Non-synaptic Mitochondria Research Articles

Effects of Chronic Hypertension on the Energy Metabolism of Cerebral Cortex Mitochondria in Normotensive and in Spontaneously Hypertensive Rats During Aging.

In this study the subcellular modifications undergone by cerebral cortex mitochondrial metabolism in chronic hypertension during aging were evaluated. The catalytic properties of regulatory energy-linked enzymes of Tricarboxylic Acid Cycle (TCA), Electron Transport Chain (ETC) and glutamate metabolism were assayed on non-synaptic mitochondria (FM, located in post-synaptic compartment) and on intra-synaptic mitochondria of pre-synaptic compartment, furtherly divided in "light" (LM) and "heavy" (HM) mitochondria, purified form cerebral cortex of normotensive Wistar Kyoto Rats (WKY) versus Spontaneously Hypertensive Rats (SHR) at 6, 12 and 18months. During physiological aging, the metabolic machinery was differently expressed in pre- and post-synaptic compartments: LM and above all HM were more affected by aging, displaying lower ETC activities. In SHR at 6months, FM and LM showed an uncoupling between TCA and ETC, likely as initial adaptive response to hypertension. During pathological aging, HM were particularly affected at 12months in SHR, as if the adaptive modifications in FM and LM at 6months granted a mitochondrial functional balance, while at 18months all the neuronal mitochondria displayed decreased metabolic fluxes versus WKY. This study describes the effects of chronic hypertension on cerebral mitochondrial energy metabolism during aging through functional proteomics of enzymes at subcellular levels, i.e. in neuronal soma and synapses. In addition, this represents the starting point to envisage an experimental physiopathological model which could be useful also for pharmacological studies, to assess drug actions during the development of age-related pathologies that could coexist and/or are provoked by chronic hypertension.

Local mitochondrial replication in the periphery of neurons requires the eEF1A1 protein and the translation of nuclear-encoded proteins

In neurons, it is commonly assumed that mitochondrial replication only occurs in the cell body, after which the mitochondria must travel to the neuron's periphery. However, while mitochondrial DNA replication has been observed to occur away from the cell body, the specific mechanisms involved remain elusive. Using EdU-labelling in mouse primary neurons, we developed a tool to determine the mitochondrial replication rate. Taking of advantage of microfluidic devices, we confirmed that mitochondrial replication also occurs locally in the periphery of neurons. To achieve this, mitochondria require de novo nuclear-encoded, but not mitochondrial-encoded protein translation. Following a proteomic screen comparing synaptic with non-synaptic mitochondria, we identified two elongation factors- eEF1A1 and TUFM- that were upregulated in synaptic mitochondria. We found that mitochondrial replication is impaired upon the downregulation of eEF1A1, and this is particularly relevant in the periphery of neurons.

Neuroanatomical zones of human traumatic brain injury reveal significant differences in protein profile and protein oxidation: Implications for secondary injury events.

Traumatic brain injury (TBI) causes significant neurological deficits and long-term degenerative changes. Primary injury in TBI entails distinct neuroanatomical zones, i.e., contusion (Ct) and pericontusion (PC). Their dynamic expansion could contribute to unpredictable neurological deterioration in patients. Molecular characterization of these zones compared with away from contusion (AC) zone is invaluable for TBI management. Using proteomics-based approach, we were able to distinguish Ct, PC and AC zones in human TBI brains. Ct was associated with structural changes (blood-brain barrier (BBB) disruption, neuroinflammation, axonal injury, demyelination and ferroptosis), while PC was associated with initial events of secondary injury (glutamate excitotoxicity, glial activation, accumulation of cytoskeleton proteins, oxidative stress, endocytosis) and AC displayed mitochondrial dysfunction that could contribute to secondary injury events and trigger long-term degenerative changes. Phosphoproteome analysis in these zones revealed that certain differentially phosphorylated proteins synergistically contribute to the injury events along with the differentially expressed proteins. Non-synaptic mitochondria (ns-mito) was associated with relatively more differentially expressed proteins (DEPs) compared to synaptosomes (Syn), while the latter displayed increased protein oxidation including tryptophan (Trp) oxidation. Proteomic analysis of immunocaptured complex I (CI) from Syn revealed increased Trp oxidation in Ct > PC > AC (vs. control). Oxidized W272 in the ND1 subunit of CI, revealed local conformational changes in ND1 and the neighboring subunits, as indicated by molecular dynamics simulation (MDS). Taken together, neuroanatomical zones in TBI show distinct protein profile and protein oxidation representing different primary and secondary injury events with potential implications for TBI pathology and neurological status of the patients.

