Irina G. Stavrovskaya

Minocycline inhibits cytochrome c release and delays progression of amyotrophic lateral sclerosis in mice

Minocycline mediates neuroprotection in experimental models of neurodegeneration. It inhibits the activity of caspase-1, caspase-3, inducible form of nitric oxide synthetase (iNOS) and p38 mitogen-activated protein kinase (MAPK). Although minocycline does not directly inhibit these enzymes, the effects may result from interference with upstream mechanisms resulting in their secondary activation. Because the above-mentioned factors are important in amyotrophic lateral sclerosis (ALS), we tested minocycline in mice with ALS. Here we report that minocycline delays disease onset and extends survival in ALS mice. Given the broad efficacy of minocycline, understanding its mechanisms of action is of great importance. We find that minocycline inhibits mitochondrial permeability-transition-mediated cytochrome c release. Minocycline-mediated inhibition of cytochrome c release is demonstrated in vivo, in cells, and in isolated mitochondria. Understanding the mechanism of action of minocycline will assist in the development and testing of more powerful and effective analogues. Because of the safety record of minocycline, and its ability to penetrate the blood–brain barrier, this drug may be a novel therapy for ALS.

Minocycline inhibits caspase-independent and -dependent mitochondrial cell death pathways in models of Huntington's disease

Minocycline is broadly protective in neurologic disease models featuring cell death and is being evaluated in clinical trials. We previously demonstrated that minocycline-mediated protection against caspase-dependent cell death related to its ability to prevent mitochondrial cytochrome c release. These results do not explain whether or how minocycline protects against caspase-independent cell death. Furthermore, there is no information on whether Smac/Diablo or apoptosis-inducing factor might play a role in chronic neurodegeneration. In a striatal cell model of Huntingtons disease and in R6/2 mice, we demonstrate the association of cell death/disease progression with the recruitment of mitochondrial caspase-independent (apoptosis-inducing factor) and caspase-dependent (Smac/Diablo and cytochrome c) triggers. We show that minocycline is a drug that directly inhibits both caspase-independent and -dependent mitochondrial cell death pathways. Furthermore, this report demonstrates recruitment of Smac/Diablo and apoptosis-inducing factor in chronic neurodegeneration. Our results further delineate the mechanism by which minocycline mediates its remarkably broad neuroprotective effects.

Chemotherapy for the Brain: The Antitumor Antibiotic Mithramycin Prolongs Survival in a Mouse Model of Huntington's Disease

Huntingtons disease (HD) is a fully penetrant autosomal-dominant inherited neurological disorder caused by expanded CAG repeats in the Huntingtin gene. Transcriptional dysfunction, excitotoxicity, and oxidative stress have all been proposed to play important roles in the pathogenesis of HD. This study was designed to explore the therapeutic potential of mithramycin, a clinically approved guanosine-cytosine-rich DNA binding antitumor antibiotic. Pharmacological treatment of a transgenic mouse model of HD (R6/2) with mithramycin extended survival by 29.1%, greater than any single agent reported to date. Increased survival was accompanied by improved motor performance and markedly delayed neuropathological sequelae. To identify the functional mechanism for the salubrious effects of mithramycin, we examined transcriptional dysfunction in R6/2 mice. Consistent with transcriptional repression playing a role in the pathogenesis of HD, we found increased methylation of lysine 9 in histone H3, a well established mechanism of gene silencing. Mithramycin treatment prevented the increase in H3 methylation observed in R6/2 mice, suggesting that the enhanced survival and neuroprotection might be attributable to the alleviation of repressed gene expression vital to neuronal function and survival. Because it is Food and Drug Administration-approved, mithramycin is a promising drug for the treatment of HD.

Prophylactic Creatine Administration Mediates Neuroprotection in Cerebral Ischemia in Mice

Creatine mediates remarkable neuroprotection in experimental models of amyotrophic lateral sclerosis, Huntingtons disease, Parkinsons disease, and traumatic brain injury. Because caspase-mediated pathways are shared functional mechanistic components in these diseases, as well as in ischemia, we evaluated the effect of creatine supplementation on an experimental stroke model. Oral creatine administration resulted in a remarkable reduction in ischemic brain infarction and neuroprotection after cerebral ischemia in mice. Postischemic caspase-3 activation and cytochrome c release were significantly reduced in creatine-treated mice. Creatine administration buffered ischemia-mediated cerebral ATP depletion. These data provide the first direct correlation between the preservation of bioenergetic cellular status and the inhibition of activation of caspase cell-death pathways in vivo. An alternative explanation to our findings is that creatine is neuroprotective through other mechanisms that are independent of mitochondrial cell-death pathways, and therefore postischemic ATP preservation is the result of tissue spearing. Given its safety record, creatine might be considered as a novel therapeutic agent for inhibition of ischemic brain injury in humans. Prophylactic creatine supplementation, similar to what is recommended for an agent such as aspirin, may be considered for patients in high stroke-risk categories.

