hypothesis is that AB induces mitochondrial dysfunction and synaptic abnormalities via its toxic effect on mitochondrial fission/fusion. Four aims will be pursued: Aim1) ADDLs induce mitochondrial dysfunction and synaptic abnormalities via its toxic effect on mitochondrial fission/fusion in vitro; Aim2) To Explore the Mechanisms of ADDL-induced DLP1 Reduction; Aim3) mutant PS1 causes mitochondrial abnormalities and neuronal dysfunction at least in part through its interaction with DLP1 and impaired balance in mitochondrial fission/fusion; Aim 4) DLP1 reduction underlies mitochondrial abnormalities and synaptic loss in vivo. PUBLIC HEALTH RELEVANCE: AB-caused synaptic dysfunction and spine loss is an early change and the most robust correlate of AD- associated cognitive deficits, however the underlying mechanism is not firmly established. It is known that mitochondria play an indispensable role in synaptic terminals and the balance of mitochondrial fission/fusion is critical for mitochondrial distribution and function. Our preliminary studies suggest the potential involvement of an impaired balance of mitochondrial fission/fusion in the pathogenesis of AD, in this application, we propose to investigate whether AB cause synaptic dysfunction and mitochondrial abnormalities via its toxic effect on the balance of mitochondrial fission and fusion.

DESCRIPTION (provided by applicant): Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause thus far identified for both familial and idiopathic Parkinson's disease (PD). The majority of LRRK2 linked cases present clinical symptoms typical of idiopathic or sporadic PD. Therefore, understanding of mechanisms underlying LRRK-induced neurodegeneration will likely provide new insights into pathogenesis of PD and open new avenues for therapeutic intervention. Compelling evidence suggest that mitochondrial dysfunction could represent a critical event in the pathogenesis of PD. However, the mechanisms underlying mitochondrial dysfunction in PD remain elusive. Given that several lines of evidence suggested the mitochondrial presence of LRRK2, a possible mechanism of LRRK2 action is at mitochondria. Mitochondria are dynamic organelles that undergo continual fission and fusion events which serve crucial physiological function and are regulated by a machinery involving large dynamin- related GTPase that exert opposing effects. These mitochondria fission and fusion proteins control not only mitochondrial number and morphology but also mitochondrial distribution and function. Notably, an altered mitochondrial dynamics plays a role in mitochondrial dysfunction in cells treated with PD-related toxins and in cells expressing pathogenic mutant PINK-1, Parkin or DJ-1, suggesting that an altered mitochondrial dynamics may be a common pathway leading to mitochondrial dysfunction during PD pathogenesis. More recent studies demonstrated a potential functional interaction between LRRK2 and PINK1/Parkin suggesting that LRRK2 could also be involved in this pathway. Indeed, in our preliminary studies, we found that overexpression of LRRK2 (G2019S) mutant causes mitochondrial fragmentation and abnormal distribution and reduced expression of DLP1/OPA1 along with mitochondrial dysfunction in SHSY-5Y human neuroblastoma cells and primary rat cortical neurons. Based on these observations, our overall hypothesis is that LRRK2 mutations cause abnormal mitochondrial dynamics which in turn causes mitochondrial ultrastructural defects, dysfunction and redistribution which adversely affects neuronal function including causing synaptic abnormalities in PD. To address this hypothesis, the following two specific aims will be pursued. Aim 1 To determine whether mutant LRRK2 cause abnormal mitochondrial fission/fusion and dysfunction in neurons;Aim 2: To determine how mutant LRRK2 affects mitochondrial fission/fusion. PUBLIC HEALTH RELEVANCE: It is of paramount significance to understand how LRRK2 mutations cause familial Parkinson disease. Based on recent development in the field and our preliminary results, we propose to investigate whether LRRK2 mutants cause mitochondrial abnormalities via its toxic effect on the balance of mitochondrial fission and fusion. The completion of this project will enable us to collect data for a more in-depth mechanistic study that may help identify novel therapeutic targets