Characterizing Sex Differences in Mitochondrial Dysfunction After Severe Traumatic Brain Injury in Mice.

Traumatic brain injury (TBI) is caused by an impact or penetrating injury to the head resulting in abnormal brain function. Mitochondrial dysfunction is an important hallmark of TBI and has been thoroughly studied in male rodent models of brain injury, but relatively little is known about these outcomes in females. These studies were designed to examine sex as a biological variable for mitochondria-related outcomes after the severe controlled cortical impact (CCI) mouse model of TBI. Synaptic and non-synaptic mitochondria were isolated from the sham- or CCI-injured cortex as well as the hippocampus ipsilateral to the craniotomy 3, 12, 24, or 48 h post-surgery, and then bioenergetics were measured. Subtle variations were observed in the timeline of mitochondrial dysfunction between sexes. Non-synaptic cortical mitochondria from injured females showed early impairment at 12 h post-CCI compared to mitochondria from injured males at 24 h post-CCI. Contrastingly, in the synaptic fraction, mitochondria from injured males showed early impairment at 12 h post-CCI, whereas mitochondria from injured females showed impairment at 24 h post-CCI. Based on bioenergetic impairments at 24 h post-CCI, synaptic and non-synaptic mitochondrial calcium loading was also measured at this time point. Consistent with bioenergetic data at 24 h, non-synaptic mitochondria from injured males had increased calcium loading compared to uninjured control, but this effect was not observed in females. Finally, histological assessment of cortical tissue sparing in each sex was measured at 7 days post-injury. There was a lack of sex-based differences in cortical tissue sparing after severe CCI. Overall, there were some subtle sex differences in mitochondrial outcomes after CCI, but these findings were not statistically significant. This study highlights the importance of utilizing both sexes when measuring mitochondrial function after severe CCI.

Chronic Fluoxetine Treatment of Socially Isolated Rats Modulates Prefrontal Cortex Proteome

Fluoxetine (Flx) is the most commonly used antidepressant to treat major depressive disorder. However, its molecular mechanisms of action are not defined as yet. A comparative proteomic approach was used to identify proteome changes in the prefrontal cortex (PFC) cytosolic and non-synaptic mitochondria (NSM)-enriched fractions of adult male Wistar rats following chronic social isolation (CSIS), a rat model of depression, and Flx treatment in CSIS and control rats, using liquid chromatography online tandem mass spectrometry. Flx reversed CSIS-induced depressive - like behavior according to preference for sucrose and immobility in the forced swim test, indicating its antidepressant effect. Flx treatment in controls led to an increase of the expression of cytosolic proteins involved in the microtubule cytoskeleton and intracellular calcium homeostasis and of enzymes involved in bioenergetic and transmembrane transport in NSM. CSIS downregulated the cytosolic proteins involved in proteasome pathway, and glutathione antioxidative system, and upregulated the expression of enzymes participating in mitochondrial-energy metabolism and transport. The presence of cytochrome c in the cytosol may suggest compromised mitochondrial membrane integrity. Flx treatment in CSIS rats downregulated protein involved in oxidative phosphorylation, such as complex III and manganese superoxide dismutase, and upregulated vesicle-mediated transport and synaptic signaling proteins in the cytosol, and neuronal calcium-binding protein 1 in NSM. Our study identified PFC modulated proteins and affected biochemical pathways that may represent potential markers/targets underlying CSIS-induced depression and effective Flx treatment, and highlights the role of protein systems involved in NSM and various metabolic pathways potentially involved in neuronal plasticity.

