All Publications


  • Precision Neurology for Parkinson's Disease: Coupling Miro1-Based Diagnosis with Drug Discovery. Movement disorders : official journal of the Movement Disorder Society Bharat, V., Wang, X. 2020

    Abstract

    Parkinson's disease (PD) is a debilitating movement disorder, significantly afflicting the aging population. Efforts to develop an effective treatment have been challenged by the lack of understanding of the pathological mechanisms underlying neurodegeneration. We have shown that Miro1, an outer mitochondrial membrane protein, situates at the intersection of the complex genetic and functional network of PD. Removing Miro1 from the surface of damaged mitochondria is a prerequisite for mitochondrial clearance via mitophagy. Parkinson's proteins PINK1, Parkin, and LRRK2 are the molecular helpers to remove Miro1 from dysfunctional mitochondria destined for mitophagy. We have found a delay in clearing Miro1 and initiating mitophagy in postmortem brains and induced pluripotent stem cell-derived neurons from PD patients harboring mutations in LRRK2, PINK1, or Parkin, or from sporadic PD patients with no known mutations. In addition, we have shown that reducing Miro1 by both genetic and pharmacological approaches can correct this Miro1 phenotype and rescue Parkinson's-relevant phenotypes in human neurons and fly PD models. These results suggest that the Miro1 defect may be a common denominator for PD, and compounds that reduce Miro1 promise a new class of drugs to battle PD. We propose to couple this Miro1 phenotype with Miro1-based drug discovery in future therapeutic studies, which could significantly improve the success of clinical trials. © 2020 International Parkinson and Movement Disorder Society.

    View details for DOI 10.1002/mds.28194

    View details for PubMedID 32710675

  • Surveillance and transportation of mitochondria in neurons. Current opinion in neurobiology Vanhauwaert, R., Bharat, V., Wang, X. 2019; 57: 87–93

    Abstract

    Neurons need to allocate and sustain mitochondria to provide adequate energy and sufficient Ca2+-buffering capacity in each sub specialization of their extensive arborizations. Damaged mitochondria, which are highly deleterious to the neuron, must be rapidly repaired or eliminated, even when they are left at terminals extremely far away from the soma. The unique shape of neurons complicates the tasks of both transporting and clearing mitochondria. Errors in the underlying molecular regulations cause detrimental neurodegeneration. Here, we review the molecular machinery and regulatory mechanisms employed to transport, distribute, and clear mitochondria in neurons, and how these fundamental regulations are compromised in neurological disorders.

    View details for PubMedID 30784981

  • Understanding how combinatorial targeting of TLRs and TNFR family costimulatory members promote enhanced T cell responses EXPERT OPINION ON BIOLOGICAL THERAPY Shinde, P., Bharat, V., Rodriguez-Oquendo, A., Zhou, B., Vella, A. T. 2018; 18 (10): 1073–83

    Abstract

    Due to the ability of pathogen-associated molecular patters and tumor necrosis factor receptor (TNFR) family costimulatory agonists to boost T cell responses, studies have combined Toll-like receptor (TLR) ligands with TNFR family costimulatory receptor agonists to induce impressive and long-lasting T cell responses. Although some studies have determined how these combinatorial vaccines promote enhanced T cell responses, much remains unknown about the mechanism used by these combinations to promote synergistic T cell responses - especially in settings of infectious diseases or cancer. Areas covered: In this review, we look in detail at the signaling pathways induced by combinatorial targeting of TLR and TNFR family costimulatory members that help them promote synergistic T cell responses. Understanding this can greatly aid the development of novel vaccine regimens that promote cellular immune responses, which is essential for treating certain infectious diseases and cancer. Expert opinion: Vaccines against some infectious diseases as well as therapeutic cancer vaccines require cellular immunity. Therefore, we evaluate here how signaling pathways induced by TLR ligand and costimulatory agonist combinations promote enhanced T cell responses during immunization with model antigens, viral pathogens, or tumor antigens. Once pathways that drive these combinatorial vaccines to boost T cell activation are identified, they can be incorporated in vaccines designed to target pathogens or cancer.

