Xin Qi - US grants

Project Summary The objective of this application is to identify biomarkers in Huntington's disease (HD) in a noninvasive way, with the hope that these insights will eventually lead to the development of tools that guide clinical diagnosis and aid drug development. HD is a fatal inherited disorder that progresses for 15-20 years after symptom onset. The mutation that causes HD is a variable expansion of CAG repeats encoding polyglutamine (polyQ) in the huntingtin (Htt) protein. As there is no therapy for this hereditary neurodegenerative disease, further effort should be made to slow the progression of neurodegeneration in patients through the definition of early therapeutic interventions. For this purpose, molecular biomarkers for monitoring disease onset, disease progression, and response to treatment need to be identified. However, the non-invasive detection of clinically useful biomarkers is a challenge faced by many research laboratories. Mitochondrial dysfunction precedes neuropathology and clinical symptoms in patients with HD, indicating that mitochondrial impairment is an early event in the cascade leading to HD pathogenesis. Thus, ability to monitor alteration of mitochondrial function during HD development may allow us to identify biomarkers for disease progression and for therapeutic response. Using targeted metabolomics, preliminary studies identified a panel of mitochondrial intermediates that were changed in the plasma of HD transgenic mice before the appearance of neuronal degeneration. Moreover, some of these mitochondrial substrates were consistently changes in patient CSF and responded to the treatment in HD mice at the early stage of disease progression. These preliminary data have led us to formulate the central hypothesis that markers reflecting mitochondrial dysfunction in peripheral blood are a characteristic pathophysiological factor of HD pathology and provide a novel and noninvasive way to monitor HD disease progression. The significance for the proposed research is that the establishment of mitochondrial signatures as a panel of candidate biomarkers will allow for further validation in a larger cohort of patients on whether these biomarkers could be used for detecting disease state and assessing the efficacy of therapies. In Aim 1, we will test the hypothesis that alteration of mitochondrial metabolic intermediates in HD patient plasma and CSF provides biomarkers amenable for tracking HD disease progression. In Aim 2, we will test the hypothesis that treatment can correct the aberrant mitochondrial intermediates in the plasma and CSF of HD transgenic mice, amenable for treatment efficacy. By the end of this study, we anticipate that we will have a method of identifying mitochondrial biomarkers to indicate HD disease progression; and that we will have a proof of principle that altered plasma and CSF mitochondrial metabolic intermediates can be used to monitor treatment efficacy of experimental drugs for HD.

PROJECT SUMMARY Parkinson's disease (PD) is characterized by a preferential loss of midbrain dopaminergic (DA) neurons. Although the mechanisms underlying PD remain elusive, ?-Synuclein (?Syn) accumulation and mitochondrial deficiency are two major changes in the brains of patients with PD. While missense mutations of ?Syn cause an early-onset autosomal dominant familial form of PD, abnormal accumulation of ?Syn is associated with sporadic PD. ?Syn contains a cryptic mitochondrial targeting sequence and is enriched in mitochondria in the striatum and substantial nigra (SN) relative to other brain regions. Pathological accumulation of ?Syn in the mitochondria of PD vulnerable brain regions is associated with mitochondrial bioenergetic defects and production of reactive oxygen species. Despite these observations showing that ?Syn-mitochondrial interactions may play a causal role in PD, the field lacks a detailed understanding of the mechanisms by which ?Syn abnormality and mitochondrial functional deficiency influence each other. To maintain normal mitochondrial health and function, cells employ a mitochondria-to-nucleus signaling pathway termed the mitochondrial unfolded protein response (UPRmt). The UPRmt monitors mitochondrial proteostasis through mitochondrial specific proteases and molecular chaperones, which facilitate folding and/or degradation of unfolded or misfolded proteins within mitochondria, and they communicate with the nucleus by retrograde signaling to activate the expression of peptide-folding related proteins. The UPRmt is an important defense mechanism for maintaining the quality of proteins within the mitochondria under stress. Defects in UPRmt have been linked to aging and neurodegeneration. Preliminary research found that the protein level of ClpP, a mitochondrial matrix protease induced during UPRmt activation, is selectively decreased in DA neurons stably expressing ?Syn wildtype (WT) or A53T mutant, and in brains of mice carrying A53T mutant. Moreover, the immunodensity of ClpP was greatly reduced in DA neurons of the SN in both ?Syn A53T transgenic mice and PD patient postmortem brains. Whereas silencing ClpP in DA neurons reduced mitochondrial bioenergetic activity and increased cell death, overexpressing ClpP abolished ?Syn-induced mitochondrial oxidative stress in cultured cells and attenuated behavioral abnormality in ?Syn A53T PD mice. Notably, we found that ?Syn WT and A53T mutant bound to ClpP and suppressed ClpP peptidase activity. Taken together, these preliminary findings have led us to formulate the central hypothesis that ?Syn causes mitochondrial bioenergetic defects and oxidative stress by suppressing ClpP and UPRmt, which results in ?Syn neuropathology. The significance of the proposed study is that a detailed characterization of the precise mechanism by which ?Syn affects mitochondrial function and neuronal survival will not only contribute to the basic understanding of disease pathogenesis, but also aid in the development of treatments for PD and other synucleinopathies.

Accumulation of alpha-synuclein (?Syn) causes degeneration of dopaminergic (DA) and non-DA neurons in Parkinson?s disease (PD) and Dementia with Lewy Bodies (DLB). ?Syn also contributes to the fibrilization of amyloid-? and tau, two key proteins in Alzheimer?s disease (AD), which suggests a key role for ?Syn toxicity in neurodegeneration. Thus, it is important to elucidate downstream effects and the factors promoting the toxic conversion of ?Syn, towards understanding the pathogenesis of and developing disease-modifying therapies for synucleinopathies. In PD and DLB, pathological ?Syn proteins enrich in mitochondria of vulnerable brain regions, where to induce mitochondrial bioenergetic defects and production of reactive oxygen species. Despite evidence suggests that ?Syn-mitochondrial interactions may play a causal role in PD and DLB, the field lacks a detailed understanding of the mechanisms by which ?Syn abnormality and mitochondrial functional deficiency influence each other. To maintain normal mitochondrial health and function, cells employ a mitochondria-to-nucleus signaling pathway termed the mitochondrial unfolded protein response (UPRmt). The UPRmt monitors mitochondrial proteostasis through mitochondrial specific proteases and molecular chaperones, which facilitate folding and/or degradation of unfolded proteins within mitochondria. They also communicate with the nucleus by retrograde signaling to activate the expression of peptide-folding related proteins. The UPRmt is an important defense mechanism for maintaining the quality of proteins within the mitochondria under stress. Defects in UPRmt have been linked to aging and neurodegeneration. Our recent work showed, for the first time, that the protein level of ClpP, a mitochondrial matrix protease induced during UPRmt activation, was decreased in neurons expressing ?Syn wildtype (WT) or A53T mutant, in brains of mice carrying A53T mutant, and in the Substantia Nigra of PD patients. The mRNA level of ClpP remained unchanged, suggesting a transcriptional independent effect. Preliminary study further found that the protein level of ClpP and not other mitochondrial proteases, decreased in the cortex of patients with DLB and mice expressing human Thy1-?Syn. These results suggest that a decrease in ClpP is a common event implicated in the pathogenesis of both PD and DLB. Whereas silencing ClpP in neurons increased a load of unfolded proteins in the mitochondria, reduced mitochondrial bioenergetic activity and increased cell death; overexpressing ClpP abolished ?Syn-induced oxidative stress in cultured cells, and attenuated ?Syn hyper-phosphorylation and behavioral abnormality in ?Syn A53T mice. Notably, we found that ?Syn bound to ClpP and suppressed ClpP peptidase activity, whereas genetic manipulation of ClpP influenced the assembly of non-toxic ?Syn tetramers that resist aggregation. Thus, our pilot findings highlight a previously unidentified interdependence between pathological ?Syn and mitochondrial protease ClpP, which results in a disturbance of mitochondrial proteostasis, leading to neuronal damage. Given that ?Syn accumulation is a common pathological hallmark of both PD and DLB, the goal of this study is to determine the role of ClpP-mediated mitochondrial proteostasis in PD and DLB at both mechanistic and therapeutic level. Built on our study supported by the NIH bridge award R56 NS105632-1A1, we will test the central hypothesis that pathological ?Syn disturbs mitochondrial proteostasis by suppressing ClpP and UPRmt, which impairs mitochondrial bioenergetic activity and promotes the toxic conversion of ?Syn, leading to ?Syn neuropathology. Our research team has the unique synergistic expertise in UPRmt, mitochondrial biology, and ?Syn neuropathology required to impact this significant area of unmet medical need. Successful completion of the proposed study will not only contribute to the basic understanding of disease pathogenesis, but it will also aid in the development of treatments for PD and DLB and even other neurodegenerative diseases in which ?Syn aggregation manifests.

