Three-dimensional object geometry of mitochondria-associated signal: 3-D analysis pipeline for two-photon image stacks of cerebrovascular endothelial mitochondria.

Increasing evidence indicates the role of mitochondrial and vascular dysfunction in aging and aging-associated pathologies; however, the exact mechanisms and chronological processes remain enigmatic. High-energy demand organs, such as the brain, depend on the health of their mitochondria and vasculature for the maintenance of normal functions, therefore representing vulnerable targets for aging. This methodology article describes an analysis pipeline for three-dimensional (3-D) mitochondria-associated signal geometry of two-photon image stacks of brain vasculature. The analysis methods allow the quantification of mitochondria-associated signals obtained in real time in their physiological environment. In addition, signal geometry results will allow the extrapolation of fission and fusion events under normal conditions, during aging, or in the presence of different pathological conditions, therefore contributing to our understanding of the role mitochondria play in a variety of aging-associated diseases with vascular etiology.NEW & NOTEWORTHY Analysis pipeline for 3-D mitochondria-associated signal geometry of two-photon image stacks of brain vasculature.

Fibrinogen in mice cerebral microvessels induces blood-brain barrier dysregulation with aging via a dynamin-related protein 1-dependent pathway.

We previously reported evidence that oxidative stress during aging leads to adverse protein profile changes of brain cortical microvessels (MVs: end arterioles, capillaries, and venules) that affect mRNA/protein stability, basement membrane integrity, and ATP synthesis capacity in mice. As an extension of our previous study, we also found that proteins which comprise the blood-brain barrier (BBB) and regulate mitochondrial quality control were also significantly decreased in the mice's cortical MVs with aging. Interestingly, the neuroinflammatory protein fibrinogen (Fgn) was increased in mice brain MVs, which corresponds with clinical reports indicating that the plasma Fgn concentration increased progressively with aging. In this study, protein-protein interaction network analysis indicated that high expression of Fgn is linked with downregulated expression of both BBB- and mitochondrial fission/fusion-related proteins in mice cortical MVs with aging. To investigate the mechanism of Fgn action, we observed that 2 mg/mL or higher concentration of human plasma Fgn changed cell morphology, induced cytotoxicity, and increased BBB permeability in primary human brain microvascular endothelial cells (HBMECs). The BBB tight junction proteins were significantly decreased with increasing concentration of human plasma Fgn in primary HBMECs. Similarly, the expression of phosphorylated dynamin-related protein 1 (pDRP1) and other mitochondrial fission/fusion-related proteins were also significantly reduced in Fgn-treated HBMECs. Interestingly, DRP1 knockdown by shRNA(h) resulted in the reduction of both BBB- and mitochondrial fission/fusion-related proteins in HBMECs. Our results suggest that elevated Fgn downregulates DRP1, leading to mitochondrial-dependent endothelial and BBB dysfunction in the brain microvasculature.

Targeting mitochondria in the aged cerebral vasculature with SS-31, a proteomic study of brain microvessels.

Cognitive impairment and dementias during aging such as Alzheimer's disease are linked to functional decline and structural alterations of the brain microvasculature. Although mechanisms leading to microvascular changes during aging are not clear, loss of mitochondria, and reduced efficiency of remaining mitochondria appear to play a major role. Pharmacological agents, such as SS-31, which target mitochondria have been shown to be effective during aging and diseases; however, the benefit to mitochondrial- and non-mitochondrial proteins in the brain microvasculature has not been examined. We tested whether attenuation of aging-associated changes in the brain microvascular proteome via targeting mitochondria represents a therapeutic option for the aging brain. We used aged male (> 18 months) C57Bl6/J mice treated with a mitochondria-targeted tetrapeptide, SS-31, or vehicle saline. Cerebral blood flow (CBF) was determined using laser speckle imaging during a 2-week treatment period. Then, isolated cortical microvessels (MVs) composed of end arterioles, capillaries, and venules were used for Orbitrap Eclipse Tribrid mass spectrometry. CBF was similar among the groups, whereas bioinformatic analysis revealed substantial differences in protein abundance of cortical MVs between SS-31 and vehicle. We identified 6267 proteins, of which 12% were mitochondria-associated. Of this 12%, 107 were significantly differentially expressed and were associated with oxidative phosphorylation, metabolism, the antioxidant defense system, or mitochondrial dynamics. Administration of SS-31 affected many non-mitochondrial proteins. Our findings suggest that mitochondria in the microvasculature represent a therapeutic target in the aging brain, and widespread changes in the proteome may underlie the rejuvenating actions of SS-31 in aging.