Progressive Mitochondrial Dysfunction of Striatal Synapses in R6/2 Mouse Model of Huntington's Disease.

Huntington's disease (HD) is a neurodegenerative disorder characterized by synaptic dysfunction and loss of white matter volume especially in the striatum of the basal ganglia and to a lesser extent in the cerebral cortex. Studies investigating heterogeneity between synaptic and non-synaptic mitochondria have revealed a pronounced vulnerability of synaptic mitochondria, which may lead to synaptic dysfunction and loss. As mitochondrial dysfunction is a hallmark of HD pathogenesis, we investigated synaptic mitochondrial function from striatum and cortex of the transgenic R6/2 mouse model of HD. We assessed mitochondrial volume, ROS production, and antioxidant levels as well as mitochondrial respiration at different pathological stages. Our results reveal that striatal synaptic mitochondria are more severely affected by HD pathology than those of the cortex. Striatal synaptosomes of R6/2 mice displayed a reduction in mitochondrial mass coinciding with increased ROS production and antioxidants levels indicating prolonged oxidative stress. Furthermore, synaptosomal oxygen consumption rates were significantly increased during depolarizing conditions, which was accompanied by a marked increase in mitochondrial proton leak of the striatal synaptosomes, indicating synaptic mitochondrial stress. Overall, our study provides new insight into the gradual changes of synaptic mitochondrial function in HD and suggests compensatory mitochondrial actions to maintain energy production in the HD brain, thereby supporting that mitochondrial dysfunction do indeed play a central role in early disease progression of HD.

Temporal Profiling of the Cortical Synaptic Mitochondrial Proteome Identifies Ageing Associated Regulators of Stability.

Synapses are particularly susceptible to the effects of advancing age, and mitochondria have long been implicated as organelles contributing to this compartmental vulnerability. Despite this, the mitochondrial molecular cascades promoting age-dependent synaptic demise remain to be elucidated. Here, we sought to examine how the synaptic mitochondrial proteome (including strongly mitochondrial associated proteins) was dynamically and temporally regulated throughout ageing to determine whether alterations in the expression of individual candidates can influence synaptic stability/morphology. Proteomic profiling of wild-type mouse cortical synaptic and non-synaptic mitochondria across the lifespan revealed significant age-dependent heterogeneity between mitochondrial subpopulations, with aged organelles exhibiting unique protein expression profiles. Recapitulation of aged synaptic mitochondrial protein expression at the Drosophila neuromuscular junction has the propensity to perturb the synaptic architecture, demonstrating that temporal regulation of the mitochondrial proteome may directly modulate the stability of the synapse in vivo.

Alcohol hangover induces nitric oxide metabolism changes by impairing NMDA receptor-PSD95-nNOS pathway

Alcohol hangover is defined as the combination of mental and physical symptoms experienced the day after a single episode of heavy drinking, starting when blood alcohol concentration approaches zero. We previously evidenced increments in free radical generation and an imbalance in antioxidant defences in non-synaptic mitochondria and synaptosomes during hangover. It is widely known that acute alcohol exposure induces changes in nitric oxide (NO) production and blocks the binding of glutamate to NMDAR in central nervous system. Our aim was to evaluate the residual effect of acute ethanol exposure (hangover) on NO metabolism and the role of NMDA receptor-PSD95-nNOS pathway in non-synaptic mitochondria and synaptosomes from mouse brain cortex. Results obtained for the synaptosomes fraction showed a 37% decrease in NO total content, a 36% decrease in NOS activity and a 19% decrease in nNOS protein expression. The in vitro addition of glutamate to synaptosomes produced a concentration-dependent enhancement of NO production which was significantly lower in samples from hangover mice than in controls for all the glutamate concentrations tested. A similar patter of response was observed for nNOS activity being decreased both in basal conditions and after glutamate addition. In addition, synaptosomes exhibited a 64% and 15% reduction in NMDA receptor subunit GluN2B and PSD-95 protein expression, respectively. Together with this, glutamate-induced calcium entry was significant decreased in synaptosomes from alcohol-treated mice. On the other hand, in non-synaptic mitochondria, no significant differences were observed in NO content, NOS activity or nNOS protein expression. The expression of iNOS remained unaltered in synaptosomes and non-synaptic mitochondria. Here we demonstrated that hangover effects on NO metabolism are strongly evidenced in synaptosomes probably due to a disruption in NMDAR/PSD-95/nNOS pathway.

