Indrapal N. Singh

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 3h 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 → †NO2 + †OH) may be particularly effective for the treatment of TBI, although the therapeutic window for this neuroprotective approach might only be 3 h.

Synergistic increases in intracellular Ca2+, and the release of MCP-1, RANTES, and IL-6 by astrocytes treated with opiates and HIV-1 Tat

Recent evidence suggests that injection drug users who abuse heroin are at increased risk of CNS complications from human immunodeficiency virus (HIV) infection. Opiate drugs may intrinsically alter the pathogenesis of HIV by directly modulating immune function and by directly modifying the CNS response to HIV. Despite this, the mechanisms by which opiates increase the neuropathogenesis of HIV are uncertain. In the present study, we describe the effect of morphine and the HIV‐1 protein toxin Tat1‐72 on astroglial function in cultures derived from ICR mice. Astroglia maintain the blood‐brain barrier and influence inflammatory signaling in the CNS. Astrocytes can express μ‐opioid receptors, and are likely targets for abused opiates, which preferentially activate μ‐opioid receptors. While Tat alone disrupts astrocyte function, when combined with morphine, Tat causes synergistic increases in [Ca2+]i. Moreover, astrocyte cultures treated with morphine and Tat showed exaggerated increases in chemokine release, including monocyte chemoattractant protein‐1 (MCP‐1) and regulated on activation, normal T cell expressed and secreted (RANTES), as well as interleukin‐6 (IL‐6). Morphine‐Tat interactions were prevented by the μ‐opioid receptor antagonist β‐funaltrexamine, or by immunoneutralizing Tat1‐72 or substituting a nontoxic, deletion mutant (TatΔ31‐61). Our findings suggest that opiates may increase the vulnerability of the CNS to viral entry (via recruitment of monocytes/macrophages) and ensuing HIV encephalitis by synergistically increasing MCP‐1 and RANTES release by astrocytes. The results further suggest that astrocytes are key intermediaries in opiate‐HIV interactions and disruptions in astroglial function and inflammatory signaling may contribute to an accelerated neuropathogenesis in HIV‐infected individuals who abuse opiates.

Peroxynitrite-Mediated Oxidative Damage to Brain Mitochondria: Protective Effects of Peroxynitrite Scavengers

Peroxynitrite‐mediated oxidative damage has been implicated in brain mitochondrial respiratory dysfunction after traumatic brain injury (TBI), which precedes the onset of neuronal loss. The aim of this study was to investigate the detrimental effects of the peroxynitrite donor SIN‐1 (3‐morpholinosydnonimine) on isolated brain mitochondria and to screen penicillamine, a stoichiometric (1:1) peroxynitrite‐scavenging agent, and tempol, a catalytic scavenger of peroxynitrite‐derived radicals, as antioxidant mitochondrial protectants. Exposure of the isolated mitochondria to SIN‐1 caused a significant dose‐dependent decrease in the respiratory control ratio and was accompanied by a significant increase in state II respiration, followed by significant decreases (P < 0.05) in states III and V. These functional alterations occurred together with significant increases in mitochondrial protein carbonyl (PC), lipid peroxidation–related 4‐hydroxynonenal (4‐HNE), and 3‐nitrotyrosine (3‐NT) content. Penicillamine hydrochloride (10 μM) partially but significantly (P < 0.05) protected against SIN‐1‐induced decreases in states III and V. However, a 2.5 μM concentration of tempol was able to significantly antagonize a 4‐fold molar excess (10 μM) concentration of SIN‐1 as effectively as were higher tempol concentrations, consistent with the likelihood that tempol works by a catalytic mechanism. The protection of mitochondrial respiration by penicillamine and tempol occurred in parallel with attenuation of PC, 4‐HNE, and 3‐NT. These results indicate that SIN‐1 causes mitochondrial oxidative damage and complex I dysfunction and that antioxidant compounds that target either peroxynitrite or its radicals may be effective mitochondrial protectants in the treatment of neural injury.

Apoptotic death of striatal neurons induced by human immunodeficiency virus-1 Tat and gp120: Differential involvement of caspase-3 and endonuclease G.

