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Biomedical subjects

Patrick G Sullivan

Publications and source records attributed to Patrick G Sullivan.

At least 19 recordsLinked to original sources
PubMed2006

The first 17 amino acids of Huntingtin modulate its sub-cellular localization, aggregation and effects on calcium homeostasis.

A truncated form of the Huntington's disease (HD) protein that contains the polyglutamine repeat, Httex1p, causes HD-like phenotypes in multiple model organisms. Molecular signatures of pathogenesis appear to involve distinct domains within this polypeptide. We studied the contribution of each domain, singly or in combination, to sub-cellular localization, aggregation and intracellular Ca2+ ([Ca2+]i) dynamics in cells. We demonstrate that sub-cellular localization is most strongly influenced by the first 17 amino acids, with this sequence critically controlling Httex1p mitochondrial localization and also promoting association with the endoplasmic reticulum (ER) and Golgi. This domain also enhances the formation of visible aggregates and together with the expanded polyQ repeat acutely disrupts [Ca2+]i levels in glutamate-challenged PC12 cells. Isolated cortical mitochondria incubated with Httex1p resulted in uncoupling and depolarization of these organelles, further supporting the idea that Httex1p-dependent mitochondrial dysfunction could be instrumental in promoting acute Ca2+ dyshomeostasis. Interestingly, neither mitochondrial nor ER associations seem to be required to promote long-term [Ca2+]i dyshomeostasis.

Animals↗
PubMed2006

Time course of post-traumatic mitochondrial oxidative damage and dysfunction in a mouse model of focal traumatic brain injury: implications for neuroprotective therapy.

In the present study, we investigate the hypothesis that mitochondrial oxidative damage and dysfunction precede the onset of neuronal loss after controlled cortical impact traumatic brain injury (TBI) in mice. Accordingly, we evaluated the time course of post-traumatic mitochondrial dysfunction in the injured cortex and hippocampus at 30 mins, 1, 3, 6, 12, 24, 48, and 72 h after severe TBI. A significant decrease in the coupling of the electron transport system with oxidative phosphorylation was observed as early as 30 mins after injury, followed by a recovery to baseline at 1 h after injury. A statistically significant (P<0.0001) decline in the respiratory control ratio was noted at 3 h, which persisted at all subsequent time-points up to 72 h after injury in both cortical and hippocampal mitochondria. Structural damage seen in purified cortical mitochondria included severely swollen mitochondria, a disruption of the cristae and rupture of outer membranes, indicative of mitochondrial permeability transition. Consistent with this finding, cortical mitochondrial calcium-buffering capacity was severely compromised by 3 h after injury, and accompanied by significant increases in mitochondrial protein oxidation and lipid peroxidation. A possible causative role for reactive nitrogen species was suggested by the rapid increase in cortical mitochondrial 3-nitrotyrosine levels shown as early as 30 mins after injury. These findings indicate that post-traumatic oxidative lipid and protein damage, mediated in part by peroxynitrite, occurs in mitochondria with concomitant ultrastructural damage and impairment of mitochondrial bioenergetics. The data also indicate that compounds which specifically scavenge peroxynitrite (ONOO(-)) or ONOO(-)-derived radicals (e.g. ONOO(-)+H(+) --> ONOOH --> (*)NO(2)+(*)OH) may be particularly effective for the treatment of TBI, although the therapeutic window for this neuroprotective approach might only be 3 h.

Animals↗
PubMed2006

Synaptic mitochondria are more susceptible to Ca2+overload than nonsynaptic mitochondria.

Mitochondria in nerve terminals are subjected to extensive Ca2+ fluxes and high energy demands, but the extent to which the synaptic mitochondria buffer Ca2+ is unclear. In this study, we identified a difference in the Ca2+ clearance ability of nonsynaptic versus synaptic mitochondrial populations enriched from rat cerebral cortex. Mitochondria were isolated using Percoll discontinuous gradients in combination with high pressure nitrogen cell disruption. Mitochondria in the nonsynaptic fraction originate from neurons and other cell types including glia, whereas mitochondria enriched from a synaptosomal fraction are predominantly neuronal and presynaptic in origin. There were no differences in respiration or initial Ca2+ loads between nonsynaptic and synaptic mitochondrial populations. Following both bolus and infusion Ca2+ addition, nonsynaptic mitochondria were able to accumulate significantly more exogenously added Ca2+ than the synaptic mitochondria before undergoing mitochondrial permeability transition, observed as a loss in mitochondrial membrane potential and decreased Ca2+ uptake. The limited ability of synaptic mitochondria to accumulate Ca2+ could result from several factors including a primary function of ATP production to support the high energy demand of presynaptic terminals, their relative isolation in comparison with the threads or clusters of mitochondria found in the soma of neurons and glia, or the older age and increased exposure to oxidative damage of synaptic versus nonsynaptic mitochondria. By more readily undergoing permeability transition, synaptic mitochondria may initiate neuron death in response to insults that elevate synaptic levels of intracellular Ca2+, consistent with the early degeneration of distal axon segments in neurodegenerative disorders.