Both environmental and genetic factors involved in the disturbance of cholesterol metabolism have been suggested as risk factors for the development of Alzheimer's disease (AD). Accumulation of cholesterol has been observed in affected brain areas from AD patients and animals, and it is associated with region-specific loss of synapses. Elevated brain cholesterol causes cognitive deficits, amyloid-? (A?) production and aggregation, and tau pathology. Despite these observations, the mechanisms that govern brain cholesterol homeostasis and influence on neurons under AD-related pathological conditions remain elusive. In particular, the field lacks knowledge on the factors that are involved in the signaling pathways of neuronal cholesterol metabolism, related to the initiation and development of AD pathology. ATAD3A belongs to a new family of eukaryotic mitochondrial AAA-ATPases. ATAD3A regulates cholesterol homeostasis and trafficking via an unknown mechanism at the mitochondria-associated ER membrane (MAM), a specialized subdomain of the ER that has the features of a lipid raft and is rich in cholesterol and sphingomyelin. Our recent work demonstrated that ATAD3A, via pathological dimerization, showed a gain-of-function that caused neurodegeneration in Huntington's disease. We further observed an enhancement of ATAD3A oligomerization in AD neuronal culture, in AD transgenic mouse brains and in AD patient postmortem hippocampus, suggesting an aberrant activity of ATAD3A in the pathogenesis of AD. We developed a novel peptide inhibitor DA1 that binds to ATAD3A to block ATAD3A dimerization. Notably, sustained treatment with DA1 reduced APP level and amyloid load, attenuated neuro-inflammation and improved short-term spatial memory in 5XFAD transgenic mice. Further, our proteomic analysis suggests that blocking ATAD3A oligomerization by DA1 treatment mainly influenced the cholesterol metabolic pathway in AD mouse brains. The treatment in AD transgenic mice improved brain cholesterol turnover and did not affect brain phospholipids levels. Moreover, we showed that DA1 treatment reduced cholesterol burden and oxidative stress in neuronal cells stably expressing APP wt or mutant. These findings highlight ATAD3A oligomerization as a previously unidentified mechanism underlying brain cholesterol disturbance and neurodegeneration in AD. Our central hypothesis is that ATAD3A oligomerization mediates amyloid pathology, leading to neurodegeneration, by impairment of brain cholesterol metabolism. The overall goal of this application is to understand ATAD3A aberrant oligomerization-mediated neuropathology in AD, and to reveal a novel therapeutic target for AD. In Aim 1, we will determine the impact of ATAD3A oligomerization on brain cholesterol homeostasis, AD pathology and behavioral deficits in AD mice. In Aim 2, we will determine whether haplosufficiency of ATAD3A in AD mice restores brain cholesterol homeostasis and reduces AD pathology. In Aim 3, we will dissect the mechanistic links between ATAD3A oligomerization and disturbance in brain cholesterol homeostasis in AD.