Browsing by Subject "Mitochondria"
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Item A Method for Real-Time Assessment of Mitochondrial Respiration Using Murine Corneal Biopsy(Association for Research in Vision and Ophthalmology, 2023-08-29) Liang, Wentao; Huang, Li; Yuan, Tian; Cheng, Rui; Takahashi, Yusuke; Moiseyev, Gennadiy P.; Karamichos, Dimitrios; Ma, Jian-XingPURPOSE: To develop and optimize a method to monitor real-time mitochondrial function by measuring the oxygen consumption rate (OCR) in murine corneal biopsy punches with a Seahorse extracellular flux analyzer. METHODS: Murine corneal biopsies were obtained using a biopsy punch immediately after euthanasia. The corneal metabolic profile was assessed using a Seahorse XFe96 pro analyzer, and mitochondrial respiration was analyzed with specific settings. RESULTS: Real-time adenosine triphosphate rate assay showed that mitochondrial oxidative phosphorylation is a major source of adenosine triphosphate production in ex vivo live murine corneal biopsies. Euthanasia methods (carbon dioxide asphyxiation vs. overdosing on anesthetic drugs) did not affect corneal OCR values. Mouse corneal biopsy punches in 1.5-mm diameter generated higher and more reproducible OCR values than those in 1.0-mm diameter. The biopsy punches from the central and off-central cornea did not show significant differences in OCR values. There was no difference in OCR reading by the tissue orientations (the epithelium side up vs. the endothelium side up). No significant differences were found in corneal OCR levels between sexes, strains (C57BL/6J vs. BALB/cJ), or ages (4, 8, and 32 weeks). Using this method, we showed that the wound healing process in the mouse cornea affected mitochondrial activity. CONCLUSIONS: The present study validated a new strategy to measure real-time mitochondrial function in fresh mouse corneal tissues. This procedure should be helpful for studies of the ex vivo live corneal metabolism in response to genetic manipulations, disease conditions, or pharmacological treatments in mouse models.Item TAARgeting Astrogliosis and Mitochondrial Dysfunction during METH Exposure and HIV-relevant Neuroinflammation(2017-12-01) Borgmann, Kathleen; Ghorpade, Anuja; Wordinger, Robert J.; Berg, Rance E.As a popular psychostimulant, methamphetamine (METH) use leads to long-lasting, strong euphoric effects. METH exacerbates the severity and onset of HIV-associated neurocognitive disorders (HAND), which affect 30-70% of the 37.6 million people globally infected with HIV. Most neurodegenerative diseases share neuroinflammation as a common pathogenic mechanism. Neuroinflammation, HIV and METH dysregulate a wide range of brain functions including neuronal signaling, glial activation, viral infection, oxidative stress and excitotoxicity. Since neuroglia determine the outcome of neurological disease, we investigate the mechanisms regulating astrocyte-mediated neurotoxicity in the context of METH and HIV comorbidity. To these ends, we examined the expression, localization and function of the novel METH astrocyte receptor, trace amine associated receptor 1 (TAAR1) in an extended METH in vitro model, which mimics chronic residual METH concentrations between binges, and HIV-associated activation. In our model, TAAR1 levels and localization to the endoplasmic reticulum and plasma membranes increased with METH and HIV-induced astrogliosis. Extended physiological METH exposure led to augmented calcium flux, a mechanism known to mediate ER and mitochondrial, and oxidative stress. METH induced dysregulation of astrocyte mitochondrial morphology by elevating mitofusin expression and inhibitory phosphorylation of dynamin-related protein-1. While METH decreased oxygen consumption and ATP levels during acute exposure, chronic treatment significantly enhanced both. Together, these changes increased expression of antioxidant proteins, augmenting the astrocyte’s oxidative capacity, but also oxidative damage. METH and HIV activation impaired excitatory amino acid transporter 2 (EAAT2) expression and activity, which were recovered by inhibition of TAAR1 with EPPTB, a TAAR1 selective antagonist. Together, these data highlight several mechanisms regulating METH/HIV-induced, astroglia-mediated neurotoxicity and the potential for astrocyte targeted intervention via TAAR1 during chronic disease. We propose that equilibrium between agonism of neuronal TAAR1 and antagonism of astrocyte TAAR1 will need to be further investigated to balance the neuroprotective benefits of TAAR1 targeting drugs in the CNS during HIV and METH comorbidity.Item Transcriptomic Signatures of Cognitive Impairment(2020-08) Silzer, Talisa K.; Phillips, Nicole R.; Barber, Robert C.; Planz, John V.; Nejtek, Vicki; Rickards, Caroline A.The dramatic shift in population demographics and rapid growth of the aging population has resulted in a drastic increase in the prevalence of complex age-related diseases. Of the most common age-related complex diseases, Alzheimer's disease (AD) remains the primary cause of dementia. The increased prevalence of this disease presents a major burden on our healthcare system and caregivers. Among the many pathophysiological hallmarks, there is a substantial amount of literature implicating mitochondrial dysfunction as an important signature of early AD pathophysiology. Use of 'omics' technologies and system-based approaches have become increasingly more common for studying AD. Though each data type (i.e. genomics, transcriptomics and proteomics) has advantages and disadvantages, transcriptomics serves as a cost-efficient method for obtaining a snapshot of functional molecular changes underlying the disease. Here we detail two distinct and innovative studies of the transcriptome using different analytical strategies to investigate the underlying molecular signatures of cognitive impairment in its varying forms. We report the first study of differential gene expression in mild cognitively impaired Mexican Americans. This study revealed 30 differentially expressed transcripts that were enriched for a number of biological processes previously implicated in AD pathophysiology. The second study is also the first of its kind (in the context of this phenotype) examining posttranscriptional methylation at functionally important sites within the mitochondrial transcriptome of individuals diagnosed with AD, progressive supranuclear palsy (PSP) or pathological aging(PA). We observed similar hypermethylation at these key sites in individuals diagnosed with tauopathies such as AD or PSP. Several nuclear-encoded genes were identified as associated and the expression of 5300 nuclear-encoded transcripts was correlated with this hypermethylation. These correlated transcripts were enriched for a number of biological and molecular processes. Though these studies are quite distinct in methodology, we observed overlap in the enriched processes identified in each respective study (i.e. mitochondrial dysfunction, chromatin binding/remodeling, protein degradation pathways, autophagy); many of these processes have been previously implicated in AD. Replication of these findings in larger cohorts and different racial/ethnic populations will be vital for gaining a more complete understanding of the molecular dysfunction that underlies cognitive impairment phenotypes.