Calcium Buffering Capacity Research Articles

Targeting Mitochondria in Fighting Cancer

During the last years, there have been a number of reports that prove involvement of mitochondria in the pathogenesis of variety of disorders including cancer and neurodegenerative diseases. Alteration of vital mitochondrial functions - production of ATP, calcium buffering capacity, abnormal production of reactive oxygen species, can be potentially responsible for pathogenesis of cancer and neurodegenerative diseases. Involvement of mitochondria in various types of cell death makes them attractive targets for tumor cell elimination. This review describes the role of mitochondria in tumor cells and how targeting of mitochondria can be used as a tool in fighting cancer.

Calcium-binding proteins and GFAP immunoreactivity alterations in murine hippocampus after 1 month of exposure to 835 MHz radiofrequency at SAR values of 1.6 and 4.0 W/kg

Widespread use of wireless mobile communication has raised concerns of adverse effect to the brain owing to the proximity during use due to the electromagnetic field emitted by mobile phones. Changes in calcium ion concentrations via binding proteins can disturb calcium homeostasis; however, the correlation between calcium-binding protein (CaBP) immunoreactivity (IR) and glial cells has not been determined with different SAR values. Different SAR values [1.6 (E1.6 group) and 4.0 (E4 group) W/kg] were applied to determine the distribution of calbindin D28-k (CB), calretinin (CR), and glial fibrillary acidic protein (GFAP) IR in murine hippocampus. Compared with sham control group, decreased CB and CR IRs, loss of CB and CR immunoreactive cells and increased GFAP IR exhibiting hypertrophic cytoplasmic processes were noted in both experimental groups. E4 group showed a prominent decrement in CB and CR IR than the E1.6 group due to down-regulation of CaBP proteins and neuronal loss. GFAP IR was more prominent in the E4 group than the E1.6 group. Decrement in the CaBPs can affect the calcium-buffering capacity leading to cell death, while increased GFAP IR and changes in astrocyte morphology, may mediate brain injury due to radiofrequency exposure.

Age-related loss of calcium buffering and selective neuronal vulnerability in Alzheimer’s disease

The reasons for the selective vulnerability of distinct neuronal populations in neurodegenerative disorders are unknown. The cholinergic neurons of the basal forebrain are vulnerable to pathology and loss early in Alzheimer's disease and in a number of other neurodegenerative disorders of the elderly. In the primate, including man, these neurons are rich in the calcium buffer calbindin-D(28K). Here, we confirm that these neurons undergo a substantial loss of calbindin in the course of normal aging and report a further loss of calbindin in Alzheimer's disease both at the level of RNA and protein. Significantly, cholinergic neurons that had lost their calbindin in the course of normal aging were those that selectively degenerated in Alzheimer's disease. Furthermore, calbindin-containing neurons were virtually resistant to the process of tangle formation, a hallmark of the disease. We conclude that the loss of calcium buffering capacity in these neurons and the resultant pathological increase in intracellular calcium are permissive to tangle formation and degeneration.

Dynamic Control of Neuronal Firing Threshold by Calcium Buffering: A New Role for Calcium Binding Proteins

We have investigated the detailed regulation of neuronal firing threshold by the cytosolic calcium buffering capacity using a combination of mathematical modeling and patch clamp recording in acute slice. Theoretical results show that, at similar free calcium concentration, increased calcium buffer concentration lowers the firing threshold of cerebellar granule cells. We show that this effect is a direct consequence of the major slowdown of calcium dynamics. Patch clamp recordings on cerebellar granule cells loaded with a high concentration of the fast calcium buffer BAPTA (15 mM) reveal alterations in the excitability threshold as compared to cells loaded with 0.15 mM BAPTA.