    View details for PubMedID 30169979

  • Capture of Dense Core Vesicles at Synapses by JNK-Dependent Phosphorylation of Synaptotagmin-4. Cell reports Bharat, V., Siebrecht, M., Burk, K., Ahmed, S., Reissner, C., Kohansal-Nodehi, M., Steubler, V., Zweckstetter, M., Ting, J. T., Dean, C. 2017; 21 (8): 2118-2133

    Abstract

    Delivery of neurotrophins and neuropeptides via long-range trafficking of dense core vesicles (DCVs) from the cell soma to nerve terminals is essential for synapse modulation and circuit function. But the mechanism by which transiting DCVs are captured at specific sites is unknown. Here, we discovered that Synaptotagmin-4 (Syt4) regulates the capture and spatial distribution of DCVs in hippocampal neurons. We found that DCVs are highly mobile and undergo long-range translocation but switch directions only at the distal ends of axons, revealing a circular trafficking pattern. Phosphorylation of serine 135 of Syt4 by JNK steers DCV trafficking by destabilizing Syt4-Kif1A interaction, leading to a transition from microtubule-dependent DCV trafficking to capture at en passant presynaptic boutons by actin. Furthermore, neuronal activity increased DCV capture via JNK-dependent phosphorylation of the S135 site of Syt4. Our data reveal a mechanism that ensures rapid, site-specific delivery of DCVs to synapses.

    View details for DOI 10.1016/j.celrep.2017.10.084

    View details for PubMedID 29166604

    View details for PubMedCentralID PMC5714612

  • EndophilinAs regulate endosomal sorting of BDNF-TrkB to mediate survival signaling in hippocampal neurons SCIENTIFIC REPORTS Burk, K., Murdoch, J. D., Freytag, S., Koenig, M., Bharat, V., Markworth, R., Burkhardt, S., Fischer, A., Dean, C. 2017; 7

    Abstract

    The sorting of activated receptors into distinct endosomal compartments is essential to activate specific signaling cascades and cellular events including growth and survival. However, the proteins involved in this sorting are not well understood. We discovered a novel role of EndophilinAs in sorting of activated BDNF-TrkB receptors into late endosomal compartments. Mice lacking all three EndophilinAs accumulate Rab7-positive late endosomes. Moreover, EndophilinAs are differentially localized to, co-traffic with, and tubulate, distinct endosomal compartments: In response to BDNF, EndophilinA2 is recruited to both early and late endosomes, EndophilinA3 is recruited to Lamp1-positive late endosomes, and co-trafficks with Rab5 and Rab7 in both the presence and absence of BDNF, while EndophilinA1 colocalizes at lower levels with endosomes. The absence of all three EndophilinAs caused TrkB to accumulate in EEA1 and Rab7-positive endosomes, and impaired BDNF-TrkB-dependent survival signaling cascades. In addition, EndophilinA triple knockout neurons exhibited increased cell death which could not be rescued by exogenous BDNF, in a neurotrophin-dependent survival assay. Thus, EndophilinAs differentially regulate activated receptor sorting via distinct endosomal compartments to promote BDNF-dependent cell survival.

    View details for DOI 10.1038/s41598-017-02202-4

    View details for Web of Science ID 000401614900007

    View details for PubMedID 28526875

    View details for PubMedCentralID PMC5438371

  • BDNF enhances spontaneous and activity-dependent neurotransmitter release at excitatory terminals but not at inhibitory terminals in hippocampal neurons. Frontiers in synaptic neuroscience Shinoda, Y., Ahmed, S., Ramachandran, B., Bharat, V., Brockelt, D., Altas, B., Dean, C. 2014; 6: 27-?