Circulating Plasma Exosomal Proteins of Either SHIV-Infected Rhesus Macaque or HIV-Infected Patient Indicates a Link to Neuropathogenesis.

Despite the suppression of human immunodeficiency virus (HIV) replication by combined antiretroviral therapy (cART), 50-60% of HIV-infected patients suffer from HIV-associated neurocognitive disorders (HAND). Studies are uncovering the role of extracellular vesicles (EVs), especially exosomes, in the central nervous system (CNS) due to HIV infection. We investigated links among circulating plasma exosomal (crExo) proteins and neuropathogenesis in simian/human immunodeficiency virus (SHIV)-infected rhesus macaques (RM) and HIV-infected and cART treated patients (Patient-Exo). Isolated EVs from SHIV-infected (SHIV-Exo) and uninfected (CTL-Exo) RM were predominantly exosomes (particle size < 150 nm). Proteomic analysis quantified 5654 proteins, of which 236 proteins (~4%) were significantly, differentially expressed (DE) between SHIV-/CTL-Exo. Interestingly, different CNS cell specific markers were abundantly expressed in crExo. Proteins involved in latent viral reactivation, neuroinflammation, neuropathology-associated interactive as well as signaling molecules were expressed at significantly higher levels in SHIV-Exo than CTL-Exo. However, proteins involved in mitochondrial biogenesis, ATP production, autophagy, endocytosis, exocytosis, and cytoskeleton organization were significantly less expressed in SHIV-Exo than CTL-Exo. Interestingly, proteins involved in oxidative stress, mitochondrial biogenesis, ATP production, and autophagy were significantly downregulated in primary human brain microvascular endothelial cells exposed with HIV+/cART+ Patient-Exo. We showed that Patient-Exo significantly increased blood-brain barrier permeability, possibly due to loss of platelet endothelial cell adhesion molecule-1 protein and actin cytoskeleton structure. Our novel findings suggest that circulating exosomal proteins expressed CNS cell markers-possibly associated with viral reactivation and neuropathogenesis-that may elucidate the etiology of HAND.

Recombinant Human Perlecan DV and Its LG3 Subdomain Are Neuroprotective and Acutely Functionally Restorative in Severe Experimental Ischemic Stroke.

Despite recent therapeutic advancements, ischemic stroke remains a major cause of death and disability. It has been previously demonstrated that ~ 85-kDa recombinant human perlecan domain V (rhPDV) binds to upregulated integrin receptors (α2ß1 and α5ß1) associated with neuroprotective and functional improvements in various animal models of acute ischemic stroke. Recombinant human perlecan laminin-like globular domain 3 (rhPDVLG3), a 21-kDa C-terminal subdomain of rhPDV, has been demonstrated to more avidly bind to the α2ß1 integrin receptor than its parent molecule and consequently was postulated to evoke significant neuroprotective and functional effects. To test this hypothesis, fifty male C57Bl/6 J mice studied in a t-MCAO model were randomly allocated to either rhPDV treatment, rhPDVLG3, or equivalent volume of PBS at the time of reperfusion in a study where all procedures and analyses were conducted blind to treatment. On post-MCAO day 7, 2,3,5-triphenyltetrazolium chloride staining of brain slices was used to quantify infarct volume. We observed that treatment with rhPDVLG3 reduced infarct volume by 65.6% (p = 0.0001), improved weight loss (p < 0.05), and improved functional outcome measures (p < 0.05) when compared to PBS controls, improvements which were generally greater in magnitude than those observed for 2 mg/kg of rhPDV. In addition, treatment with 6 mg/kg of rhPDVLG3 was observed to significantly reduce mortality due to stroke in one model, an outcome not previously observed for rhPDV. Our initial findings suggest that treatment with rhPDVLG3 provides significant improvement in neuroprotective and functional outcomes in experimental stroke models and that further investigation of rhPDVLG3 as a novel neuroprotective therapy for patients with stroke is warranted.