Clinically approved heterocyclics act on a mitochondrial target and reduce stroke-induced pathology.

Substantial evidence indicates that mitochondria are a major checkpoint in several pathways leading to neuronal cell death, but discerning critical propagation stages from downstream consequences has been difficult. The mitochondrial permeability transition (mPT) may be critical in stroke-related injury. To address this hypothesis, identify potential therapeutics, and screen for new uses for established drugs with known toxicity, 1,040 FDA-approved drugs and other bioactive compounds were tested as potential mPT inhibitors. We report the identification of 28 structurally related drugs, including tricyclic antidepressants and antipsychotics, capable of delaying the mPT. Clinically achievable doses of one drug in this general structural class that inhibits mPT, promethazine, were protective in both in vitro and mouse models of stroke. Specifically, promethazine protected primary neuronal cultures subjected to oxygen-glucose deprivation and reduced infarct size and neurological impairment in mice subjected to middle cerebral artery occlusion/reperfusion. These results, in conjunction with new insights provided to older studies, (a) suggest a class of safe, tolerable drugs for stroke and neurodegeneration; (b) provide new tools for understanding mitochondrial roles in neuronal cell death; (c) demonstrate the clinical/experimental value of screening collections of bioactive compounds enriched in clinically available agents; and (d) provide discovery-based evidence that mPT is an essential, causative event in stroke-related injury.

Methazolamide and Melatonin Inhibit Mitochondrial Cytochrome C Release and Are Neuroprotective in Experimental Models of Ischemic Injury

BACKGROUND AND PURPOSE The identification of a neuroprotective drug for stroke remains elusive. Given that mitochondria play a key role both in maintaining cellular energetic homeostasis and in triggering the activation of cell death pathways, we evaluated the efficacy of newly identified inhibitors of cytochrome c release in hypoxia/ischemia induced cell death. We demonstrate that methazolamide and melatonin are protective in cellular and in vivo models of neuronal hypoxia. METHODS The effects of methazolamide and melatonin were tested in oxygen/glucose deprivation-induced death of primary cerebrocortical neurons. Mitochondrial membrane potential, release of apoptogenic mitochondrial factors, pro-IL-1beta processing, and activation of caspase -1 and -3 were evaluated. Methazolamide and melatonin were also studied in a middle cerebral artery occlusion mouse model. Infarct volume, neurological function, and biochemical events were examined in the absence or presence of the 2 drugs. RESULTS Methazolamide and melatonin inhibit oxygen/glucose deprivation-induced cell death, loss of mitochondrial membrane potential, release of mitochondrial factors, pro-IL-1beta processing, and activation of caspase-1 and -3 in primary cerebrocortical neurons. Furthermore, they decrease infarct size and improve neurological scores after middle cerebral artery occlusion in mice. CONCLUSIONS We demonstrate that methazolamide and melatonin are neuroprotective against cerebral ischemia and provide evidence of the effectiveness of a mitochondrial-based drug screen in identifying neuroprotective drugs. Given the proven human safety of melatonin and methazolamide, and their ability to cross the blood-brain-barrier, these drugs are attractive as potential novel therapies for ischemic injury.

Soluble, Prefibrillar α-Synuclein Oligomers Promote Complex I-dependent, Ca2+-induced Mitochondrial Dysfunction

Background: Mitochondrial dysfunction and aggregation of α-synuclein both contribute to Parkinson disease. Results: Prefibrillar α-synuclein oligomers reduce the Ca2+ retention time of isolated mitochondria respiring with complex I but not II substrates. Conclusion: Oligomeric α-synuclein promotes mitochondrial dysfunction in a Ca2+- and respiratory substrate-dependent manner. Significance: The Ca2+-dependence of α-synucleins effects may contribute to selective neuronal vulnerability in Parkinson disease. α-Synuclein (αSyn) aggregation and mitochondrial dysfunction both contribute to the pathogenesis of Parkinson disease (PD). Although recent studies have suggested that mitochondrial association of αSyn may disrupt mitochondrial function, it is unclear what aggregation state of αSyn is most damaging to mitochondria and what conditions promote or inhibit the effect of toxic αSyn species. Because the neuronal populations most vulnerable in PD are characterized by large cytosolic Ca2+ oscillations that burden mitochondria, we examined mitochondrial Ca2+ stress in an in vitro system comprising isolated mitochondria and purified recombinant human αSyn in various aggregation states. Using fluorimetry to simultaneously measure four mitochondrial parameters, we observed that soluble, prefibrillar αSyn oligomers, but not monomeric or fibrillar αSyn, decreased the retention time of exogenously added Ca2+, promoted Ca2+-induced mitochondrial swelling and depolarization, and accelerated cytochrome c release. Inhibition of the permeability transition pore rescued these αSyn-induced changes in mitochondrial parameters. Interestingly, the mitotoxic effects of αSyn were specifically dependent upon both electron flow through complex I and mitochondrial uptake of exogenous Ca2+. Our results suggest that soluble prefibrillar αSyn oligomers recapitulate several mitochondrial phenotypes previously observed in animal and cell models of PD: complex I dysfunction, altered membrane potential, disrupted Ca2+ homeostasis, and enhanced cytochrome c release. These data reveal how the association of oligomeric αSyn with mitochondria can be detrimental to the function of cells with high Ca2+-handling requirements.