    Abstract

    Brain-derived neurotrophic factor (BDNF) is widely reported to enhance synaptic vesicle (SV) exocytosis and neurotransmitter release. But it is still unclear whether BDNF enhances SV recycling at excitatory terminals only, or at both excitatory and inhibitory terminals. In the present study, in a direct comparison using cultured rat hippocampal neurons, we demonstrate that BDNF enhances both spontaneous and activity-dependent neurotransmitter release from excitatory terminals, but not from inhibitory terminals. BDNF treatment for 5 min or 48 h increased both spontaneous and activity-induced anti-synaptotagmin1 (SYT1) antibody uptake at excitatory terminals marked with vGluT1. Conversely, BDNF treatment did not enhance spontaneous or activity-induced uptake of anti-SYT1 antibodies in inhibitory terminals marked with vGAT. Time-lapse imaging of FM1-43 dye destaining in excitatory and inhibitory terminals visualized by post-hoc immunostaining of vGluT1 and vGAT also showed the same result: The rate of spontaneous and activity-induced destaining was increased by BDNF at excitatory synapses, but not at inhibitory synapses. These data demonstrate that BDNF enhances SV exocytosis in excitatory but not inhibitory terminals. Moreover, BDNF enhanced evoked SV exocytosis, even if vesicles were loaded under spontaneous vesicle recycling conditions. Thus, BDNF enhances both spontaneous and activity-dependent neurotransmitter release on both short and long time-scales, by the same mechanism.

    View details for DOI 10.3389/fnsyn.2014.00027

    View details for PubMedID 25426063

    View details for PubMedCentralID PMC4226143

  • Axonal and dendritic synaptotagmin isoforms revealed by a pHluorin-syt functional screen MOLECULAR BIOLOGY OF THE CELL Dean, C., Dunning, F. M., Liu, H., Bomba-Warczak, E., Martens, H., Bharat, V., Ahmed, S., Chapman, E. R. 2012; 23 (9): 1715-1727

    Abstract

    The synaptotagmins (syts) are a family of molecules that regulate membrane fusion. There are 17 mammalian syt isoforms, most of which are expressed in the brain. However, little is known regarding the subcellular location and function of the majority of these syts in neurons, largely due to a lack of isoform-specific antibodies. Here we generated pHluorin-syt constructs harboring a luminal domain pH sensor, which reports localization, pH of organelles to which syts are targeted, and the kinetics and sites of exocytosis and endocytosis. Of interest, only syt-1 and 2 are targeted to synaptic vesicles, whereas other isoforms selectively recycle in dendrites (syt-3 and 11), axons (syt-5, 7, 10, and 17), or both axons and dendrites (syt-4, 6, 9, and 12), where they undergo exocytosis and endocytosis with distinctive kinetics. Hence most syt isoforms localize to distinct secretory organelles in both axons and dendrites and may regulate neuropeptide/neurotrophin release to modulate neuronal function.

    View details for DOI 10.1091/mbc.E11-08-0707

    View details for Web of Science ID 000306244000011

    View details for PubMedID 22398727

    View details for PubMedCentralID PMC3338438

  • A mitochondrial membrane-bridging machinery mediates signal transduction of intramitochondrial oxidation. Nature metabolism Li, L., Conradson, D. M., Bharat, V., Kim, M. J., Hsieh, C., Minhas, P. S., Papakyrikos, A. M., Durairaj, A. S., Ludlam, A., Andreasson, K. I., Partridge, L., Cianfrocco, M. A., Wang, X. 2021

    Abstract

    Mitochondria are the main site for generating reactive oxygen species, which are key players in diverse biological processes. However, the molecular pathways of redox signal transduction from the matrix to the cytosol are poorly defined. Here we report an inside-out redox signal of mitochondria. Cysteine oxidation of MIC60, an inner mitochondrial membrane protein, triggers the formation of disulfide bonds and the physical association of MIC60 with Miro, an outer mitochondrial membrane protein. The oxidative structural change of this membrane-crossing complex ultimately elicits cellular responses that delay mitophagy, impair cellular respiration and cause oxidative stress. Blocking the MIC60-Miro interaction or reducing either protein, genetically or pharmacologically, extends lifespan and health-span of healthy fruit flies, and benefits multiple models of Parkinson's disease and Friedreich's ataxia. Our discovery provides a molecular basis for common treatment strategies against oxidative stress.