Calcium‐dependent mitochondrial function and dysfunction in neurons

Calcium is an extraordinarily versatile signaling ion, encoding cellular responses to a wide variety of external stimuli. In neurons, mitochondria can accumulate enormous amounts of calcium, with the consequence that mitochondrial calcium uptake, sequestration and release play pivotal roles in orchestrating calcium-dependent responses as diverse as gene transcription and cell death. In this review, we consider the basic chemistry of calcium as a 'sticky' cation, which leads to extremely high bound/free ratios, and discuss areas of current interest or controversy. Topics addressed include methodologies for measuring local intracellular calcium, mitochondrial calcium buffering and loading capacity, mitochondrially directed spatial calcium gradients, and the role of calcium overload-dependent mitochondrial dysfunction in glutamate-evoked excitotoxic injury and neurodegeneration. Finally, we consider the relationship between delayed calcium de-regulation, the mitochondrial permeability transition and the generation of reactive oxygen species, and propose a unified view of the 'source specificity' and 'calcium overload' models of N-methyl-d-aspartate (NMDA) receptor-dependent excitotoxicity. Non-NMDA receptor mechanisms of excitotoxicity are discussed briefly.

Amyotrophic Lateral Sclerosis and Excitotoxicity: From Pathological Mechanism to Therapeutic Target

Glutamate-induced excitotoxicity is responsible for neuronal death in acute neurological conditions as well as in chronic neurodegeneration. In this review, we give an overview of the contribution of excitotoxicity in the pathogenesis of amyotrophic lateral sclerosis (ALS). The selective motor neuron death that is the hallmark of this neurodegenerative disease seems to be related to a number of intrinsic characteristics of these neurons. Most of these characteristics relate to calcium entry and calcium handling in the motor neurons as intracellular free calcium concentrations increase quickly due to a high glutamate-induced calcium influx in combination with a low calcium buffering capacity. The high calcium influx is because of the presence of GluR2 lacking, calcium-permeable AMPA receptors while a low expression of calcium binding proteins explains the low calcium buffering capacity. In the absence of these proteins, mitochondria play an important role to remove calcium from the cytoplasm. While all of these characteristics make at least a subpopulation of motor neurons intrinsically very prone to AMPA receptor mediated excitotoxicity, this vulnerability is further increased by the disease process. Mutated genes as well as unknown factors do not only influence the intrinsic characteristics of the motor neurons, but also the properties of the surrounding astrocytes. In conclusion, excitotoxicity remains an intriguing pathological pathway that could not only explain the selectivity of the motor neuron death but also the role of surrounding non-neuronal cells in ALS. In addition, excitotoxicity is also an interesting drug-able target as indicated by the only FDA-approved drug, riluzole, as well as by a number of ongoing clinical trials.

Mitochondrial protection after traumatic brain injury by scavenging lipid peroxyl radicals

Mitochondrial dysfunction after traumatic brain injury (TBI) is manifested by increased levels of oxidative damage, loss of respiratory functions and diminished ability to buffer cytosolic calcium. This study investigated the detrimental effects of lipid peroxyl radicals (LOO(*)) and lipid peroxidation (LP) in brain mitochondria after TBI by examining the protective effects of U-83836E, a potent and selective scavenger of LOO(*) radicals. Male CF1 mice were subjected to severe controlled cortical impact TBI (CCI-TBI) and treated with either vehicle or U-83836E initiated i.v. at 15 min post-injury. Calcium (Ca(++)) buffering capacity and respiratory function were measured in isolated cortical mitochondrial samples taken from the ipsilateral hemisphere at 3 and 12 h post-TBI, respectively. In vehicle-treated injured mice, the cortical mitochondrial Ca(++) buffering capacity was reduced by 60% at 3 h post-injury (p < 0.001) and the respiratory control ratio was decreased by 27% at 12 h post-TBI, relative to sham, non-injured mice. U-83836E treatment significantly (p < 0.05) preserved Ca(++) buffering capacity and attenuated the reduction in respiratory control ratio values. Consistent with the functional effects of U-83836E being as a result of an attenuation of mitochondrial oxidative damage, the compound significantly (p < 0.001) reduced LP-generated 4-hydroxynonenal levels in both cortical homogenates and mitochondria at both 3 and 12 h post-TBI. Unexpectedly, U-83836E also reduced peroxynitrite-generated 3-nitrotyrosine in parallel with the reduction in 4-hydroxynonenal. The results demonstrate that LOO(*) radicals contribute to secondary brain mitochondrial dysfunction after TBI by propagating LP and protein nitrative damage in cellular and mitochondrial membranes.