Tianeptine Enhances Energy-related Processes in the Hippocampal Non-synaptic Mitochondria in a Rat Model of Depression

Tianeptine (Tian) has been widely used in treating mood and anxiety disorders, and recently as a nootropic to improve cognitive performance. However, its mechanisms of action are insufficiently clear. We used a comparative proteomic approach to identify sub-proteome changes in hippocampal cytosol and non-synaptic mitochondria (NSM) following chronic Tian treatment (3 weeks, 10 mg/kg/day) of adult male Wistar rats and rats exposed to chronic social isolation stress (CSIS) (6 weeks), an animal model of depression. Behavioural assessment of depressive and anxiety-like behaviours was based on sucrose preference, forced swim test and marble burying. Selected differently expressed proteins were validated by Western blot and/or immunohistochemical analysis. Tian normalized the behavioural alternations induced by CSIS, indicating its antidepressant and anxiolytic efficacy. Proteomic data showed that Tian increased the expression of proteasome system elements and redox system enzymes, enhanced energy metabolism and increased glyceraldehyde-3-phosphate dehydrogenase expression bound to NSM in control rats. Tian-treatment of CSIS-stressed rats resulted in a minor suppression of the increase in proteasome elements and antioxidative enzymes, except for an increase in Cu-Zn superoxide dismutase, and increased the level of Lactate dehydrogenase. Our results indicate on an increased NSM functionality in controls and suppression of the CSIS-induced impairment of NSM functionality by Tian treatment as well as on the CSIS-caused discrepancy in Tian effects relative to controls.

Social isolation stress-resilient rats reveal energy shift from glycolysis to oxidative phosphorylation in hippocampal nonsynaptic mitochondria

AimsTo examine the differences in the hippocampal proteome profiles of resilience or susceptibility to chronic social isolation (CSIS), animal model of depression, and to identify biomarkers that can distinguish the two. Main methodsComparative subproteomic approach was used to identify changes in hippocampal cytosol and nonsynaptic mitochondria (NSM) of CSIS-resilient compared to CSIS-sensitive or control rats. The resilient and sensitive phenotypes of CSIS rats were distinguished based on their sucrose preference values. Selected proteins were validated by Western blot or immunofluorescence. Key findingsPredominantly down-regulated processes such as cytosolic cytoskeleton organization, the calcium signaling pathway, ubiquitin proteasome degradation, redox system, malate/aspartate shuttling and glutamate metabolism in CSIS-resilient compared to CSIS-sensitive rats were found. Decreased protein expression of glycolytic enzymes with simultaneous increased expression of Aco2 involved in tricarboxylic acid cycle and expression of several subunits composing oxidative phosphorylation involved enzymes (Uqcrc2, Atp5f1a, Atp5f1b) were found, indicating shift in energy production from glycolysis to oxidative phosphorylation in NSM. The four-fold higher level of mitochondrial glyceraldehyde-3-phosphate dehydrogenase of resilient rats indicated its transfer from the cytosol to the NSM. An increased level of transketolase along with the reduced pyruvate kinase level suggested an activated pentose phosphate pathway in CSIS-resilient relative to control rats. Cytosolic up-regulated CSIS proteins were implicated in antioxidative and proteasomal systems, while down-regulated NSM protein was involved in oxidative phosphorylation. SignificanceThe identified altered activated pathways and potential biomarkers enhance understanding of molecular mechanisms underlying resilience or susceptibility to CSIS, crucial in developing new therapeutic strategies.

