Comparing effects of CDK inhibition and E2F1/2 ablation on neuronal cell death pathways in vitro and after traumatic brain injury.

Traumatic brain injury (TBI) activates multiple neuronal cell death mechanisms, leading to post-traumatic neuronal loss and neurological deficits. TBI-induced cell cycle activation (CCA) in post-mitotic neurons causes regulated cell death involving cyclin-dependent kinase (CDK) activation and initiation of an E2F transcription factor-mediated pro-apoptotic program. Here we examine the mechanisms of CCA-dependent neuronal apoptosis in primary neurons in vitro and in mice exposed to controlled cortical impact (CCI). In contrast to our prior work demonstrating robust neuroprotective effects by CDK inhibitors after TBI, examination of neuronal apoptotic mechanisms in E2F1-/-/E2F2-/- or E2F2-/- transgenic mice following CCI suggests that E2F1 and/or E2F2 likely play only a modest role in neuronal cell loss after brain trauma. To elucidate more critical CCA molecular pathways involved in post-traumatic neuronal cell death, we investigated the neuroprotective effects and mechanisms of the potent CDK inhibitor CR8 in a DNA damage model of cell death in primary cortical neurons. CR8 treatment significantly reduced caspase activation and cleavage of caspase substrates, attenuating neuronal cell death. CR8 neuroprotective effects appeared to reflect inhibition of multiple pathways converging on the mitochondrion, including injury-induced elevation of pro-apoptotic Bcl-2 homology region 3 (BH3)-only proteins Puma and Noxa, thereby attenuating mitochondrial permeabilization and release of cytochrome c and AIF, with reduction of both caspase-dependent and -independent apoptosis. CR8 administration also limited injury-induced deficits in mitochondrial respiration. These neuroprotective effects may be explained by CR8-mediated inhibition of key upstream injury responses, including attenuation of c-Jun phosphorylation/activation as well as inhibition of p53 transactivation of BH3-only targets.

The Putative Drp1 Inhibitor mdivi-1 Is a Reversible Mitochondrial Complex I Inhibitor that Modulates Reactive Oxygen Species.

Mitochondrial fission mediated by the GTPase dynamin-related protein 1 (Drp1) is an attractive drug target in numerous maladies that range from heart disease to neurodegenerative disorders. The compound mdivi-1 is widely reported to inhibit Drp1-dependent fission, elongate mitochondria, and mitigate brain injury. Here, we show that mdivi-1 reversibly inhibits mitochondrial complex I-dependent O2 consumption and reverse electron transfer-mediated reactive oxygen species (ROS) production at concentrations (e.g., 50 µM) used to target mitochondrial fission. Respiratory inhibition is rescued by bypassing complex I using yeast NADH dehydrogenase Ndi1. Unexpectedly, respiratory impairment by mdivi-1 occurs without mitochondrial elongation, is not mimicked by Drp1 deletion, and is observed in Drp1-deficient fibroblasts. In addition, mdivi-1 poorly inhibits recombinant Drp1 GTPase activity (Ki > 1.2 mM). Overall, these results suggest that mdivi-1 is not a specific Drp1 inhibitor. The ability of mdivi-1 to reversibly inhibit complex I and modify mitochondrial ROS production may contribute to effects observed in disease models.

Mitochondrial E3 ubiquitin ligase MARCH5 controls mitochondrial fission and cell sensitivity to stress-induced apoptosis through regulation of MiD49 protein.

Ubiquitin- and proteasome-dependent outer mitochondrial membrane (OMM)-associated degradation (OMMAD) is critical for mitochondrial and cellular homeostasis. However, the scope and molecular mechanisms of the OMMAD pathways are still not well understood. We report that the OMM-associated E3 ubiquitin ligase MARCH5 controls dynamin-related protein 1 (Drp1)-dependent mitochondrial fission and cell sensitivity to stress-induced apoptosis. MARCH5 knockout selectively inhibited ubiquitination and proteasomal degradation of MiD49, a mitochondrial receptor of Drp1, and consequently led to mitochondrial fragmentation. Mitochondrial fragmentation in MARCH5(-/-) cells was not associated with inhibition of mitochondrial fusion or bioenergetic defects, supporting the possibility that MARCH5 is a negative regulator of mitochondrial fission. Both MARCH5 re-expression and MiD49 knockout in MARCH5(-/-) cells reversed mitochondrial fragmentation and reduced sensitivity to stress-induced apoptosis. These findings and data showing MARCH5-dependent degradation of MiD49 upon stress support the possibility that MARCH5 regulation of MiD49 is a novel mechanism controlling mitochondrial fission and, consequently, the cellular response to stress.