Adenosine Triphosphate↗
PubMed2005

Cryopreservation of brain mitochondria: a novel methodology for functional studies.

Often, comparative studies involving large number of animals or human post-mortem tissue samples are precluded, especially those requiring structurally and functionally intact cells and/or organelles. The ability to 'bank' such samples for storage and restore or 'reanimate' them at a later time without causing damage to the structure and/or function becomes imperative. However, to date, such attempts have produced conflicting results. We here demonstrate for the first time that isolated rat brain mitochondria can be successfully cryopreserved and restored for later use. We added a well characterized cryoprotectant 10% (v/v) dimethyl sulfoxide (DMSO) to purified rat cortical mitochondria and allowed them to cool at a uniform rate of approximately 1 degree C/min and stored them at -80 degrees C. Freshly isolated as well as reanimated brain mitochondria were analyzed for respiration. Structural integrity of cryopreserved mitochondria was also verified by electron microscopy. Mitochondrial membrane marker levels were assessed along with cytochrome c levels. Intact structure and function of the cryopreserved brain mitochondria observed allows us the opportunity to store mitochondria for longer periods of time as well as perform metabolic studies as needed. This will considerably expand the time-frame required for carrying out functional analysis in large comparative studies.

Animals↗
PubMed2005

Interventions with neuroprotective agents: novel targets and opportunities.

Several antiepileptic drugs have demonstrated neuroprotective properties in animal models by inhibiting the components of the excitotoxic cascade. The process of neurodegeneration appears to be dependent on the mitochondria, with their central role as regulators of both energy metabolism and apoptotic pathways during seizures. Excess cytosolic Ca(2+) during periods of excitotoxicity disrupts homeostasis in the mitochondria, causing inhibition of adenosine triphosphate synthesis and increased production of reactive oxygen species, with resultant oxidative damage, leading to neuronal cell death. The Ca(2+)-mediated mitochondrial dysfunction in epilepsy may be the main target for neuroprotective intervention by antiepileptic drugs.

Animals↗
PubMed2005

Mitochondrial aging and dysfunction in Alzheimer's disease.

Disruptions in energy metabolism have been suggested to be a prominent feature, perhaps even a fundamental component, of Alzheimer's disease (AD). These abnormalities in cerebral metabolism precede the onset of neurological dysfunction as well as gross neuropathology of AD. These changes may stem from inhibition of mitochondrial enzymes including pyruvate dehydrogenase, cytochrome c oxidase, and alpha-ketoglutarate dehydrogenase. Several lines of evidence also suggest a role for oxidative stress in the neuropathology associated with the disease state. Because mitochondria are the major site of free radical production in cells, they are also a primary target for oxidative damage and subsequent dysfunction. This link between mitochondrial dysfunction and the pathophysiology of AD is supported by several lines of evidence.

Aging↗
PubMed2005

The uncoupling agent 2,4-dinitrophenol improves mitochondrial homeostasis following striatal quinolinic acid injections.

It is now generally accepted that excitotoxic cell death involves bioenergetic failure resulting from the cycling of Ca2+ and the generation of reactive oxygen species (ROS) by mitochondria. Both Ca2+ cycling and ROS formation by mitochondria are dependent on the mitochondrial membrane potential (Deltapsi(m)) that results from the proton gradient that is generated across the inner membrane. Mitochondrial uncoupling refers to a condition in which protons cross the inner membrane back into the matrix while bypassing the ATP synthase. As a consequence of this "short-circuit," there is a reduction in Deltapsi(m). We have previously demonstrated that animals treated with the classic uncoupling agent 2,4-dinitrophenol (DNP) show significant protection against brain damage following striatal injections of the NMDA agonist quinolinic acid (QA). In an effort to elucidate the mechanism of neuroprotection, we have assessed the effects of DNP on several parameters of mitochondrial function caused by QA. The results presented herein demonstrate that treatment with DNP attenuates QA-induced increases in mitochondrial Ca2+ levels and ROS formation and also improves mitochondrial respiration. Our findings indicate that DNP may confer protection against acute brain injury involving excitotoxic pathways by mechanisms that maintain mitochondrial function.