Inhibitors of Cytochrome c Release with Therapeutic Potential for Huntington's Disease

Release of mitochondrial cytochrome c resulting in downstream activation of cell death pathways has been suggested to play a role in neurologic diseases featuring cell death. However, the specific biologic importance of cytochrome c release has not been demonstrated in Huntingtons disease (HD). To evaluate the role of cytochrome c release, we screened a drug library to identify new inhibitors of cytochrome c release from mitochondria. Drugs effective at the level of purified mitochondria were evaluated in a cellular model of HD. As proof of principle, one drug was chosen for in depth evaluation in vitro and a transgenic mouse model of HD. Our findings demonstrate the utility of mitochondrial screening to identify inhibitors of cell death and provide further support for the important functional role of cytochrome c release in HD. Given that many of these compounds have been approved by the Food and Drug Administration for clinical usage and cross the blood–brain barrier, these drugs may lead to trials in patients.

Nortriptyline Protects Mitochondria and Reduces Cerebral Ischemia/Hypoxia Injury

Background and Purpose— Nortriptyline, an antidepressant, was identified as a strong inhibitor of mitochondrial permeability transition by our screening of a library of 1040 drugs. Because mitochondrial permeability transition and consequent mitochondrial dysfunction have been implicated in acute neuronal death, we proposed to investigate the possible neuroprotective effects of nortriptyline in cerebral ischemia. Methods— The effects of nortriptyline were first studied in oxygen/glucose deprivation-induced death of primary cerebrocortical neurons, a cellular model of cerebral ischemia. Mitochondrial membrane potential, mitochondrial factor release, and caspase 3 activation were evaluated after its treatment. Nortriptyline was also studied in a mouse model, which was established by occlusion of the middle cerebral artery. The infarct volume, neurological function, and biochemical events were examined in the absence or the presence of nortriptyline. Results— Nortriptyline inhibits oxygen/glucose deprivation-induced cell death, loss of mitochondrial membrane potential, downstream release of mitochondrial factors, and activation of caspase 3 in primary cerebrocortical neurons. Furthermore, it decreases infarct size and improves neurological scores after middle cerebral artery occlusion in mice. Conclusions— The ability of nortriptyline to inhibit mitochondrial factor release and caspase activation and further protect the animals correlates to its inhibitory effect on mitochondrial permeability transition in isolated mitochondria. This study indicated that nortriptyline is neuroprotective against cerebral ischemia. It also suggested mitochondrial permeability transition might be a valuable therapeutic target for acute neurodegeneration.

Neuroprotective mechanisms of creatine occur in the absence of mitochondrial creatine kinase.

There is substantial evidence that creatine administration exerts neuroprotective effects both in vitro and in vivo. The precise mechanisms for these neuroprotective effects however are as yet unclear. We investigated whether creatine administration could exert neuroprotective effects in mice deficient in ubiquitous mitochondrial creatine kinase (UbMi-CK). UbMi-CK-deficient mice showed increased sensitivity to 1-methyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced dopamine depletion and loss of tyrosine hydroxylase (TH) stained neurons. Isolated mitochondria from these mice showed no alterations in calcium retention, oxygen utilization, membrane potential, or swelling in response to a calcium challenge. Creatine administration significantly increased brain concentrations of both creatine and PCr in the UbMi-CK knockout mice. Creatine administration to the UbMi-CK-deficient mice exerted significant neuroprotective effects against MPTP toxicity that were comparable in magnitude to those seen in wild-type mice. These results suggest that the neuroprotective effects of creatine are not mediated by an effect on UbMi-CK to inhibit the mitochondrial permeability transition, and are more likely to be mediated by maintenance of appropriate ATP/ADP and PCr/Cr levels.