Detrimental effects of transient cerebral ischemia on middle cerebral artery mitochondria in female rats.

Mitochondrial numbers and dynamics in brain blood vessels differ between young male and female rats under physiological conditions, but how these differences are affected by stroke is unclear. In males, we found that mitochondrial numbers, possibly due to mitochondrial fission, in large middle cerebral arteries (MCAs) increased following transient middle cerebral artery occlusion (tMCAO). However, mitochondrial effects of stroke on MCAs of female rats have not been studied. To address this disparity, we conducted morphological, biochemical, and functional studies using electron microscopy, Western blot, mitochondrial respiration, and Ca2+ sparks activity measurements in MCAs of female, naïve or sham Sprague-Dawley rats before and 48 h after 90 min of tMCAO. Adverse changes in mitochondrial characteristics and the relationship between mitochondria and sarcoplasmic reticulum (SR) in MCAs were present on both sides. However, mitochondria and mitochondrial/SR associations were often within the range of normal appearance. Mitochondrial protein levels were similar between ipsilateral (ipsi) and contralateral (contra) sides. Nonrespiratory oxygen consumption, maximal respiration, and spare respiratory capacity were similar between ipsi and contra but were reduced compared with sham. Basal respiration, proton leak, and ATP production were similar among MCAs. Ca2+ sparks activity increased in sham and ipsi MCAs exposed to a mitochondrial ATP-sensitive potassium channel opener: diazoxide. Our results show that tMCAO has effects on mitochondria in MCAs on both the ipsi and contra sides. Mitochondrial responses of cerebral arteries to tMCAO in females are substantially different from responses seen previously in male rats suggesting the need for specific sex-based therapies.NEW & NOTEWORTHY We propose that differences in mitochondrial characteristics of males and females, including mitochondrial morphology, respiration, and calcium sparks activity contribute to sex differences in protective and repair mechanisms in response to transient ischemia-reperfusion.

Neuropathology and virus in brain of SARS-CoV-2 infected non-human primates.

Neurological manifestations are a significant complication of coronavirus disease (COVID-19), but underlying mechanisms aren't well understood. The development of animal models that recapitulate the neuropathological findings of autopsied brain tissue from patients who died from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are critical for elucidating the neuropathogenesis of infection and disease. Here, we show neuroinflammation, microhemorrhages, brain hypoxia, and neuropathology that is consistent with hypoxic-ischemic injury in SARS-CoV-2 infected non-human primates (NHPs), including evidence of neuron degeneration and apoptosis. Importantly, this is seen among infected animals that do not develop severe respiratory disease, which may provide insight into neurological symptoms associated with "long COVID". Sparse virus is detected in brain endothelial cells but does not associate with the severity of central nervous system (CNS) injury. We anticipate our findings will advance our current understanding of the neuropathogenesis of SARS-CoV-2 infection and demonstrate SARS-CoV-2 infected NHPs are a highly relevant animal model for investigating COVID-19 neuropathogenesis among human subjects.

A Central Role for TRPM4 in Ca2+-Signal Amplification and Vasoconstriction.

Transient receptor potential melastatin-4 (TRPM4) is activated by an increase in intracellular Ca2+ concentration and is expressed on smooth muscle cells (SMCs). It is implicated in the myogenic constriction of cerebral arteries. We hypothesized that TRPM4 has a general role in intracellular Ca2+ signal amplification in a wide range of blood vessels. TRPM4 function was tested with the TRPM4 antagonist 9-phenanthrol and the TRPM4 activator A23187 on the cardiovascular responses of the rat, in vivo and in isolated basilar, mesenteric, and skeletal muscle arteries. TRPM4 inhibition by 9-phenanthrol resulted in hypotension and a decreased heart rate in the rat. TRPM4 inhibition completely antagonized myogenic tone development and norepinephrine-evoked vasoconstriction, and depolarization (high extracellular KCl concentration) evoked vasoconstriction in a wide range of peripheral arteries. Vasorelaxation caused by TRPM4 inhibition was accompanied by a significant decrease in intracellular Ca2+ concentration, suggesting an inhibition of Ca2+ signal amplification. Immunohistochemistry confirmed TRPM4 expression in the smooth muscle cells of the peripheral arteries. Finally, TRPM4 activation by the Ca2+ ionophore A23187 was competitively inhibited by 9-phenanthrol. In summary, TRPM4 was identified as an essential Ca2+-amplifying channel in peripheral arteries, contributing to both myogenic tone and agonist responses. These results suggest an important role for TRPM4 in the circulation. The modulation of TRPM4 activity may be a therapeutic target for hypertension. Furthermore, the Ca2+ ionophore A23187 was identified as the first high-affinity (nanomolar) direct activator of TRPM4, acting on the 9-phenanthrol binding site.