2,4-Dinitrophenol↗
PubMed2005

Spatial and temporal characteristics of neurodegeneration after controlled cortical impact in mice: more than a focal brain injury.

The present study examined the neuropathology of the lateral controlled cortical impact (CCI) traumatic brain injury (TBI) model in mice utilizing the de Olmos silver staining method that selectively identifies degenerating neurons and their processes. The time course of ipsilateral and contralateral neurodegeneration was assessed at 6, 24, 48, 72, and 168 h after a severe (1.0 mm, 3.5 M/sec) injury in young adult CF-1 mice. At 6 hrs, neurodegeneration was apparent in all layers of the ipsilateral cortex at the epicenter of the injury. A low level of degeneration was also detected within the outer molecular layer of the underlying hippocampal dentate gyrus and to the mossy fiber projections in the CA3 pyramidal subregions. A time-dependent increase in cortical and hippocampal neurodegeneration was observed between 6 and 72 hrs post-injury. At 24 h, neurodegeneration was apparent in the CA1 and CA3 pyramidal and dentate gyral granule neurons and in the dorsolateral portions of the thalamus. Image analysis disclosed that the overall volume of ipsilateral silver staining was maximal at 48 h. In the case of the hippocampus, staining was generalized at 48 and 72 h, indicative of damage to all of the major afferent pathways: perforant path, mossy fibers and Schaffer collaterals as well as the efferent CA1 pyramidal axons. The hippocampal neurodegeneration was preceded by a significant increase in the levels of calpain-mediated breakdown products of the cytoskeletal protein alpha-spectrin that began at 6 h, and persisted out to 72 h post-injury. Damage to the corpus callosal fibers was observed as early as 24 h. An anterior to posterior examination of neurodegeneration showed that the cortical damage included the visual cortex. At 168 h (7 days), neurodegeneration in the ipsilateral cortex and hippocampus had largely abated except for ongoing staining in the cortical areas surrounding the contusion lesion and in hippocampal mossy fiber projections. Callosal and thalamic neurodegeneration was also very intense. This more complete neuropathological examination of the CCI model shows that the associated damage is much more widespread than previously appreciated. The extent of ipsilateral and contralateral neurodegeneration provides a more complete anatomical correlate for the cognitive and motor dysfunction seen in this paradigm and suggests that visual disturbances are also likely to be involved in the post-CCI neurological deficits.

Animals↗
PubMed2004

Nitrogen disruption of synaptoneurosomes: an alternative method to isolate brain mitochondria.

Mitochondria are known to be localized in synaptic and non-synaptic compartments in the brain. Synaptoneurosomes, which contain high numbers of mitochondria, may act as a major contaminant of currently used isolation techniques. Currently, there is no method employed to successfully disrupt synaptoneurosomes and isolate both synaptic and non-synaptic mitochondria without structural or functional damage. A novel method is reported here for disruption of synaptoneurosomes and isolation of total brain mitochondria from synaptic and non-synaptic sources using a nitrogen decompression technique. Nitrogen gas was dissolved into crude mitochondrial preparations and maintained under constant, moderate pressure. After a short incubation, the pressure was released causing the nitrogen to come out of solution as growing bubbles, which ruptures cellular and synaptoneurosomal membranes. Mitochondria isolated using this rapid technique were bioenergetically competent and exhibited functional characteristics comparable to mitochondria isolated using traditional techniques. This nitrogen decompression technique will allow for further characterization of synaptic pools of mitochondria, which are almost exclusively neuronal in origin.

Animals↗
PubMed2004

Intrinsic differences in brain and spinal cord mitochondria: Implication for therapeutic interventions.

It is well known that regions of the CNS differentially respond to insults. After brain injury, cyclosporine A reduces damage but is ineffective following spinal cord injury. We address this disparity by assessing several parameters of mitochondrial physiology in the normal neocortex and spinal cord. In situ measurements of O(2) (-.) production, lipid peroxidation, and mitochondrial DNA oxidation revealed significantly higher levels in spinal cord vs. neocortical neurons. Real-time PCR demonstrated differences in mitochondrial transcripts coupled with decreases in complex I enzyme activity and respiration in spinal cord mitochondria. The threshold for calcium-induced mitochondrial permeability transition was substantially reduced in spinal cord vs. neocortex and modulated by lipid peroxidation. These intrinsic differences may provide a pivotal target for strategies to ameliorate neuronal damage following injury, and this imbalance in oxidative stress may contribute to the susceptibility of spinal cord motor neurons in neuropathologies such as amyotrophic lateral sclerosis.