Premature synaptic mitochondrial dysfunction in the hippocampus during aging contributes to memory loss

Aging is a process characterized by cognitive impairment and mitochondrial dysfunction. In neurons, these organelles are classified as synaptic and non-synaptic mitochondria depending on their localization. Interestingly, synaptic mitochondria from the cerebral cortex accumulate more damage and are more sensitive to swelling than non-synaptic mitochondria. The hippocampus is fundamental for learning and memory, synaptic processes with high energy demand. However, it is unknown if functional differences are found in synaptic and non-synaptic hippocampal mitochondria; and whether this could contribute to memory loss during aging. In this study, we used 3, 6, 12 and 18 month-old (mo) mice to evaluate hippocampal memory and the function of both synaptic and non-synaptic mitochondria. Our results indicate that recognition memory is impaired from 12mo, whereas spatial memory is impaired at 18mo. This was accompanied by a differential function of synaptic and non-synaptic mitochondria. Interestingly, we observed premature dysfunction of synaptic mitochondria at 12mo, indicated by increased ROS generation, reduced ATP production and higher sensitivity to calcium overload, an effect that is not observed in non-synaptic mitochondria. In addition, at 18mo both mitochondrial populations showed bioenergetic defects, but synaptic mitochondria were prone to swelling than non-synaptic mitochondria. Finally, we treated 2, 11, and 17mo mice with MitoQ or Curcumin (Cc) for 5 weeks, to determine if the prevention of synaptic mitochondrial dysfunction could attenuate memory loss. Our results indicate that reducing synaptic mitochondrial dysfunction is sufficient to decrease age-associated cognitive impairment. In conclusion, our results indicate that age-related alterations in ATP produced by synaptic mitochondria are correlated with decreases in spatial and object recognition memory and propose that the maintenance of functional synaptic mitochondria is critical to prevent memory loss during aging.

Fractionated mitochondrial magnetic separation for isolation of synaptic mitochondria from brain tissue

While mitochondria maintain essential cellular functions, such as energy production, calcium homeostasis, and regulating programmed cellular death, they also play a major role in pathophysiology of many neurological disorders. Furthermore, several neurodegenerative diseases are closely linked with synaptic damage and synaptic mitochondrial dysfunction. Unfortunately, the ability to assess mitochondrial dysfunction and the efficacy of mitochondrial-targeted therapies in experimental models of neurodegenerative disease and CNS injury is limited by current mitochondrial isolation techniques. Density gradient ultracentrifugation (UC) is currently the only technique that can separate synaptic and non-synaptic mitochondrial sub-populations, though small brain regions cannot be assayed due to low mitochondrial yield. To address this limitation, we used fractionated mitochondrial magnetic separation (FMMS), employing magnetic anti-Tom22 antibodies, to develop a novel strategy for isolation of functional synaptic and non-synaptic mitochondria from mouse cortex and hippocampus without the usage of UC. We compared the yield and functionality of mitochondria derived using FMMS to those derived by UC. FMMS produced 3x more synaptic mitochondrial protein yield compared to UC from the same amount of tissue, a mouse hippocampus. FMMS also has increased sensitivity, compared to UC separation, to measure decreased mitochondrial respiration, demonstrated in a paradigm of mild closed head injury. Taken together, FMMS enables improved brain-derived mitochondrial yield for mitochondrial assessments and better detection of mitochondrial impairment in CNS injury and neurodegenerative disease.