Hypoglossal motor neurons display a reduced calcium increase after axotomy in mice with upregulated parvalbumin

Motor neurons that exhibit differences in vulnerability to degeneration have been identified in motor neuron disease and in its animal models. The oculomotor and hypoglossal neurons are regarded as the prototypes of the resistant and susceptible cell types, respectively. Because an increase in the level of intracellular calcium has been proposed as a feature amplifying degenerative processes, we earlier studied the calcium increase in these motor neurons after axotomy in Balb/c mice and demonstrated a correlation between the susceptibility to degeneration and the intracellular calcium increase, with an inverse relation with the calcium buffering capacity, characterized by the parvalbumin or calbindin-D(28k) content. Because the differential susceptibility of the cells might also be attributed to their different cellular environments, in the present experiments, with the aim of verifying directly that a higher calcium buffering capacity is indeed responsible for the enhanced resistance, motor neurons were studied in their original milieu in mice with a genetically increased parvalbumin level. The changes in intracellular calcium level of the hypoglossal and oculomotor neurons after axotomy were studied electron microscopically at a 21-day interval after axotomy, during which time no significant calcium increase was detected in the hypoglossal motor neurons, the response being similar to that of the oculomotor neurons. The hypoglossal motor neurons of the parental mice, used as positive controls, exhibited a transient, significant elevation of calcium. These data provide more direct evidence of the protective role of parvalbumin against the degeneration mediated by a calcium increase in the acute injury of motor neurons.

Selective neuronal vulnerability to oxidative stress in the brain

Oxidative stress (OS), caused by the imbalance between the generation and detoxification of reactive oxygen and nitrogen species (ROS/RNS), plays an important role in brain aging, neurodegenerative diseases, and other related adverse conditions, such as ischemia. While ROS/RNS serve as signaling molecules at physiological levels, an excessive amount of these molecules leads to oxidative modification and, therefore, dysfunction of proteins, nucleic acids, and lipids. The response of neurons to this pervasive stress, however, is not uniform in the brain. While many brain neurons can cope with a rise in OS, there are select populations of neurons in the brain that are vulnerable. Because of their selective vulnerability, these neurons are usually the first to exhibit functional decline and cell death during normal aging, or in age-associated neurodegenerative diseases, such as Alzheimer's disease. Understanding the molecular and cellular mechanisms of selective neuronal vulnerability (SNV) to OS is important in the development of future intervention approaches to protect such vulnerable neurons from the stresses of the aging process and the pathological states that lead to neurodegeneration. In this review, the currently known molecular and cellular factors that contribute to SNV to OS are summarized. Included among the major underlying factors are high intrinsic OS, high demand for ROS/RNS-based signaling, low ATP production, mitochondrial dysfunction, and high inflammatory response in vulnerable neurons. The contribution to the selective vulnerability of neurons to OS by other intrinsic or extrinsic factors, such as deficient DNA damage repair, low calcium-buffering capacity, and glutamate excitotoxicity, are also discussed.

Loss of PINK1 Function Promotes Mitophagy through Effects on Oxidative Stress and Mitochondrial Fission

Mitochondrial dysregulation is strongly implicated in Parkinson disease. Mutations in PTEN-induced kinase 1 (PINK1) are associated with familial parkinsonism and neuropsychiatric disorders. Although overexpressed PINK1 is neuroprotective, less is known about neuronal responses to loss of PINK1 function. We found that stable knockdown of PINK1 induced mitochondrial fragmentation and autophagy in SH-SY5Y cells, which was reversed by the reintroduction of an RNA interference (RNAi)-resistant plasmid for PINK1. Moreover, stable or transient overexpression of wild-type PINK1 increased mitochondrial interconnectivity and suppressed toxin-induced autophagy/mitophagy. Mitochondrial oxidant production played an essential role in triggering mitochondrial fragmentation and autophagy in PINK1 shRNA lines. Autophagy/mitophagy served a protective role in limiting cell death, and overexpressing Parkin further enhanced this protective mitophagic response. The dominant negative Drp1 mutant inhibited both fission and mitophagy in PINK1-deficient cells. Interestingly, RNAi knockdown of autophagy proteins Atg7 and LC3/Atg8 also decreased mitochondrial fragmentation without affecting oxidative stress, suggesting active involvement of autophagy in morphologic remodeling of mitochondria for clearance. To summarize, loss of PINK1 function elicits oxidative stress and mitochondrial turnover coordinated by the autophagic and fission/fusion machineries. Furthermore, PINK1 and Parkin may cooperate through different mechanisms to maintain mitochondrial homeostasis.