Human immunodeficiency virus-1 (HIV-1) infection affects the striatum, resulting in gliosis and neuronal losses. To determine whether HIV-1 proteins induce striatal neurotoxicity through an apoptotic mechanism, mouse striatal neurons isolated on embryonic day 15 and the effects of HIV-1 Tat1–72 and gp120 on survival were assessed in vitro. Mitochondrial release of cytochrome c, caspase-3 activation, and neuron survival, as well as an alternative apoptotic pathway involving endonuclease G (endo G), were assessed at 4 h, 24 h, 48 h, and/or 72 h using enzyme assays and immunoblotting. Both HIV-1 Tat and gp120 significantly increased caspase-3 activation in a concentration-dependent manner in striatal neurons at 4 h following continuous exposure in vitro. Tat1–72 and gp120 caused significant neuronal losses at 48 h and/or 72 h. Tat1–72 increased cytochrome c release, and caspase-3 and endo G activation at 4 h, 24 h, and/or 72 h. By contrast, gp120 increased caspase-3 activation, but failed to increase cytochrome c or endo G levels in the cytoplasm at 4 h, 24 h, and/or 72 h. The cell permeant caspase inhibitor Z-DEVD-FMK significantly attenuated gp120-induced, but not Tat1–72-induced, neuronal death, suggesting that gp120 acts in large part through the activation of caspase(s), whereas Tat1–72-induced neurotoxicity was accompanied by activating an alternative pathway involving endo G. Thus, although Tat1–72 and gp120 induced significant neurotoxicity, the nature of the apoptotic events preceding death differed. Collectively, our findings suggest that HIV-1 proteins are intrinsically toxic to striatal neurons and the pathogenesis is mediated through separate actions involving both caspase-3 and endo G.

Comparative neuroprotective effects of cyclosporin A and NIM811, a nonimmunosuppressive cyclosporin A analog, following traumatic brain injury

Earlier experiments have shown that cyclosporin A (CsA) and its non-calcineurin inhibitory analog NIM811 attenuate mitochondrial dysfunction after experimental traumatic brain injury (TBI). Presently, we compared the neuroprotective effects of previously determined mitochondrial protective doses of CsA (20 mg/kg intraperitoneally) and NIM811 (10 mg/kg intraperitoneally) when administered at 15 mins postinjury in preventing cytoskeletal (α-spectrin) degradation, neuro-degeneration, and neurological dysfunction after severe (1.0 mm) controlled cortical impact (CCI) TBI in mice. In a first set of experiments, we analyzed calpain-mediated α-spectrin proteolysis at 24 h postinjury. Both NIM811 and CsA significantly attenuated the increased α-spectrin breakdown products observed in vehicle-treated animals (P < 0.005). In a second set of experiments, treatment of animals with either NIM811 or CsA at 15 mins and again at 24 h postinjury attenuated motor function impairment at 48 h and 7 days (P < 0.005) and neurodegeneration at 7 days postinjury (P < 0.0001). Delayed administration of NIM811 out to 12 h was still able to significantly reduce α-spectrin degradation. These results show that the neuroprotective mechanism of CsA involves maintenance of mitochondrial integrity and that calcineurin inhibition plays little or no role because the non-calcineurin inhibitory analog, NIM811, is as effective as CsA.

Neuroprotective effects of tempol, a catalytic scavenger of peroxynitrite-derived free radicals, in a mouse traumatic brain injury model.

We examined the ability of tempol, a catalytic scavenger of peroxynitrite (PN)-derived free radicals, to reduce cortical oxidative damage, mitochondrial dysfunction, calpain-mediated cytoskeletal (α-spectrin) degradation, and neurodegeneration, and to improve behavioral recovery after a severe (depth 1.0u2009mm), unilateral controlled cortical impact traumatic brain injury (CCI-TBI) in male CF-1 mice. Administration of a single 300u2009mg/kg intraperitoneal dose of tempol 15u2009mins after TBI produced a complete suppression of PN-mediated oxidative damage (3-nitrotyrosine, 3NT) in injured cortical tissue at 1u2009h after injury. Identical tempol dosing maintained respiratory function and attenuated 3NT in isolated cortical mitochondria at 12u2009h after injury, the peak of mitochondrial dysfunction. Multiple dosing with tempol (300u2009mg/kg intraperitoneally at 15u2009mins, 3, 6, 9, and 12u2009h) also suppressed α-spectrin degradation by 45% at its 24u2009h post-injury peak. The same dosing regimen improved 48u2009h motor function and produced a significant, but limited (17.4%, P<0.05), decrease in hemispheric neurodegeneration at 7 days. These results are consistent with a mechanistic link between PN-mediated oxidative damage to brain mitochondria, calpain-mediated proteolytic damage, and neurodegeneration. However, the modest neuroprotective effect of tempol suggests that multitarget combination strategies may be needed to interfere with posttraumatic secondary injury to a degree worthy of clinical translation.