    View details for DOI 10.1038/s42255-021-00443-2

    View details for PubMedID 34504353

  • Mitochondrial Defects in Fibroblasts of Pathogenic MAPT Patients. Frontiers in cell and developmental biology Bharat, V., Hsieh, C. H., Wang, X. 2021; 9: 765408

    Abstract

    Mutations in MAPT gene cause multiple neurological disorders, including frontal temporal lobar degeneration and parkinsonism. Increasing evidence indicates impaired mitochondrial homeostasis and mitophagy in patients and disease models of pathogenic MAPT. Here, using MAPT patients' fibroblasts as a model, we report that disease-causing MAPT mutations compromise early events of mitophagy. By employing biochemical and mitochondrial assays we discover that upon mitochondrial depolarization, the recruitment of LRRK2 and Parkin to mitochondria and degradation of the outer mitochondrial membrane protein Miro1 are disrupted. Using high resolution electron microscopy, we reveal that the contact of mitochondrial membranes with ER and cytoskeleton tracks is dissociated following mitochondrial damage. This membrane dissociation is blocked by a pathogenic MAPT mutation. Furthermore, we provide evidence showing that tau protein, which is encoded by MAPT gene, interacts with Miro1 protein, and this interaction is abolished by pathogenic MAPT mutations. Lastly, treating fibroblasts of a MAPT patient with a small molecule promotes Miro1 degradation following depolarization. Altogether, our results show molecular defects in a peripheral tissue of patients and suggest that targeting mitochondrial quality control may have a broad application for future therapeutic intervention.

    View details for DOI 10.3389/fcell.2021.765408

    View details for PubMedID 34805172

    View details for PubMedCentralID PMC8595217

  • Global survey-based assessment of lifestyle changes during the COVID-19 pandemic. PloS one Agarwal, P., Kaushik, A., Sarkar, S., Rao, D., Mukherjee, N., Bharat, V., Das, S., Saha, A. K. 2021; 16 (8): e0255399

    Abstract

    Along with the major impact on public health, the COVID-19 outbreak has caused unprecedented concerns ranging from sudden loss of employment to mental stress and anxiety. We implemented a survey-based data collection platform to characterize how the COVID-19 pandemic has affected the socio-economic, physical and mental health conditions of individuals. We focused on three broad areas, namely, changes in social interaction during home confinement, economic impact and their health status. We identified a substantial increase in virtual interaction among individuals, which might be a way to alleviate the sudden unprecedented mental health burden, exacerbated by general awareness about viral infections or other manifestations associated with them. The majority of participants (85%) lived with one or more companions and unemployment issues did not affect 91% of the total survey takers, which was one of the crucial consequences of the pandemic. Nevertheless, measures such as an increased frequency of technology-aided distant social interaction, focus on physical fitness and leisure activities were adopted as coping mechanisms during this period of home isolation. Collectively, these metrics provide a succinct and informative summary of the socio-economic and health impact of the COVID-19 pandemic on the individuals. Findings from our study reflect that continuous surveillance of the psychological consequences for outbreaks should become routine as part of preparedness efforts worldwide. Given the limitations of analyzing the large number of variables, we have made the raw data publicly available on the OMF ME/CFS Data Center server to facilitate further analyses (https://igenomed.stanford.edu/dataset/survey-study-on-lifestyle-changes-during-covid-19-pandemic).

    View details for DOI 10.1371/journal.pone.0255399

    View details for PubMedID 34388151

  • Miro1 Impairment in a Parkinson's At-Risk Cohort. Frontiers in molecular neuroscience Nguyen, D., Bharat, V., Conradson, D. M., Nandakishore, P., Wang, X. 2021; 14: 734273

    Abstract

    There is a lack of reliable molecular markers for Parkinson's disease (PD) patients and at-risk individuals. The detection of the pre-symptomatic population of PD will empower more effective clinical intervention to delay or prevent disease onset. We have previously found that the mitochondrial protein Miro1 is resistant to mitochondrial depolarization-induced degradation in fibroblasts from a large number of PD patients and several at-risk individuals. Therefore, Miro1 has the potential to molecularly label PD populations. In order to determine whether Miro1 could serve as a molecular marker for the risk of PD, here we examine the Miro1 response to mitochondrial depolarization by biochemical approaches in induced pluripotent stem cells from a cohort of at-risk individuals. Our results show that the Miro1 phenotype is significantly associated with PD risk. We propose that Miro1 is a promising molecular marker for detecting both PD and at-risk populations. Tracking this Miro1 marker could aid in diagnosis and Miro1-based drug discoveries.

    View details for DOI 10.3389/fnmol.2021.734273

    View details for PubMedID 34434090