Xinglong Wang, PhD - US grants

DESCRIPTION (provided by applicant): Mitochondrial dysfunction plays a prominent role in the pathogenesis of Parkinson's disease (PD). Mitochondria are dynamics organelles that undergo continual fission and fusion events which serve crucial physiological function. Increasing evidence demonstrated abnormal mitochondrial dynamics in PD and PD models, suggesting that an altered balance in mitochondrial fission/fusion and impaired mitochondrial quality control was likely a common mechanism leading to mitochondrial and neuronal dysfunction/degeneration critical to the pathogenesis of PD. Mutations in VPS35 cause autosomal dominant PD. VPS35 is a key component of the retromer complex, which is important for endosome-to-golgi and endosome-to-plasma membrane sorting and many signaling events. Recent studies found the localization of VPS35 on mitochondria and its involvement in inter-organelle communication between mitochondria and other organelles. In our preliminary studies, we confirmed the mitochondrial localization of VPS35 in both human neuroblastoma cells and human brain hippocampal neurons. We further found that overexpression of wild-type VPS35 in neurons caused significant changes of mitochondrial dynamics, which became more severe in neurons expressing PD- associated VPS35 mutant D620N. More importantly, we found that VPS35 physically interacted with DLP1, a key regulator of mitochondrial dynamics, which was enhanced by PD-associated mutation. All these exciting findings strongly suggest that VPS35 were involved in the regulation of mitochondrial dynamics which may be impaired by VPS35 PD associated mutations and detailed investigation into the potential role of PS1 in mitochondrial function and dynamics is warranted. Our proposed study will be the first mechanistic study investigating the effect of the pathogenic VPS35 PD mutations on mitochondrial dynamics/function and neuronal function and will likely reveal a novel role of VPS35 in the regulation of mitochondrial dynamics/function. In addition, our proposed studies will also provide novel insights into the contribution of retromer to various cellular processes an signaling pathways.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the leading cause of dementia in the elderly, characterized by neurofibrillary tangles, senile plaques and a progressive loss of neuronal cells in selective brain regions. Mitochondrial dysfunction is a prominent and early feature of the disease, although the underlying mechanism is still not clear. Mitochondria are dynamics organelles that undergo continual fission and fusion events which serve crucial physiological function. Our recent studies demonstrated that an altered balance in mitochondrial fission and fusion was likely an important mechanism leading to mitochondrial and synaptic/neuronal dysfunction in AD brain. Mutations in presenilins (PS) cause early-onset familial form of AD (FAD). PS1 is found in mitochondria and mutant PS1 affects mitochondrial function and transport. Our preliminary studies revealed that PS1 knockout (KO) primary neurons demonstrated significant changes in mitochondria morphology, distribution and movement which could be prevented by co-expression of wild-type PS1, but not FAD-causing PS1 mutant, suggesting that presenilins are involved in the regulation of mitochondrial dynamics which may be impaired by PS1 FAD mutations. Most importantly we found that PS1 physically interacted with DLP1, a key regulator of both mitochondrial fission and distribution. These studies suggest that a detailed investigation into the potential role of PS1 in mitochondrial function and dynamics is warranted. Based on our preliminary studies, we hypothesize that FAD-associated PS1 mutants cause impaired regulation of mitochondrial dynamics through specific interaction with DLP1 which causes mitochondrial dysfunction and redistribution which adversely affects neuronal functions including causing synaptic abnormalities in AD. To begin to address this hypothesis, the following specific aims will be pursued: 1) to determine the effect of FAD-associated PS1 mutants on mitochondria dynamics; 2) to determine whether PS1-DLP1 interaction mediates the effects of FAD-associated PS1 mutants on mitochondrial dynamics.