Mitochondrial protection after traumatic brain injury by scavenging lipid peroxyl radicals

J. Neurochem. (2010) 114, 271–280.

Attenuation of acute mitochondrial dysfunction after traumatic brain injury in mice by NIM811, a non-immunosuppressive cyclosporin A analog.

Following traumatic brain injury (TBI), mitochondrial function becomes compromised. Mitochondrial dysfunction is characterized by intra-mitochondrial Ca(2+) accumulation, induction of oxidative damage, and mitochondrial permeability transition (mPT). Experimental studies show that cyclosporin A (CsA) inhibits mPT. However, CsA also inhibits calcineurin. In the present study, we conducted a dose-response analysis of NIM811, a non-calcineurin inhibitory CsA analog, on mitochondrial dysfunction following TBI in mice, and compared the effects of the optimal dose of NIM811 (10 mg/kg i.p.) against an optimized dose of CsA (20 mg/kg i.p.). Male CF-1 mice were subjected to severe TBI utilizing the controlled cortical impact model. Mitochondrial respiration was assessed from animals treated with either NIM811, CsA, or vehicle 15 min post-injury. The respiratory control ratio (RCR) of mitochondria from vehicle-treated animals was significantly (p<0.01) lower at 3 or 12 h post-TBI, relative to shams. Treatment of animals with either NIM811 or CsA significantly (p<0.03) attenuated this reduction. Consistent with this finding, both NIM811 and CsA significantly reduced lipid peroxidative and protein nitrative damage to mitochondria at 12 h post-TBI. These results showing the ability of NIM811 to fully duplicate the mitochondrial protective efficacy of CsA supports the conclusion that inhibition of the mPT may be sufficient to explain CsAs protective effects.

PATHOBIOLOGY OF DYNORPHINS IN TRAUMA AND DISEASE

Dynorphins, endogenous opioid neuropeptides derived from the prodynorphin gene, are involved in a variety of normative physiologic functions including antinociception and neuroendocrine signaling, and may be protective to neurons and oligodendroglia via their opioid receptor-mediated effects. However, under experimental or pathophysiological conditions in which dynorphin levels are substantially elevated, these peptides are excitotoxic largely through actions at glutamate receptors. Because the excitotoxic actions of dynorphins require supraphysiological concentrations or prolonged tissue exposure, there has likely been little evolutionary pressure to ameliorate the maladaptive, non-opioid receptor mediated consequences of dynorphins. Thus, dynorphins can have protective and/or proapoptotic actions in neurons and glia, and the net effect may depend upon the distribution of receptors in a particular region and the amount of dynorphin released. Increased prodynorphin gene expression is observed in several disease states and disruptions in dynorphin processing can accompany pathophysiological situations. Aberrant processing may contribute to the net negative effects of dysregulated dynorphin production by tilting the balance towards dynorphin derivatives that are toxic to neurons and/or oligodendroglia. Evidence outlined in this review suggests that a variety of CNS pathologies alter dynorphin biogenesis. Such alterations are likely maladaptive and contribute to secondary injury and the pathogenesis of disease.

Preferential vulnerability of astroglia and glial precursors to combined opioid and HIV-1 Tat exposure in vitro.

Human immunodeficiency virus (HIV)‐1 infection can cause characteristic neural defects such as progressive motor dysfunction, striatal pathology and gliosis. Recent evidence suggests that HIV‐induced pathogenesis is exacerbated by heroin abuse and that the synergistic neurotoxicity is a direct effect of heroin on the CNS, an alarming observation considering the high incidence of HIV infection with injection drug abuse. Although HIV infection results in neurodegeneration, neurons themselves are not directly infected. Instead, HIV affects microglia and astroglia, which subsequently contributes to the neurodegenerative changes. Opioid receptors are widely expressed by macroglia and macroglial precursors, and the activation of µ‐opioid receptors can modulate programmed cell death, as well as the response of neural cells to cytotoxic insults. For this reason, we questioned whether opioid drugs might modify the vulnerability of macroglia and macroglial precursors to HIV‐1 Tat protein. To address this problem, the effects of morphine and/or HIV Tat1−72 on the viability of macroglia and macroglial precursors were assessed in mixed‐glial cultures derived from mouse striatum. Our findings indicate that sustained exposure to morphine and Tat1−72 viral protein induces the preferential death of glial precursors and some astrocytes. Moreover, the increased cell death is mediated by µ‐opioid receptors and accompanied by the activation of caspase‐3. Our results imply that opiates can enhance the cytotoxicity of HIV‐1 Tat through direct actions on glial precursors and/or astroglia, suggesting novel cellular targets for HIV–opiate interactions.