Effects of aging on protein expression in mice brain microvessels: ROS scavengers, mRNA/protein stability, glycolytic enzymes, mitochondrial complexes, and basement membrane components.

Differentially expressed (DE) proteins in the cortical microvessels (MVs) of young, middle-aged, and old male and female mice were evaluated using discovery-based proteomics analysis (> 4,200 quantified proteins/group). Most DE proteins (> 90%) showed no significant differences between the sexes; however, some significant DE proteins showing sexual differences in MVs decreased from young (8.3%), to middle-aged (3.7%), to old (0.5%) mice. Therefore, we combined male and female data for age-dependent comparisons but noted sex differences for examination. Key proteins involved in the oxidative stress response, mRNA or protein stability, basement membrane (BM) composition, aerobic glycolysis, and mitochondrial function were significantly altered with aging. Relative abundance of superoxide dismutase-1/-2, catalase and thioredoxin were reduced with aging. Proteins participating in either mRNA degradation or pre-mRNA splicing were significantly increased in old mice MVs, whereas protein stabilizing proteins decreased. Glycolytic proteins were not affected in middle age, but the relative abundance of these proteins decreased in MVs of old mice. Although most of the 41 examined proteins composing mitochondrial complexes I-V were reduced in old mice, six of these proteins showed a significant reduction in middle-aged mice, but the relative abundance increased in fourteen proteins. Nidogen, collagen, and laminin family members as well as perlecan showed differing patterns during aging, indicating BM reorganization starting in middle age. We suggest that increased oxidative stress during aging leads to adverse protein profile changes of brain cortical MVs that affect mRNA/protein stability, BM integrity, and ATP synthesis capacity.

Transcriptome analysis reveals sexual disparities in gene expression in rat brain microvessels.

Sex is an important determinant of brain microvessels (MVs) function and susceptibility to cerebrovascular and neurological diseases, but underlying mechanisms are unclear. Using high throughput RNA sequencing analysis, we examined differentially expressed (DE) genes in brain MVs from young, male, and female rats. Bioinformatics analysis of the 23,786 identified genes indicates that 298 (1.2%) genes were DE using False Discovery Rate criteria (FDR; p < 0.05), of which 119 (40%) and 179 (60%) genes were abundantly expressed in male and female MVs, respectively. Nucleic acid binding, enzyme modulator, and transcription factor were the top three DE genes, which were more highly expressed in male than female MVs. Synthesis of glycosylphosphatidylinositol (GPI), biosynthesis of GPI-anchored proteins, steroid and cholesterol synthesis, were the top three significantly enriched canonical pathways in male MVs. In contrast, respiratory chain, ribosome, and 3 ́-UTR-mediated translational regulation were the top three enriched canonical pathways in female MVs. Different gene functions of MVs were validated by proteomic analysis and western blotting. Our novel findings reveal major sex disparities in gene expression and canonical pathways of MVs and these differences provide a foundation to study the underlying mechanisms and consequences of sex-dependent differences in cerebrovascular and other neurological diseases.

Latent HIV-Exosomes Induce Mitochondrial Hyperfusion Due to Loss of Phosphorylated Dynamin-Related Protein 1 in Brain Endothelium.