Functional Differences between Synaptic Mitochondria from the Striatum and the Cerebral Cortex

Mitochondrial dysfunction has been shown to play a major role in neurodegenerative disorders such as Huntington's disease, Alzheimer's disease and Parkinson's disease. In these and other neurodegenerative disorders, disruption of synaptic connectivity and impaired neuronal signaling are among the early signs. When looking for potential causes of neurodegeneration, specific attention is drawn to the function of synaptic mitochondria, as the energy supply from mitochondria is crucial for normal synaptic function. Mitochondrial heterogeneity between synaptic and non-synaptic mitochondria has been described, but very little is known about possible differences between synaptic mitochondria from different brain regions. The striatum and the cerebral cortex are often affected in neurodegenerative disorders. In this study we therefore used isolated nerve terminals (synaptosomes) from female mice, striatum and cerebral cortex, to investigate differences in synaptic mitochondrial function between these two brain regions. We analyzed mitochondrial mass, citrate synthase activity, general metabolic activity and mitochondrial respiration in resting as well as veratridine-activated synaptosomes using glucose and/or pyruvate as substrate. We found higher mitochondrial oxygen consumption rate in both resting and activated cortical synaptosomes compared to striatal synaptosomes, especially when using pyruvate as a substrate. The higher oxygen consumption rate was not caused by differences in mitochondrial content, but instead corresponded with a higher proton leak in the cortical synaptic mitochondria compared to the striatal synaptic mitochondria. Our results show that the synaptic mitochondria of the striatum and cortex differently regulate respiration both in response to activation and variations in substrate conditions.

Changes in motor function and brain cortex mitochondrial active oxygen species production in aged mice.

Neuronal ageing is a complex physiological process, associated to metabolic and motor changes. In this study, 3 and 17 months old male Swiss mice were used. Aged mice exhibited a significant reduction in motor performance and walking footprint pattern. Synaptosomes and mitochondrial fractions were isolated from mouse brain cortex. Active oxygen species and cardiolipin content were measured in both subcellular fractions. Synaptosomal acetylcholinesterase activity was measured in both animal age groups. Results showed that superoxide levels were 42.9% lower in synaptosomes from old mice as compared with young animals, while no changes were observed in non-synaptic mitochondria. Succinate-glutamate dependent H2O2 production rate was 27.5% decreased in non-synaptic mitochondria from aged mice. Cardiolipin content was 21% decreased in synaptosomes from 17-months old animals, while no changes were observed in non-synaptic mitochondria. Acetylcholinesterase activity decreased 16% in 17-months old mice, as compared with young animals. Age-related alterations in neuronal function could be associated with changes in active oxygen species at synapses, with parallel motor deficiencies.

Synaptic Mitochondria are More Susceptible to Traumatic Brain Injury-induced Oxidative Damage and Respiratory Dysfunction than Non-synaptic Mitochondria

Traumatic brain injury (TBI) results in mitochondrial dysfunction and induction of lipid peroxidation (LP). Lipid peroxidation-derived neurotoxic aldehydes such as 4-HNE and acrolein bind to mitochondrial proteins, inducing additional oxidative damage and further exacerbating mitochondrial dysfunction and LP. Mitochondria are heterogeneous, consisting of both synaptic and non-synaptic populations. Synaptic mitochondria are reported to be more vulnerable to injury; however, this is the first study to characterize the temporal profile of synaptic and non-synaptic mitochondria following TBI, including investigation of respiratory dysfunction and oxidative damage to mitochondrial proteins between 3 and 120 h following injury. These results indicate that synaptic mitochondria are indeed the more vulnerable population, showing both more rapid and severe impairments than non-synaptic mitochondria. By 24 h, synaptic respiration is significantly impaired compared to synaptic sham, whereas non-synaptic respiration does not decline significantly until 48 h. Decreases in respiration are associated with increases in oxidative damage to synaptic and non-synaptic mitochondrial proteins at 48 h and 72 h, respectively. These results indicate that the therapeutic window for mitochondria-targeted pharmacological neuroprotectants to prevent respiratory dysfunction is shorter for the more vulnerable synaptic mitochondria than for the non-synaptic population.