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS), also called Lou Gehrig's disease, is the most common of the five motor neuron diseases characterized by progressive neurodegeneration of motor neurons in the brain stem and spinal cord. Currently, there is no cure or effective treatment for ALS. The cause of disease is unknown in the majority of ALS cases. Less than 10% of ALS cases are familial, involving mutations in several genes such as SOD1 and TARDBP. The protein encoded by TARDBP, i.e. TAR DNA-binding protein 43 (TDP-43), was identified as a major component of the histopathological hallmark, i.e., neuronal ubiquitinated inclusions, of degenerating neurons in most forms of ALS and increasing evidence suggests a critical role of TDP-43 in diverse neurodegenerative diseases including ALS and frontotemporal lobar degeneration (FTLD). Unfortunately, how TDP-43 mutant causes neurodegeneration is poorly understood. Interestingly, in our preliminary studies, we also observed significant impairment of mitochondrial bioenergetics in motor neuronal cell lines expressing mutant TDP-43. As mitochondrial dysfunction plays a prominent role in ALS, further more detailed studies should be performed to assess the effect of mutant TDP-43 on mitochondrial function in primary motor neurons in vitro and in vivo, and explore potential underlying mechanisms by which mutant TDP- 43 cause mitochondrial dysfunction. TDP-43 translocates from the nucleus to cytoplasm in of ALS and frontotemporal lobar degeneration (FTLD-U). Unfortunately, few attempt has been taken to investigate its subcellular organelle target(s). Excitingly, our pilot studies found that TDP-43 could be present in the matrix of mitochondria. And, more importantly, TDP-43 interacts with a matrix facing protein critical for mitochondrial electron transport chain, and binds mitochondrial genome encoded mRNA, indicating a direct role of TDP-43 in regulating mitochondrial function. All these exciting finding strongly suggest that TDP-43 may impair mitochondrial function through its specific localization in mitochondria which adversely affects neuronal functions in ALS. Thus, it is important to investigate how TDP-43 is taken up by mitochondria and test whether TDP-43 mitochondrial localization is required for its toxicity on mitochondria and neurons. Our proposed study will be the first systematic and mechanistic study of TDP-43 mitochondrial import as well as TDP-43 induced mitochondrial dysfunction. Our proposed studies will reveal a novel role of TDP-43 in the regulation of mitochondrial function and likely provide novel therapeutic targets for ALS.

PROJECT SUMMARY Mitochondrial dysfunction and neuromuscular junction (NMJ) decline are both prominent features in amyotrophic lateral sclerosis (ALS), and implicated in the onset and progression of ALS. However, whether and how mitochondrial dysfunction plays a role in NMJ decline in ALS is not clear. Constant mitochondrial fission and fusion dynamics are essential for both mitochondrial morphology and function. We and others recently demonstrated that the impaired mitochondrial fission and fusion dynamics was likely an important mechanism leading to mitochondrial dysfunction and neurodegeneration in ALS and other various major neurodegenerative diseases. Interestingly, in our preliminary studies, we found that mitochondria became highly fragmented in spinal cord motor neurons of ALS patients and the widely studied transgenic SOD1 G93A mouse model of ALS (SOD1G93A mice). Mitofusin 2 (Mfn2), the key regulator of mitochondrial fusion, was further found to be reduced in spinal cords of ALS patients and SOD1G93A mice. Excitingly, our newly identified Mfn2 degradation pathway via calpain was found activated in SOD1G93A mice. Mfn2 deficiency in motor neurons caused mitochondrial fragmentation, NMJ denervation and neuronal death, whereas forced expression of Mfn2 in neurons was sufficient to completely abolish mitochondrial fragmentation, NMJ decline and related skeletal muscle atrophy in SOD1G93A mice even at the endstage. These exciting and promising preliminary studies suggest that a detailed investigation into the potential role of Mfn2 in the maintenance of NMJs and related skeletal muscles in ALS is warranted. The following specific aims will be pursued: 1) To perform detailed assessments of motor function, skeletal muscles, motor neurons and NMJs in Mfn2/SOD1G93A mice; 2) To elucidate the molecular mechanism underlying Mfn2 reduction in SOD1G93A mice and validate calpain-mediated Mfn2 degradation as a therapeutic target; 3) To explore the pathways by which neuronal Mfn2 protects NMJs in Mfn2/SOD1G93A mice. This will be first systematic and mechanistic in vivo study using novel mouse models, a promising novel synthetic therapeutic peptide and cross-disciplinary approaches to investigate the role of mitochondrial dynamics in the maintenance of NMJs and function of motor neuron and muscle in ALS. Our proposed studies of the impact of mitochondrial dynamics on NMJs as a functional unit of nerve and muscle in the context of ALS will has both scientific (new insight into mechanisms underlying mitochondrial dysfunction and NMJ decline in ALS) and translational (provide novel therapeutic approaches to delay or reverse NMJ decline and associated skeletal muscle atrophy in ALS) significance.