Mitochondria as targets for chemotherapy

Mitochondrial malfunctioning is implicated in the pathogenesis of a variety of disorders, including cancer and multiple neurodegenerative diseases, such as Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and Huntington's disease. Disturbance of mitochondrial vital functions, e.g., production of ATP, calcium buffering capacity, and generation of reactive oxygen species, can be potentially involved in disease pathogenesis. Neurological disorders caused by mitochondrial deterioration are often associated with cell loss within specific brain regions. In contrast, mitochondrial alterations in tumor cells and the "Warburg effect" might lead to cell survival and resistance of tumor cells to chemotherapy. This review is devoted to the role of mitochondria in neurodegeneration and tumor formation, and describes how targeting of mitochondria can be beneficial in the therapy of these diseases, which affect a large human population.

Altered Structure Of The Cerebellar Granule Cell Layer Of Mice Lacking Calretinin

Calcium binding proteins, such as calretinin, are abundantly expressed in distinctive patterns in the central nervous system, but their physiological functions remain poorly understood. Calretinin is highly expressed in cerebellar granule cells and calretinin deficient mice exhibit alterations in motor coordination. Using confocal microscopy, we demonstrate that the cerebellar cortex of calretinin deficient mice exhibit a significantly decreased granule cells density. Interestingly, it has been shown that the migration of granule cells is tightly associated with intracellular calcium oscillations . Therefore, we hypothesize that an alteration of these calcium oscillations in calretinin deficient mice could be involved in the observed morphological alterations. To test this assumption, we are currently developping two strategies. First, using confocal microscopy and cerebellar microexplant culture, we are studying calcium oscillations occuring during granule cell migration in the wild type control and calretinin knock-out mice. This allow us to characterize the impact of variations in calcium buffering capacity over neuronal development and on the generation of the calcium oscillations observed during the granule cell migration. On the other hand, we are developping a theoretical model to study the impact of calcium buffering modifications on the dynamics underlying the observed calcium oscillations . This dedicated computational model will shed light on the possible mechanism responsible for the modulation, by calretinin, of calcium oscillations during the granule cell migration.

Synaptic vesicle pool size, release probability and synaptic depression are sensitive to Ca2+ buffering capacity in the developing rat calyx of Held

The calyx of Held, a specialized synaptic terminal in the medial nucleus of the trapezoid body, undergoes a series of changes during postnatal development that prepares this synapse for reliable high frequency firing. These changes reduce short-term synaptic depression during tetanic stimulation and thereby prevent action potential failures during a stimulus train. We measured presynaptic membrane capacitance changes in calyces from young postnatal day 5-7 (p5-7) or older (p10-12) rat pups to examine the effect of calcium buffer capacity on vesicle pool size and the efficiency of exocytosis. Vesicle pool size was sensitive to the choice and concentration of exogenous Ca2+ buffer, and this sensitivity was much stronger in younger animals. Pool size and exocytosis efficiency in p5-7 calyces were depressed by 0.2 mM EGTA to a greater extent than with 0.05 mM BAPTA, even though BAPTA is a 100-fold faster Ca2+ buffer. However, this was not the case for p10-12 calyces. With 5 mM EGTA, exocytosis efficiency was reduced to a much larger extent in young calyces compared to older calyces. Depression of exocytosis using pairs of 10-ms depolarizations was reduced by 0.2 mM EGTA compared to 0.05 mM BAPTA to a similar extent in both age groups. These results indicate a developmentally regulated heterogeneity in the sensitivity of different vesicle pools to Ca2+ buffer capacity. We propose that, during development, a population of vesicles that are tightly coupled to Ca2+ channels expands at the expense of vesicles more distant from Ca2+ channels.