Low micromolar concentrations of the superoxide probe MitoSOX uncouple neural mitochondria and inhibit complex IV.

MitoSOX Red is a fluorescent probe used for the detection of mitochondrial reactive oxygen species by live cell imaging. The lipophilic, positively charged triphenylphosphonium moiety within MitoSOX concentrates the superoxide-sensitive dihydroethidium conjugate within the mitochondrial matrix. Here we investigated whether common MitoSOX imaging protocols influence mitochondrial bioenergetic function in primary rat cortical neurons and microglial cell lines. MitoSOX dose-dependently uncoupled neuronal respiration, whether present continuously in the assay medium or washed following a ten minute loading protocol. Concentrations of 5-10µM MitoSOX caused severe loss of ATP synthesis-linked respiration. Redistribution of MitoSOX to the cytoplasm and nucleus occurred concomitant to mitochondrial uncoupling. MitoSOX also dose-dependently decreased the maximal respiration rate and this impairment could not be rescued by delivery of a complex IV specific substrate, revealing complex IV inhibition. As in neurons, loading microglial cells with MitoSOX at low micromolar concentrations resulted in uncoupled mitochondria with reduced respiratory capacity whereas submicromolar MitoSOX had no adverse effects. The MitoSOX parent compound dihydroethidium also caused mitochondrial uncoupling and respiratory inhibition at low micromolar concentrations. However, these effects were abrogated by pre-incubating dihydroethidium with cation exchange beads to remove positively charged oxidation products, which would otherwise by sequestered by polarized mitochondria. Collectively, our results suggest that the matrix accumulation of MitoSOX or dihydroethidium oxidation products causes mitochondrial uncoupling and inhibition of complex IV. Because MitoSOX is inherently capable of causing severe mitochondrial dysfunction with the potential to alter superoxide production, its use therefore requires careful optimization in imaging protocols.

Transient assembly of F-actin on the outer mitochondrial membrane contributes to mitochondrial fission.

In addition to established membrane remodeling roles in various cellular locations, actin has recently emerged as a participant in mitochondrial fission. However, the underlying mechanisms of its participation remain largely unknown. We report that transient de novo F-actin assembly on the mitochondria occurs upon induction of mitochondrial fission and F-actin accumulates on the mitochondria without forming detectable submitochondrial foci. Impairing mitochondrial division through Drp1 knockout or inhibition prolonged the time of mitochondrial accumulation of F-actin and also led to abnormal mitochondrial accumulation of the actin regulatory factors cortactin, cofilin, and Arp2/3 complexes, suggesting that disassembly of mitochondrial F-actin depends on Drp1 activity. Furthermore, down-regulation of actin regulatory proteins led to elongation of mitochondria, associated with mitochondrial accumulation of Drp1. In addition, depletion of cortactin inhibited Mfn2 down-regulation- or FCCP-induced mitochondrial fragmentation. These data indicate that the dynamic assembly and disassembly of F-actin on the mitochondria participates in Drp1-mediated mitochondrial fission.

Photoactivatable green fluorescent protein-based visualization and quantification of mitochondrial fusion and mitochondrial network complexity in living cells.