DESCRIPTION (provided by applicant): Multiple lines of evidence indicate that mitochondrial dysfunction plays a critical role in the pathogenesis of Alzheimer disease; however, the underlying molecular mechanism and its role in AD pathogenesis remain poorly understood. Mitochondria are dynamic organelles that undergo continuous fission and fusion. Our recent in vitro studies suggest that abnormal mitochondrial dynamics likely contributes to mitochondrial dysfunction and synaptic/neuronal dysfunction in AD. Based on these studies, we hypothesized that impaired balance in mitochondrial fission/fusion plays a critical role in the pathogenesis of AD by causing excessive mitochondrial fragmentation and redistribution as well as causes mitochondrial ultrastructural defects and dysfunction which in turn adversely affects neuronal functions including synaptic dysfunction and cognitive/behavioral deficits in AD. However, due to the limitation of in vitro cell culture models, it remains to be determined whether it is mitochondrial fragmentation or elongation that occurs in vivo since swollen mitochondrial in AD or APP mice may demonstrate increased size and/or length. Moreover, it also remains to be determined whether abnormal mitochondrial dynamics is causally involved in mitochondrial/synaptic dysfunction and cognitive deficits in APP mice in vivo. Our preliminary results demonstrated mitochondrial dysfunction, fragmented mitochondria, and decreased expression of mitochondrial fission/fusion proteins in the hippocampus of 3 month-old CRND8 APP transgenic mice, suggesting an altered mitochondrial dynamics early in the disease process. More importantly, we were able to enhance mitochondrial fusion in vivo by overexpressing Mfn2 expression in hippocampus, which enables us to address these critical gaps in our knowledge in vivo. Therefore, we propose to cross these Mfn2 mice with CRND8 APP transgenic mice and determine how normalization of mitochondrial dynamics will affect mitochondrial function, and neuronal/synaptic dysfunction and pathological/behavioral/cognitive deficits in CRND8 mice. The goal is to obtain a definite answer on the involvement of mitochondrial fragmentation or elongation in APP mice and to determine the causal role of mitochondrial dynamics in mitochondrial/neuronal dysfunction and cognitive/behavioral deficits in AD mouse models, which will also serve as a proof-of-concept study for Mfn2-directed therapy for AD. To complement the in vivo studies, we will determine the causal involvement of abnormal mitochondrial dynamics in Abeta-induced mitochondrial/neuronal function and further pursue mechanisms underlying Abeta-induced changes in mitochondrial dynamics and how Mfn2 overexpression rescues Abeta-induced mitochondrial deficits.

DESCRIPTION (provided by applicant): Compelling evidence suggests that both genetic factors and environmental factors play important roles in the pathogenesis of PD. Over 13 genetic loci have been identified in the familial forms of PD. Aging and a number of neurotoxins, including MPTP and certain pesticides, are known risk factors of human parkinsonia. However, the underlying molecular mechanism of the disease-associated environmental factors, particularly, their interaction with genetic factors in PD pathogenesis, remains poorly understood. It is thought that epigenetic changes are mediators between genetic determinants and aging or environmental toxins. In a preliminary study, we determined the global methylation status of 80,000-110,000 highly informative CpG sites in brain tissue from a series of sporadic PD patient and well-matched control patients utilizing novel sequencing technique and we found that multiple genes involved in neurogenesis, particularly the ones in the wnt signaling pathway, were hypermethylated in PD brains. Consistent with this notion, genome-wide expression analysis of SH-SY5Y cells treated with sub-lethal dosage of MPP+ revealed reduced expression of genes in wnt pathways along with increased expression of cytokine genes. These findings demonstrate that specific functional pathways, such as the wnt pathway, are epigenetically dysregulated during the pathogenesis and progression of PD. Our preliminary results support an important role of epigenetic regulation in PD pathogenesis. Based on these exciting preliminary data, we hypothesize that environmental risk factors contribute significantly to initiation and progression of PD via, at least partially, an epigenetic- involved mechanism. In this study, we propose to determine the functional pathways dysregulated by DNA methylation in PD patient brains. We will also determine whether known environmental toxins induce similar changes of DNA methylation in mouse models. Finally, we will select the wnt pathway as a target to determine the role of its dysregulation in the pathogenesis of PD as proof of concept. Successful completion of the study will provide novel insights into the mechanisms through which environmental factors contribute to PD pathogenesis and identify genes or pathways as potential targets for future development of therapeutic reagents.

DESCRIPTION (provided by applicant): The goal of the Neurodegeneration Training Program (NTP) is to provide rigorous pre-doctoral training in various aspects of neurodegeneration and mechanisms of diseases involving neurodegeneration. An outstanding pool of trainees will combine with world-class faculty to investigate a wide range of neurodegeneration-related topics spanning research areas such as protein structure, cell and molecular biology and in vitro and in vivo models of disease. The NTP spans departments, schools and institutions to include multiple departments at Case Western Reserve University, particularly the School of Medicine, and its affiliated institutions, University Hospitals Case Medical Center (UHCMC), The Louis Stokes Cleveland Veteran's Administration Medical Center (VAMC), and the Cleveland Clinic Foundation (CCF, including the Lerner Research Institute). All of these institutions are within walking distance of each other and this rich training environment enjoys very active basic science and clinical activities and state of the art resources to enrich the training of pre- doctoal trainees as they engage in basic and/or translational research in the field of neurodegeneration. Training in the NTP involves NTP course work, formal and informal seminars, an annual retreat, and a research experience resulting in scholarly publications. A unique component of this training program is the inclusion of a required 1 semester, half day per week, mentored experience in a neurodegenerative disease clinic. The combination of didactic and experiential training opportunities afforded by the NTP will provide trainees a solid foundation for a future career in the scientific inquiry of neurodegeneration.