Destabilization of the cytosolic calcium level and the death of cardiomyocytes in the presence of derivatives of long-chain fatty acids

By means of fluorescent microscopy, long-chain fatty acid derivatives, myristoylcarnitine and palmitoylcarnitine, were shown to exert the most toxic effect on rat ventricular cardiomyocytes. The addition of 20-50 µ M acylcarnitines increased calcium concentration in cytoplasm ((Ca 2+ ) i ) and caused cell death after a lag-period of 4-8 min. This effect was independent of extracellular calcium level and Ca 2+ inhibitors of L-type channels. Free myristic and palmitic acids at concentrations of 300-500 µ M had little effect on (Ca 2+ ) i within 30 min. We suggest that the toxic effect is due to the activation of calcium channels of sarcoplasmic reticulum by acylcarnitines and/or arising acyl-CoA. Mitochondria play a role of calcium-buffer system under these con- ditions. The calcium capacity of the buffer determines the duration of the lag-period. Phosphate increases the calcium capacity of mitochondria and the lag-period. In the presence of rotenone and oligomycin, the elevation of (Ca 2+ ) i after the addition of acylcarnitines occurs without the lag-period. The exhaustion of the mitochondrial calcium-buffer capacity or significant depolarization of mitochondria leads to a rapid release of calcium from mitochondria and cell death. Thus, the activation of reticular calcium channels is the main reason of the toxicity of myristoylcarnitine and palmitoylcarnitine.

Cyclophilin D deficiency attenuates mitochondrial and neuronal perturbation and ameliorates learning and memory in Alzheimer's disease

Cyclophilin D (CypD, encoded by Ppif) is an integral part of the mitochondrial permeability transition pore, whose opening leads to cell death. Here we show that interaction of CypD with mitochondrial amyloid-beta protein (Abeta) potentiates mitochondrial, neuronal and synaptic stress. The CypD-deficient cortical mitochondria are resistant to Abeta- and Ca(2+)-induced mitochondrial swelling and permeability transition. Additionally, they have an increased calcium buffering capacity and generate fewer mitochondrial reactive oxygen species. Furthermore, the absence of CypD protects neurons from Abeta- and oxidative stress-induced cell death. Notably, CypD deficiency substantially improves learning and memory and synaptic function in an Alzheimer's disease mouse model and alleviates Abeta-mediated reduction of long-term potentiation. Thus, the CypD-mediated mitochondrial permeability transition pore is directly linked to the cellular and synaptic perturbations observed in the pathogenesis of Alzheimer's disease. Blockade of CypD may be a therapeutic strategy in Alzheimer's disease.

Decline in age‐dependent, MK801‐induced injury coincides with developmental switch in parvalbumin expression: Somatosensory and motor cortex

MK801-induced activation of caspase-3 is developmentally regulated, peaking at postnatal day (P) 7 and decreasing with increasing postnatal age thereafter. Further, at P7, cells displaying activation of caspase-3 lack expression of calcium binding proteins (CaBPs). To further explore this relationship, we investigated postnatal expression of calbindin (CB), calretinin (CR) and parvalbumin (PV) in two brain regions susceptible to MK801-induced injury, the somatosensory cortex (S1) and layer II/III of motor cortex (M1/M2). Expression of CB and especially PV was low to absent prior to P7 but substantially increased from P7 through to P21 and adulthood. In contrast, CR expression was more variable at early developmental ages, stabilized to lower levels after P7 and showed a marked decline by P21. The results suggest that not only does calcium buffering capacity increase developmentally but also acquisition of enhanced buffering may be one mechanism by which neurons survive agent-induced alterations in calcium homeostasis.