Damage to the cerebral vascular endothelium is a critical initiating event in the development of HIV-1-associated neurocognitive disorders. To study the role of mitochondria in cerebral endothelial dysfunction, we investigated how exosomes, isolated from both cell lines with integrated provirus and HIV-1 infected primary cells (HIV-exosomes), accelerate the dysfunction of primary human brain microvascular endothelial cells (HBMVECs) by inducing mitochondrial hyperfusion, and reducing the expression of phosphorylated endothelial nitric oxide synthase (p-eNOS). The quantitative analysis of the extracellular vesicles (EVs) indicates that the isolated EVs were predominantly exosomes. It was further supported by the detection of exosomal markers, and the absence of large EV-related protein in the isolated EVs. The exosomes were readily taken up by primary HBMVECs. HIV-exosomes induce cellular and mitochondrial superoxide production but reduce mitochondrial membrane potential in HBMVECs. HIV-exosomes increase mitochondrial hyperfusion, possibly due to loss of phosphorylated dynamin-related protein 1 (p-DRP1). HIV-exosomes, containing the HIV-Tat protein, and viral Tat protein reduce the expression of p-DRP1 and p-eNOS, and accelerate brain endothelial dysfunction. Finally, exosomes isolated from HIV-1 infected primary human peripheral blood mononuclear cells (hPBMCs) produce more exosomes than uninfected controls and reduce both p-DRP1 and p-eNOS expressions in primary HBMVECs. Our novel findings reveal the significant role of HIV-exosomes on dysregulation of mitochondrial function, which induces adverse changes in the function of the brain microvascular endothelium.

Sexual differences in mitochondrial and related proteins in rat cerebral microvessels: A proteomic approach.

Sex differences in mitochondrial numbers and function are present in large cerebral arteries, but it is unclear whether these differences extend to the microcirculation. We performed an assessment of mitochondria-related proteins in cerebral microvessels (MVs) isolated from young, male and female, Sprague-Dawley rats. MVs composed of arterioles, capillaries, and venules were isolated from the cerebrum and used to perform a 3 versus 3 quantitative, multiplexed proteomics experiment utilizing tandem mass tags (TMT), coupled with liquid chromatography/mass spectrometry (LC/MS). MS data and bioinformatic analyses were performed using Proteome Discoverer version 2.2 and Ingenuity Pathway Analysis. We identified a total of 1969 proteins, of which 1871 were quantified by TMT labels. Sixty-four proteins were expressed significantly (p < 0.05) higher in female samples compared with male samples. Females expressed more mitochondrial proteins involved in energy production, mitochondrial membrane structure, anti-oxidant enzyme proteins, and those involved in fatty acid oxidation. Conversely, males had higher expression levels of mitochondria-destructive proteins. Our findings reveal, for the first time, the full extent of sexual dimorphism in the mitochondrial metabolic protein profiles of MVs, which may contribute to sex-dependent cerebrovascular and neurological pathologies.

Perlecan Domain-V Enhances Neurogenic Brain Repair After Stroke in Mice.

The extracellular matrix fragment perlecan domain V is neuroprotective and functionally restorative following experimental stroke. As neurogenesis is an important component of chronic post-stroke repair, and previous studies have implicated perlecan in developmental neurogenesis, we hypothesized that domain V could have a broad therapeutic window by enhancing neurogenesis after stroke. We demonstrated that domain V is chronically increased in the brains of human stroke patients, suggesting that it is present during post-stroke neurogenic periods. Furthermore, perlecan deficient mice had significantly less neuroblast precursor cells after experimental stroke. Seven-day delayed domain V administration enhanced neurogenesis and restored peri-infarct excitatory synaptic drive to neocortical layer 2/3 pyramidal neurons after experimental stroke. Domain V's effects were inhibited by blockade of α2ß1 integrin, suggesting the importance of α2ß1 integrin to neurogenesis and domain V neurogenic effects. Our results demonstrate that perlecan plays a previously unrecognized role in post-stroke neurogenesis and that delayed DV administration after experimental stroke enhances neurogenesis and improves recovery in an α2ß1 integrin-mediated fashion. We conclude that domain V is a clinically relevant neuroprotective and neuroreparative novel stroke therapy with a broad therapeutic window.

Chronic imaging of mitochondria in the murine cerebral vasculature using in vivo two-photon microscopy.