PROJECT SUMMARY Alzheimer's disease (AD) is the leading cause of dementia in the elderly, characterized by neurofibrillary tangles, senile plaques and a progressive loss of neuronal cells in neocortex and hippocampus. Currently, there is no effective treatment for AD. Genetic mutations in TAR DNA-binding protein 43 (TDP-43) cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD, the second most common form of early-onset dementia), and the increased presence of TDP-43 in the cytoplasm is a prominent histopathological feature of degenerating neurons in more than half of AD patients. Despite an expanding body of evidence suggests that TDP-43 may be ?the third protein? playing a distinct role in the pathogenesis of AD or related dementia, in addition to amyloid beta (A?) and tau, the molecular pathomechanisms of TDP-43 remain elusive. Interestingly, in our preliminary studies, we found that TDP-43 became highly associated with mitochondria in AD patients, neurons treated with A? and APP/PS1 (5XFAD) transgenic mice for AD. Based on identified motifs critical for TDP-43 mitochondrial localization, our most recent study revealed that the suppression of TDP-43 mitochondrial localization was sufficient to prevent TDP-43-induced neuronal loss, and improve behavioral performances in TDP-43 transgenic mice, indicating mitochondria as important mediators for TDP-43 neurotoxicity. Excitingly, the inhibition of TDP-43 mitochondrial localization could significantly alleviate neuronal death and behavioral deficits in 5XFAD mice well after symptom onset. These exciting and promising preliminary studies suggest that a detailed investigation into the potential role of mitochondria- associated TDP-43 in AD and related dementia is warranted. Using both cultured neuronal and transgenic mouse models for AD and related dementia, this study will test the feasibility of targeting mitochondria- associated TDP-43 as a novel therapeutic approach for AD and related dementia. The increased presence of TDP-43 in the cytoplasm is a prominent common histopathological feature of degenerating neurons in various major neurodegenerative diseases including AD, FTD and ALS. Our proposed studies of mitochondria- associated TDP-43 and its connection with the generally believed AD culprit A? will have very broad scientific and translational significance.

PROJECT SUMMARY Alzheimer's disease (AD) is the most common dementia in the elderly characterized by neurofibrillary tangles, senile plaques and a progressive loss of brain neurons. Compared with senile plaques, neurofibrillary tangles have a better correlation with the severity of cognitive impairment in AD. As intracellular lesions, neurofibrillary are largely composed of hyperphosphorylated microtubule-associated protein tau. Not surprisingly, considerable efforts have been devoted to tau-based AD drug development though the pathomechanism underlying tau toxicity remains largely unknown. Mitochondrial dysfunction and neuroinflammation are prominent early pathological features of AD and have been increasingly implicated as critical factors for AD pathogenesis. Despite both mitochondrial dysfunction and neuroinflammation have been repeatedly reported in animal models of tauopathies, there is limited study of their interplay. Interestingly, in our preliminary studies, we found that Mfn2, the mitochondrial outer membrane protein regulating mitochondrial morphology and association with endoplasmic reticulum, was significantly reduced in the widely used PS19 tau transgenic mice for AD and related tauopathies. Excitingly, the overexpression of Mfn2 in neurons is sufficient to remarkably suppress tau phosphorylation, mitochondrial dysfunction, neuroinflammation, neuronal loss and behavioral deficits in PS19 mice. In addition, lipopolysaccharide-induced neuroinflammation and even sudden death could also be greatly suppressed by overexpressing Mfn2 in neurons, together implying neuronal Mfn2 as a crucial mediator for both mitochondrial dysfunction and neuroinflammation. These exciting and promising preliminary studies suggest that a detailed investigation into the potential role of Mfn2 as a point of convergence for mitochondrial dysfunction and neuroinflammation in AD and related tauopathies is warranted. Using novel transgenic mouse models and a promising synthetic therapeutic peptide inhibiting Mfn2 degradation, this study will not only study whether and how Mfn2 regulates mitochondrial dysfunction and neuroinflammation, but also test the feasibility of targeting Mfn2 as a novel therapeutic approach against tau toxicity. Tau pathology is a prominent common histopathological feature of various major neurodegenerative diseases including but not limited to AD. Our proposed studies of Mfn2 and its convergent role in mitochondrial dysfunction and neuroinflammation will have very broad scientific and translational significance.