P3-402: Age-related loss of calbindin identifies basal forebrain cholinergic neurons destined to degenerate in Alzheimer's disease

The reasons for the vulnerability of select neuronal populations in Alzheimer's Disease (AD) remain unknown. We have used the basal forebrain cholinergic neurons (BFCN), which are vulnerable to degeneration early in the course of AD, to determine of the causes of selective neuronal vulnerability. We have observed a selective loss of the calcium binding protein calbindin-D28K from the human BFCN in the course of normal aging. The calcium dysregulation caused by the loss of calbindin may contribute to the loss of BFCN in AD. In the present set of experiments, we investigated whether the BFCN which degenerate in AD are those which lose calbindin in the course of aging. Immunohistochemical methods were used in postmortem brains from young (<65 years), old and AD cases. Cell counting techniques were used to determine the numbers of immunoreactive neurons. The number of choline acetyltransferase (ChAT) and low affinity neurotrophin receptor (p75NTR) immunoreactive BFCN remained constant in old individuals when compared with the young and displayed a significant decrease in AD. In contrast, the number of calbindin-positive BFCN showed a significant decrease in normal aged individuals when compared with the young and remained constant in AD. The percentage of calbindin-positive BFCN in the aged was significantly lower when compared with the young. Of great interest, the percentage of the remaining BFCN in AD which contained calbindin immunoreactivity displayed a significant increase when compared with the normal aged, indicating that the majority of surviving BFCN in AD are calbindin-positive. Western blot analysis showed no increase in the total levels of calbinidin protein in AD basal forebrain, suggesting that the maintained calbindin immunoreactivity in AD BFCN is not due to reactive upregulation. These observations indicate that the loss of cholinergic neurons in AD occurs preferentially in the BFCN which lose their calbindin in the course of normal aging and that the BFCN which contain calbindin survive the neurodegenerative process. It is likely that loss of calbindin during aging deprives the BFCN from the calcium buffering capacity of this protein, leaving them vulnerable to degeneration due to calcium toxicity in AD.

Distribution of parvalbumin and calretinin immunoreactive interneurons in motor cortex from multiple sclerosis post-mortem tissue

Parvalbumin (PV) and calretinin (CR) are calcium binding proteins (CBP's) expressed in discrete GABAergic interneuron populations in the human cortex. CBP's are known to buffer calcium concentrations and protect neurons from increases in intracellular calcium. Perturbations in intracellular calcium can activate proteolytic enzymes including calpain, leading to deleterious effects to axons. Ca++-mediated mechanisms have been found to be associated with axonal pathology in MS and the restructuring of calcium channels has been shown to occur in experimental autoimmune encephalomyelitis (EAE) as well as multiple sclerosis tissue. Previous data indicates a reduction in the expression of the parvalbumin gene as well as reduced extension of neurites on parvalbumin expressing interneurons within multiple sclerosis normal appearing grey matter (NAGM). Modifications in interneuron parvalbumin or calretinin levels could change calcium buffering capacity, as well as the way these cells respond to neuronal insults. The present study was designed to compare CBP immunoreactive neurons in normal and multiple sclerosis post-mortem NAGM. To this end, we utilized immunofluorescent staining and high resolution confocal microscopy to map regions of the human motor cortex, and characterize layer specific CBP distribution in the normal and multiple sclerosis motor cortex. Our results indicate a significant reduction in the number of PV interneurons within layer 2 of the multiple sclerosis primary motor cortex with no concurrent change in number of calretinin positive neurons.

The Interplay between Calcium and the in Vitro Lectin and Chaperone Activities of Calreticulin

The ER resident protein calreticulin fulfills at least two important roles. It works as a chaperone preventing Golgi exit of non-native protein species and enhancing protein folding efficiency in either N-glycan-dependent, lectin chaperone, or classical chaperone, N-glycan-independent, modes and is one of the main calcium buffers in the cell. This last feature is independent from the lectin chaperone properties of the protein as this last activity is also observed in a CRT fragment lacking calcium buffer capacity. Here we study the interplay between calcium and the lectin and chaperone activities of CRT. The affinity of CRT for monoglucosylated glycans measured in solution by equilibrium dialysis and fluorescence anisotropy was not affected by the absence of calcium. Binding of CRT to monoglucosylated neoglycoproteins displaying either native or molten globule-like conformations was also independent of calcium concentration. Moreover, calcium and monoglucosylated glycans stabilized the CRT structure in an apparent additive, independent manner when the protein was subjected to thermal or urea-induced denaturation. In addition, the ability of CRT to decrease the level of aggregation of a chemically denatured monoglucosylated and nonglycosylated protein was also independent of calcium level.

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.