Technological improvements in microscopy and the development of mitochondria-specific imaging molecular tools have illuminated the dynamic rearrangements of these essential organelles. These rearrangements are mainly the result of two opposing processes: mitochondrial fusion and mitochondrial fission. Consistent with this, in addition to mitochondrial motility, these two processes are major factors determining the overall degree of continuity of the mitochondrial network, as well as the average size of mitochondria within the cell. In this chapter, we detail the use of advanced confocal microscopy and mitochondrial matrix-targeted photoactivatable green fluorescent protein (mito-PAGFP) for the investigation of mitochondrial dynamics. We focus on direct visualization and quantification of mitochondrial fusion and mitochondrial network complexity in living mammalian cells. These assays were instrumental in important recent discoveries within the field of mitochondrial biology, including the role of mitochondrial fusion in the activation of mitochondrial steps in apoptosis, participation of Bcl-2 family proteins in mitochondrial morphogenesis, and stress-induced mitochondrial hyperfusion. We present some basic directions that should be helpful in designing mito-PAGFP-based experiments. Furthermore, since analyses of mitochondrial fusion using mito-PAGFP-based assays rely on time-lapse imaging, critical parameters of time-lapse microscopy and cell preparation are also discussed.

Use of potentiometric fluorophores in the measurement of mitochondrial reactive oxygen species.

Mitochondrial reactive oxygen species (ROS) are implicated in signal transduction, inflammation, neurodegenerative disorders, and normal aging. Net ROS release by isolated brain mitochondria derived from a mixture of neurons and glia is readily quantified using fluorescent dyes. Measuring intracellular ROS in intact neurons or glia and assigning the origin to mitochondria are far more difficult. In recent years, the proton-motive force crucial to mitochondrial function has been exploited to target a variety of compounds to the highly negative mitochondrial matrix using the lipophilic triphenylphosphonium cation (TPP(+)) as a "delivery" conjugate. Among these, MitoSOX Red, also called mito-hydroethidine or mito-dihydroethidium, is prevalently used for mitochondrial ROS estimation. Although the TPP(+) moiety of MitoSOX enables the manyfold accumulation of ROS-sensitive hydroethidine in the mitochondrial matrix, the membrane potential sensitivity conferred by TPP(+) creates a daunting set of challenges not often considered in the application of this dye. This chapter provides recommendations and cautionary notes on the use of potentiometric fluorescent indicators for the approximation of mitochondrial ROS in live neurons, with principles that can be extrapolated to nonneuronal cell types. It is concluded that mitochondrial membrane potential changes render accurate estimation of mitochondrial ROS using MitoSOX difficult to impossible. Consequently, knowledge of mitochondrial membrane potential is essential to the application of potentiometric fluorophores for the measurement of intramitochondrial ROS.

The N-terminal helix of Bcl-xL targets mitochondria.

Anti- and pro-apoptotic Bcl-2 family members regulate the mitochondrial phase of apoptotic cell death. The mitochondrial targeting mechanisms of Bcl-2 family proteins are tightly regulated. Known outer mitochondrial membrane targeting sequences include the C-terminal tail and central helical hairpin. Bcl-xL also localizes to the inner mitochondrial membrane, but these targeting sequences are unknown. Here we investigate the possibility that the N-terminus of Bcl-xL also contains mitochondrial targeting information. Amino acid residues 1-28 of Bcl-xL fused to EGFP are sufficient to target mitochondria. Although positive charges and helical propensity are required for targeting, similar to import sequences the N-terminus is not sufficient for efficient mitochondrial import.

N-terminally cleaved Bcl-xL mediates ischemia-induced neuronal death.

Transient global ischemia in rats induces delayed death of hippocampal CA1 neurons. Early events include caspase activation, cleavage of anti-death Bcl-2 family proteins and large mitochondrial channel activity. However, whether these events have a causal role in ischemia-induced neuronal death is unclear. We found that the Bcl-2 and Bcl-x(L) inhibitor ABT-737, which enhances death of tumor cells, protected rats against neuronal death in a clinically relevant model of brain ischemia. Bcl-x(L) is prominently expressed in adult neurons and can be cleaved by caspases to generate a pro-death fragment, ΔN-Bcl-x(L). We found that ABT-737 administered before or after ischemia inhibited ΔN-Bcl-x(L)-induced mitochondrial channel activity and neuronal death. To establish a causal role for ΔN-Bcl-x(L), we generated knock-in mice expressing a caspase-resistant form of Bcl-x(L). The knock-in mice exhibited markedly reduced mitochondrial channel activity and reduced vulnerability to ischemia-induced neuronal death. These findings suggest that truncated Bcl-x(L) could be a potentially important therapeutic target in ischemic brain injury.