Mitochondria are important regulators of cerebral vascular function in health and disease, but progress in understanding their roles has been hindered by methodological limitations. We report the first in vivo imaging of mitochondria specific to the cerebral endothelium in real time in the same mouse for extended periods. Mice expressing Dendra2 fluorescent protein in mitochondria (mito-Dendra2) in the cerebral vascular endothelium were generated by breeding PhAM-floxed and Tie2-Cre mice. We used mito-Dendra2 expression, cranial window implantation, and two-photon microscopy to visualize mitochondria in the cerebral vascular endothelium of mice. Immunohistochemistry and mitochondrial staining were used to confirm the localization of the mitochondrial signal to endothelial cells and the specificity of mito-Dendra2 to mitochondria. Mito-Dendra2 and Rhodamine B-conjugated dextran allowed simultaneous determinations of mitochondrial density, vessel diameters, area, and mitochondria-to-vessel ratio in vivo, repeatedly, in the same mouse. Endothelial expression of mito-Dendra2 was confirmed in vitro on brain slices and aorta. In addition, we observed an overlapping mito-Dendra2 and Chromeo mitochondrial staining of cultured brain microvascular endothelial cells. Repeated imaging of the same location in the cerebral microcirculation in the same mouse demonstrated stability of mito-Dendra2. While the overall mitochondrial signal was stable over time, mitochondria within the same endothelial cell were mobile. In conclusion, our results indicate that the mito-Dendra2 signal and vascular parameters are suitable for real-time and longitudinal examination of mitochondria in vivo in the cerebral vasculature of mice.NEW & NOTEWORTHY We introduce an innovative in vivo approach to study mitochondria in the cerebral circulation in their physiological environment by demonstrating the feasibility of long-term imaging and three-dimensional reconstruction. We postulate that the appropriate combination of Cre/Lox system and two-photon microscopy will contribute to a better understanding of the role of mitochondria in not only endothelium but also the different cell types of the cerebral circulation.

Review of Alterations in Perlecan-Associated Vascular Risk Factors in Dementia.

Perlecan is a heparan sulfate proteoglycan protein in the extracellular matrix that structurally and biochemically supports the cerebrovasculature by dynamically responding to changes in cerebral blood flow. These changes in perlecan expression seem to be contradictory, ranging from neuroprotective and angiogenic to thrombotic and linked to lipid retention. This review investigates perlecan's influence on risk factors such as diabetes, hypertension, and amyloid that effect Vascular contributions to Cognitive Impairment and Dementia (VCID). VCID, a comorbidity with diverse etiology in sporadic Alzheimer's disease (AD), is thought to be a major factor that drives the overall clinical burden of dementia. Accordingly, changes in perlecan expression and distribution in response to VCID appears to be injury, risk factor, location, sex, age, and perlecan domain dependent. While great effort has been made to understand the role of perlecan in VCID, additional studies are needed to increase our understanding of perlecan's role in health and in cerebrovascular disease.

Effects of prolonged type 2 diabetes on mitochondrial function in cerebral blood vessels.

One of the major characteristics of hyperglycemic states such as type 2 diabetes is increased reactive oxygen species (ROS) generation. Since mitochondria are a major source of ROS, it is vital to understand the involvement of these organelles in the pathogenesis of ROS-mediated conditions. Therefore, we investigated mitochondrial function and ROS production in cerebral blood vessels of 21-wk-old Zucker diabetic fatty obese rats and their lean controls. We have previously shown that in the early stages of insulin resistance, and short periods of type 2 diabetes mellitus, only mild differences exist in mitochondrial function. In the present study, we examined mitochondrial respiration, mitochondrial protein expression, and ROS production in large-surface cerebral arteries. We used 21-wk-old animals exposed to peak glucose levels for 7 wk and compared them with our previous studies on younger diabetic animals. We found that the same segments of mitochondrial respiration (basal respiration and proton leak) were diminished in diabetic groups as they were in younger diabetic animals. Levels of rattin, a rat humanin analog, tended to decrease in the diabetic group but did not reach statistical significance (P = 0.08). Other mitochondrial proteins were unaffected, which might indicate the existence of compensatory mechanisms with extension of this relatively mild form of diabetes. Superoxide levels were significantly higher in large cerebral vessels of diabetic animals compared with the control group. In conclusion, prolonged dietary diabetes leads to stabilization, rather than deterioration, of metabolic status in the cerebral circulation, despite continued overproduction of ROS.NEW & NOTEWORTHY We have characterized for the first time the dynamics of mitochondrial function during the progression of type 2 diabetes mellitus with regard to mitochondrial respiration, protein expression, and reactive oxygen species production. In addition, this is the first measurement of rattin levels in the cerebral vasculature, which could potentially lead to novel treatment options.