PROJECT SUMMARY/ABSTRACT Compelling evidence suggest that mitochondrial dysfunction is an early prominent feature in susceptible neurons in the brain of patients with Alzheimer's disease and plays a critical role in the pathogenesis of AD. Mitochondria are dynamic organelles that undergo continual fission and fusion events. Most recent studies from multiple groups including ours suggested a likely involvement of abnormal mitochondrial dynamics in AD brain. Indeed, overexpression of APP mutant or A? treatment induces profound mitochondrial fragmentation, ultrastructural deficits and altered distribution which are likely causally involved in A?-induced spine loss and synaptic abnormalities in hippocampal neurons. A?-induced changes in mitochondrial dynamics and distribution are also early events in animal models of AD. However, molecular mechanisms underlying A?-induced abnormal mitochondrial dynamics remains to be determined. Mitochondrial dynamics and function may be modulated by calcium signaling. Notably, a critical role of intracellular calcium dysregulation in the pathogenesis of AD has long been postulated. It is well established that A? oligomers induce a rapid and sustained increase in intracellular calcium in neurons which mediate A?-induced neuronal abnormalities including spine loss and synaptic dysfunction likely through the activation of calcium- dependent signaling molecules such as calpain and calcineurin. Importantly, our pilot study demonstrated that soluble A? oligomers induce elevation in cytosolic calcium which precedes mitochondrial fragmentation, and elevated mitochondrial calcium coincides with a wave of rapid decrease in mitochondrial length, suggesting that A?-induced aberrant calcium signaling is involved in the modulation of mitochondrial dynamics. This is likely through the modulation of mitochondrial fission/fusion proteins since our preliminary studies demonstrated that ADDLs caused reduction in DLP1/OPA1/Mfn1/2 and dephosphorylation of DLP1 at Ser637. Therefore, we hypothesize that A?- induced aberrant calcium signaling causes posttranslational changes (i.e., modifications and/or degradation) in mitochondrial fission/fusion proteins that leads to abnormal mitochondrial dynamics. To test this hypothesis, we will characterized the causal role of aberrant calcium signaling in mediating A?-induced mitochondrial fragmentation and abnormal mitochondrial transport/distribution in details both in vitro and in vivo. Our proposed studies will provide mechanistic insights into mitochondrial dynamic abnormalities in AD by linking two important deficits (i.e., calcium dyshomeostasis and mitochondrial dysfunction) involved in the pathogenesis of AD, which could serve as foundation for future drug development.

PROJECT SUMMARY/ABSTRACT Compelling evidence suggest that mitochondrial dysfunction is an early prominent feature in susceptible neurons in the brain of patients with Alzheimer's disease and plays a critical role in the pathogenesis of AD, yet the underlying molecular mechanism remains incompletely understood. Studies from my group and several other groups demonstrated reduced PGC-1? expression and impaired mitochondrial biogenesis signaling in hippocampus from AD patients and in APP transgenic mouse models. We demonstrated that APPswe mutant causes reduced PGC-1? and impaired mitochondrial biogenesis in neurons likely through the PKA/CREB pathway. Our preliminary studies demonstrated that PGC-1? overexpression restores the mitochondrial biogenesis signaling, alleviates mitochondrial deficits such as abnormal mitochondrial dynamics/distribution and leads to improved mitochondrial function in neuronal cells. These studies suggest that impaired mitochondrial biogenesis signaling plays a critical role in mitochondrial dysfunction which adversely affected neuronal function and contributed to the pathogenesis of AD. It remains to be determined whether and how impaired mitochondrial biogenesis is causally involved in mitochondrial/neuronal dysfunction and cognitive deficits in animal models of AD in vivo. Based on studies in other neurodegenerative diseases and our preliminary studies, we developed the working hypothesis that PGC-1? overexpression to restore mitochondrial biogenesis plays a neuroprotective role in animal models of AD. A recent study reported a negative impact of PGC- 1? overexpression on mitochondrial function and AD-related deficits in one APP Tg mouse model. However, due to the important concerns such as a lack of mitochondrial biogenesis activation on this negative study, the critical role of PGC-1? and mitochondrial biogenesis in the pathogenesis of AD in vivo remains unanswered. Given the critical role of PGC-1? and mitochondrial biogenesis in mitochondrial/neuronal function, in our opinion, it actually makes a stronger case for a more rigorous characterization of the effects of PGC-1? in AD mouse models to resolve the controversy which will likely pave the way for developing treatment targeting mitochondrial biogenesis in AD. Based on the literature and our preliminary study, we also propose to explore the mechanism underlying the effects of PGC-1? on mitochondrial dynamics and axonal transport which will reveal novel regulatory mechanisms between mitochondrial biogenesis and dynamics/transport and suggest the central role of PGC-1? in rescuing mitochondrial deficits broader than biogenesis in AD.