Aldehydes↗
PubMed2004

Proteasome inhibition alters neural mitochondrial homeostasis and mitochondria turnover.

Inhibition of proteasome activity occurs in normal aging and in a wide variety of neurodegenerative conditions including Alzheimer's disease and Parkinson's disease. Although each of these conditions is also associated with mitochondrial dysfunction potentially mediated by proteasome inhibition, the relationship between proteasome inhibition and the loss of mitochondrial homeostasis in each of these conditions has not been fully elucidated. In this study, we conducted experimentation in order to begin to develop a more complete understanding of the effects proteasome inhibition has on neural mitochondrial homeostasis. Mitochondria within neural SH-SY5Y cells exposed to low level proteasome inhibition possessed similar morphological features and similar rates of electron transport chain activity under basal conditions as compared with untreated neural cultures of equal passage number. Despite such similarities, maximal complex I and complex II activities were dramatically reduced in neural cells subject to proteasome inhibition. Proteasome inhibition also increased mitochondrial reactive oxygen species production, reduced intramitochondrial protein translation, and increased cellular dependence on glycolysis. Finally, whereas proteasome inhibition generated cells that consistently possessed mitochondria located in close proximity to lysosomes with mitochondria present in the cellular debris located within autophagosomes, increased levels of lipofuscin suggest that impairments in mitochondrial turnover may occur following proteasome inhibition. Taken together, these data demonstrate that proteasome inhibition dramatically alters specific aspects of neural mitochondrial homeostasis and alters lysosomal-mediated degradation of mitochondria with both of these alterations potentially contributing to aging and age-related disease in the nervous system.

Animals↗
PubMed2004

The ketogenic diet increases mitochondrial uncoupling protein levels and activity.

Fatty acids are known to enhance mitochondrial uncoupling protein (UCP) activity. We asked whether a high-fat ketogenic diet (KD) increases UCP levels and activity in hippocampi of juvenile mice. Maximum mitochondrial respiration rates were significantly (p < 0.001) higher in KD- versus standard diet (SD)-treated animals, indicating increased UCP-mediated proton conductance that can reduce reactive oxygen species (ROS) production. Western blots showed significant (p < 0.05) or borderline significant increases in UCP2, UCP4, and UCP5 protein levels, and increased immunoreactivity to these three UCP isoforms was most prominently seen in the dentate gyrus of KD-fed mice. Finally, we found that oligomycin-induced ROS production was significantly (p < 0.05) lower in KD-fed mice than in SD controls. Collectively, our data suggest that a KD may exert neuroprotective effects by diminishing ROS production through activation of mitochondrial UCPs.

Animals↗
PubMed2004

Mitochondrial damage and dysfunction in traumatic brain injury.

The enduring cognitive deficits and histopathology associated with traumatic brain injury (TBI) may arise from damage to mitochondrial populations, which initiates the metabolic dysfunction observed in clinical and experimental TBI. The anecdotal evidence for in vivo structural damage to mitochondria corroborates metabolic and physiologic dysfunction, which depletes substrates and promotes free radical generation. Excessive calcium pathology differentially disrupts the heterogeneous mitochondrial population, such that calcium sensitivity increases after TBI. The ongoing pathology may escalate to include protein and DNA oxidation that impacts mitochondrial function and promotes cell death. Thus, in vivo TBI damages, if not eliminates, mitochondrial populations depending on injury severity, with the remaining population left to provide metabolic support for survival or repair in the wake of cellular pathology. With a considerable understanding of post-injury mitochondrial populations, therapeutic interventions targeted to the mitochondria may delay or prevent secondary cascades that lead to long-term cell death and neurobehavioral disability.

Journal Article↗
PubMed2004

Brain region-specific, age-related, alterations in mitochondrial responses to elevated calcium.