Free radical production and antioxidant status in brain cortex non-synaptic mitochondria and synaptosomes at alcohol hangover onset

Alcohol hangover (AH) is the pathophysiological state after a binge-like drinking. We have previously demonstrated that AH induced bioenergetics impairments in a total fresh mitochondrial fraction in brain cortex and cerebellum. The aim of this work was to determine free radical production and antioxidant systems in non-synaptic mitochondria and synaptosomes in control and hangover animals. Superoxide production was not modified in non-synaptic mitochondria while a 17.5% increase was observed in synaptosomes. A similar response was observed for cardiolipin content as no changes were evidenced in non-synaptic mitochondria while a 55% decrease in cardiolipin content was found in synaptosomes. Hydrogen peroxide production was 3-fold increased in non-synaptic mitochondria and 4-fold increased in synaptosomes. In the presence of deprenyl, synaptosomal H2O2 production was 67% decreased in the AH condition. Hydrogen peroxide generation was not affected by deprenyl addition in non-synaptic mitochondria from AH mice. MAO activity was 57% increased in non-synaptic mitochondria and 3-fold increased in synaptosomes. Catalase activity was 40% and 50% decreased in non-synaptic mitochondria and synaptosomes, respectively. Superoxide dismutase was 60% decreased in non-synaptic mitochondria and 80% increased in synaptosomal fractions. On the other hand, GSH (glutathione) content was 43% and 17% decreased in synaptosomes and cytosol. GSH-related enzymes were mostly affected in synaptosomes fractions by AH condition. Acetylcholinesterase activity in synaptosomes was 11% increased due to AH. The present work reveals that AH provokes an imbalance in the cellular redox homeostasis mainly affecting mitochondria present in synaptic terminals.

Mitochondrial energy metabolism of rat hippocampus after treatment with the antidepressants desipramine and fluoxetine

Alterations in mitochondrial functions have been hypothesized to participate in the pathogenesis of depression, because brain bioenergetic abnormalities have been detected in depressed patients by neuroimaging in vivo studies. However, this hypothesis is not clearly demonstrated in experimental studies: some suggest that antidepressants are inhibitors of mitochondrial metabolism, while others observe the opposite.In this study, the effects of 21-day treatment with desipramine (15 mg/kg) and fluoxetine (10 mg/kg) were examined on the energy metabolism of rat hippocampus, evaluating the catalytic activity of regulatory enzymes of mitochondrial energy-yielding metabolic pathways. Because of the micro-heterogeneity of brain mitochondria, we have distinguished between (a) non-synaptic mitochondria (FM) of neuronal perikaryon (post-synaptic compartment) and (b) intra-synaptic light (LM) and heavy (HM) mitochondria (pre-synaptic compartment).Desipramine and fluoxetine changed the catalytic activity of specific enzymes in the different types of mitochondria: (a) in FM, both drugs enhanced cytochrome oxidase and glutamate dehydrogenase, (b) in LM, the overall bioenergetics was unaffected and (c) in HM only desipramine increased malate dehydrogenase and decreased the activities of Electron Transport Chain Complexes.These results integrate the pharmacodynamic features of desipramine and fluoxetine at subcellular level, overcoming the previous conflicting data about the effects of antidepressants on brain energy metabolism, mainly referred to whole brain homogenates or to bulk of cerebral mitochondria. With the differentiation in non-synaptic and intra-synaptic mitochondria, this study demonstrates that desipramine and fluoxetine lead to adjustments in the mitochondrial bioenergetics respect to the energy requirements of pre- and post-synaptic compartments.

Chronic fluoxetine treatment directs energy metabolism towards the citric acid cycle and oxidative phosphorylation in rat hippocampal nonsynaptic mitochondria