Measuring Respiration in Isolated Murine Brain Mitochondria: Implications for Mechanistic Stroke Studies.

Measuring mitochondrial respiration in brain tissue is very critical in understanding the physiology and pathology of the central nervous system. Particularly, measurement of respiration in isolated mitochondria provides the advantage over the whole cells or tissues as the changes in respiratory function are intrinsic to mitochondrial structures rather than the cellular signaling that regulates mitochondria. Moreover, a high-throughput technique for measuring mitochondrial respiration minimizes the experimental time and the sample-to-sample variation. Here, we provide a detailed protocol for measuring respiration in isolated brain non-synaptosomal mitochondria using Agilent Seahorse XFe24 Analyzer. We optimized the protocol for the amount of mitochondria and concentrations of ADP, oligomycin, and trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP) for measuring respiratory parameters for complex I-mediated respiration. In addition, we measured complex II-mediated respiratory parameters. We observed that 10 µg of mitochondrial protein per well, ADP concentrations ranging between 2.5 and 10 mmol/L along with 5 µmol/L of oligomycin, and 5 µmol/L of FCCP are ideal for measuring the complex I-mediated respiration in isolated mouse brain mitochondria. Furthermore, we determined that 2.5 µg of mitochondrial protein per well is ideal for measuring complex II-mediated respiration. Notably, we provide a discussion of logical analysis of data and how the assay could be utilized to design mechanistic studies for experimental stroke. In conclusion, we provide detailed experimental design for measurement of various respiratory parameters in isolated brain mitochondria utilizing a novel high-throughput technique along with interpretation and analysis of data.

Cerebrovascular function and mitochondrial bioenergetics after ischemia-reperfusion in male rats.

The underlying factors promoting increased mitochondrial proteins, mtDNA, and dilation to mitochondrial-specific agents in male rats following tMCAO are not fully elucidated. Our goal was to determine the morphological and functional effects of ischemia/reperfusion (I/R) on mitochondria using electron microscopy, Western blot, mitochondrial oxygen consumption rate (OCR), and Ca2+ sparks activity measurements in middle cerebral arteries (MCAs) from male Sprague Dawley rats (Naïve, tMCAO, Sham). We found a greatly increased OCR in ipsilateral MCAs (IPSI) compared with contralateral (CONTRA), Sham, and Naïve MCAs. Consistent with our earlier findings, the expression of Mitofusin-2 and OPA-1 was significantly decreased in IPSI arteries compared with Sham and Naïve. Mitochondrial morphology was disrupted in vascular smooth muscle, but morphology with normal and perhaps greater numbers of mitochondria were observed in IPSI compared with CONTRA MCAs. Consistently, there were significantly fewer baseline Ca2+ events in IPSI MCAs compared with CONTRA, Sham, and Naïve. Mitochondrial depolarization significantly increased Ca2+ sparks activity in the IPSI, Sham, Naïve, but not in the CONTRA group. Our data indicate that altered mitochondrial structure and function occur in MCAs exposed to I/R and that these changes impact not only OCR but Ca2+ sparks activity in both IPSI and CONTRA MCAs.

Measurement of respiratory function in isolated cardiac mitochondria using Seahorse XFe24 Analyzer: applications for aging research.

Mitochondria play a critical role in the cardiomyocyte physiology by generating majority of the ATP required for the contraction/relaxation through oxidative phosphorylation (OXPHOS). Aging is a major risk factor for cardiovascular diseases (CVD) and mitochondrial dysfunction has been proposed as potential cause of aging. Recent technological innovations in Seahorse XFe24 Analyzer enhanced the detection sensitivity of oxygen consumption rate and proton flux to advance our ability study mitochondrial function. Studies of the respiratory function tests in the isolated mitochondria have the advantages to detect specific defects in the mitochondrial protein function and evaluate the direct mitochondrial effects of therapeutic/pharmacological agents. Here, we provide the protocols for studying the respiratory function of isolated murine cardiac mitochondria by measuring oxygen consumption rate using Seahorse XFe24 Analyzer. In addition, we provide details about experimental design, measurement of various respiratory parameters along with interpretation and analysis of data.