RESEARCH EDUCATION COMPONENT PROJECT SUMMARY The Research Education Component (REC) is aimed at fulfilling two critical needs in the Alzheimer's disease (AD) and Alzheimer's disease related dementias (ADRD) community: 1) the number of current researchers and clinical specialists in AD/ADRD are clearly inadequate to meet the needs of the rapid increase in the AD population in the US and around world and 2) while we have made advances in bridging the knowledge gaps in understanding neurodegenerative mechanisms through many years of research, advances in clinical care are still lacking. As such, new scientists trained in the breadth of approaches with deep clinical appreciation are required to promote progress in translational research in the AD/ADRD field and to develop innovative and effective strategies for the diagnosis and the treatment. The REC program has been specifically designed for selected Program Participants to provide rigorous scientific research training in the AD/ADRD field along with extensive clinical exposure. The REC specifically aims to train talented and promising junior faculty and research associates and supports their career growth with individualized Research Plan and Mentoring Plan with clearly articulated goals. The REC emphasizes rigorous scientific training in fundamental aspects of neurodegeneration and mechanisms of diseases involving AD/ADRD and clinical perspectives through classroom teaching, AD/ADRD- related laboratory research and mentored neurodegenerative clinical experiences combined with educational forums that address important topics in clinical and translational AD/ADRD research and career growth. The REC has identified a group of diverse promising candidates who will be encouraged to develop new competencies under the mentorship of a faculty Mentoring committee composed of outstanding Program Faculty including both clinical and basic researchers. The REC will also utilize the vast educational and training resources available through CADRC-associated institutions including formal programs, training grants, and other educational resources to enable the identified participants to advance their research careers. This REC program will not only provide the necessary infrastructure for the training of new AD/ADRD-related clinical/basic researchers, but also stimulate interdepartmental/interdisciplinary collaborations among CADRC clinical and basic science researchers to serve the overall goal to accelerate collaborative health services, interventional science, and translational neurobehavioral science research specifically in AD/ADRD, augmenting activities of both the CADRC and national and international efforts.

PROJECT SUMMARY/ABSTRACT Compelling evidence suggests that mitochondrial dysfunction is an early feature in susceptible neurons in the brains of patients with Alzheimer?s disease (AD) and plays a critical role in pathogenesis, yet the underlying molecular mechanisms remain incompletely understood. Phosphodiesterases (PDEs) are a superfamily of enzymes responsible for the hydrolysis of cAMP and cGMP, second messengers that regulate important cellular functions. Interestingly, recent studies demonstrated that cAMP/cGMP-PKA/PKG signaling is involved in the regulation of mitochondrial dynamics and expression/assembly of key enzymes in the electron transport chain (ETC) and mitochondrial respiration. Among the many PDEs, PDE2A is the most highly expressed PDE in the hippocampus and frontal/temporal cortex, brain regions vulnerable to AD. Our preliminary studies found increased PDE2A expression in the brains of AD patients and APP/PS1 mice (an AD model), accompanied by decreased cAMP and cGMP in both cytosol and mitochondria matrix, implicating the potential involvement of an aberrant PDE2A-cAMP/cGMP signaling in the pathogenesis of AD. Multiple studies, including ours, demonstrated cognitive enhancing effect of PDE2A inhibitors, although the underlying mechanism remains elusive. In this regard, our preliminary studies revealed that PDE2A overexpression impaired mitochondrial function accompanied by extensive mitochondrial fragmentation. Importantly, A?-induced mitochondrial fragmentation and respiratory deficits could be rescued by a PDE2A inhibitor, suggesting mitochondrial dynamics and function could be mechanism of action for PDE2A to influence cognition. Based on these studies, we hypothesized that aberrant PDE2A signaling caused mitochondrial dysfunction which adversely impacted neuronal/synaptic function and caused pathological/cognitive deficits in AD. Novel animal models with PDE2A conditional knockout in the forebrain will be crossed with different AD transgenic mouse models and carefully characterized. The role of PDE2A2, the PDE2A isoform uniquely localized to mitochondria, in brain function and behavior in AD mouse models will also be determined. Finally, based on the literature and our preliminary study, we propose to explore the mechanism underlying the effects of aberrant PDE2A expression on mitochondrial dysfunction with a focus on mitochondrial dynamics and the expression/assembly of mitochondrial ETC complexes. Our proposed studies will provide mechanistic insights into molecular mechanisms underlying mitochondrial dysfunction in AD and deepen our understanding of PDE2A in the regulation of cognition in the brain. The successful completion of this study will likely pave the way for future drug development of PDE2A inhibitors, specifically for the mitochondrial PDE2A2 isoform, as a promising treatment for AD.