Fluoxetine (Flx) is the principal treatment for depression; however, the precise mechanisms of its actions remain elusive. Our aim was to identify protein expression changes within rat hippocampus regulated by chronic Flx treatment versus vehicle-controls using proteomics.Fluoxetine-hydrohloride (15mg/kg) was administered daily to adult male Wistar rats for 3weeks, and cytosolic and nonsynaptic mitochondrial hippocampal proteomes were analyzed. All differentially expressed proteins were functionally annotated according to biological process and molecular function using Uniprot and Blast2GO. Our comparative study revealed that in cytosolic and nonsynaptic mitochondrial fractions, 60 and 3 proteins respectively, were down-regulated, and 23 and 60 proteins, respectively, were up-regulated. Proteins differentially regulated in cytosolic and nonsynaptic mitochondrial fractions were primarily related to cellular and metabolic processes. Of the identified proteins, the expressions of calretinin and parvalbumine were confirmed. The predominant molecular functions of differentially expressed proteins in both cell hippocampal fractions were binding and catalytic activity. Most differentially expressed proteins in nonsynaptic mitochondria were catalytic enzymes involved in the pyruvate metabolism, citric acid cycle, oxidative phosphorylation, ATP synthesis, ATP transduction and glutamate metabolism. Results indicate that chronic Flx treatment may influence proteins involved in calcium signaling, cytoskeletal structure, chaperone system and stimulates energy metabolism via the upregulation of GAPDH expression in cytoplasm, as well as directing energy metabolism toward the citric acid cycle and oxidative phosphorylation in nonsynaptic mitochondria. This approach provides new insight into the chronic effects of Flx treatment on protein expression in a key brain region associated with stress response and memory.

Synaptic Mitochondria Sustain More Damage than Non-Synaptic Mitochondria after Traumatic Brain Injury and Are Protected by Cyclosporine A.

Currently, there are no Food and Drug Administration (FDA)-approved pharmacotherapies for the treatment of those with traumatic brain injury (TBI). As central mediators of the secondary injury cascade, mitochondria are promising therapeutic targets for prevention of cellular death and dysfunction after TBI. One of the most promising and extensively studied mitochondrial targeted TBI therapies is inhibition of the mitochondrial permeability transition pore (mPTP) by the FDA-approved drug, cyclosporine A (CsA). A number of studies have evaluated the effects of CsA on total brain mitochondria after TBI; however, no study has investigated the effects of CsA on isolated synaptic and non-synaptic mitochondria. Synaptic mitochondria are considered essential for proper neurotransmission and synaptic plasticity, and their dysfunction has been implicated in neurodegeneration. Synaptic and non-synaptic mitochondria have heterogeneous characteristics, but their heterogeneity can be masked in total mitochondrial (synaptic and non-synaptic) preparations. Therefore, it is essential that mitochondria targeted pharmacotherapies, such as CsA, be evaluated in both populations. This is the first study to examine the effects of CsA on isolated synaptic and non-synaptic mitochondria after experimental TBI. We conclude that synaptic mitochondria sustain more damage than non-synaptic mitochondria 24 h after severe controlled cortical impact injury (CCI), and that intraperitoneal administration of CsA (20 mg/kg) 15 min after injury improves synaptic and non-synaptic respiration, with a significant improvement being seen in the more severely impaired synaptic population. As such, CsA remains a promising neuroprotective candidate for the treatment of those with TBI.

Brain cortex mitochondrial bioenergetics in synaptosomes and non-synaptic mitochondria during aging.

Alterations in mitochondrial bioenergetics have been associated with brain aging. In order to evaluate the susceptibility of brain cortex synaptosomes and non-synaptic mitochondria to aging-dependent dysfunction, male Swiss mice of 3 or 17 months old were used. Mitochondrial function was evaluated by oxygen consumption, mitochondrial membrane potential and respiratory complexes activity, together with UCP-2 protein expression. Basal respiration and respiration driving proton leak were decreased by 26 and 33 % in synaptosomes from 17-months old mice, but spare respiratory capacity was not modified by aging. Succinate supported state 3 respiratory rate was decreased by 45 % in brain cortex non-synaptic mitochondria from 17-month-old mice, as compared with young animals, but respiratory control was not affected. Synaptosomal mitochondria would be susceptible to undergo calcium-induced depolarization in 17 months-old mice, while non-synaptic mitochondria would not be affected by calcium overload. UCP-2 was significantly up-regulated in both synaptosomal and submitochondrial membranes from 17-months old mice, compared to young animals. UCP-2 upregulation seems to be a possible mechanism by which mitochondria would be resistant to suffer oxidative damage during aging.