An age-related Ca(2+) dysregulation and increased production of reactive oxygen species (ROS) may contribute to late-onset neurodegenerative disorders. These alterations are often attributed to impaired mitochondrial function yet few studies have directly examined mitochondria isolated from various regions of the aged brain. The purpose of this study was to examine Ca(2+)-buffering and ROS production in mitochondria isolated from Fischer 344 rats ranging in age from 4 to 25 months. Mitchondria isolated from the cortex of the 25 month rat brain exhibited greater rates of ROS production and mitochondrial swelling in response to increasing Ca(2+) loads as compared to mitochondria isolated from younger (4, 13 month) animals. The increased swelling is indicative of opening of the mitochondrial permeability transition pore indicating impaired Ca(2+) buffering/cycling in aged animals. These age-related differences were not observed in mitochondria isolated from cerebellum. Together, these results demonstrate region specific, age-related, alterations in mitochondrial responses to Ca(2+).

Adaptation, Physiological↗
PubMed2004

The mitochondrial uncoupling agent 2,4-dinitrophenol improves mitochondrial function, attenuates oxidative damage, and increases white matter sparing in the contused spinal cord.

The purpose of this study was to investigate the potential neuroprotective efficacy of the mitochondrial uncoupler 2,4-dinitrophenol (DNP) in rats following a mild to moderate spinal cord contusion injury. Animals received intraperitoneal injections of vehicle (DMSO) or 5 mg/mL of DNP prior to injury. Twenty-four hours following surgery, mitochondrial function was assessed in mitochondria isolated from spinal cord synaptosomes. In addition, synaptosomes were used to measure indicators of reactive oxygen species formation, lipid peroxidation, and protein oxidation. Relative to vehicle-treated animals, pretreatment with DNP maintained mitochondrial bioenergetics and significantly decreased reactive oxygen species levels, lipid peroxidation, and protein carbonyl content following spinal cord injury. Furthermore, pretreatment with DNP significantly increased the amount of remaining white matter at the injury epicenter 6 weeks after injury. These results indicate that treatment with mitochondrial uncoupling agents may provide a novel approach for the treatment of secondary injury following spinal cord contusion.

2,4-Dinitrophenol↗
PubMed2004

Lack of long-term histopathologic changes in brain and skeletal muscle of mice treated with a ketogenic diet.

Although there is increasing awareness of adverse effects associated with use of the high-fat ketogenic diet, very little is known regarding its long-term clinical consequences, especially in relation to cardiovascular health. Recent reports have highlighted rare but significant cardiac problems in patients treated with the ketogenic diet. Given the inherent limitations in conducting detailed pathologic assessments in patients, we asked whether histologic changes might develop in the brain and skeletal muscle of mice fed a high-fat diet for 2 to 3 months. We found no evidence of gross morphologic or histochemical alterations in muscle or brain after administration of the ketogenic diet. Further, there was no abnormal lipid storage or mitochondrial enzymatic staining. Our data suggest that patients chronically treated with a ketogenic diet are not likely to develop a lipid myopathy or neuronal inclusions.

Animals↗
PubMed2003

Modulation of mitochondrial function by endogenous Zn2+ pools.

Recent evidence suggests that intracellular Zn(2+) accumulation contributes to the neuronal injury that occurs in epilepsy or ischemia in certain brain regions, including hippocampus, amygdala, and cortex. Although most attention has been given to the vesicular Zn(2+) that is released into the synaptic space and may gain entry to postsynaptic neurons, recent studies have highlighted pools of intracellular Zn(2+) that are mobilized in response to stimulation. One such Zn(2+) pool is likely bound to cytosolic proteins, like metallothioneins. Applying imaging techniques to cultured cortical neurons, this study provides novel evidence for the presence of a mitochondrial pool distinct from the cytosolic protein or ligand-bound pool. These pools can be pharmacologically mobilized largely independently of each other, with Zn(2+) release from one resulting in apparent net Zn(2+) transfer to the other. Further studies found evidence for complex and potent effects of Zn(2+) on isolated brain mitochondria. Submicromolar levels, comparable to those that might occur on strong mobilization of intracellular compartments, induced membrane depolarization (loss of Deltapsi(m)), increases in currents across the mitochondrial inner membrane as detected by direct patch clamp recording of mitoplasts, increased O(2) consumption and decreased reactive oxygen species (ROS) generation, whereas higher levels decreased O(2) consumption and increased ROS generation. Finally, strong mobilization of protein-bound Zn(2+) appeared to induce partial loss of Deltapsi(m), suggesting that movement of Zn(2+) between cytosolic and mitochondrial pools might be of functional significance in intact neurons.

Animals↗