Yulia Kushnareva

Nitric oxide‐induced mitochondrial fission is regulated by dynamin‐related GTPases in neurons

Mitochondria are present as tubular organelles in neuronal projections. Here, we report that mitochondria undergo profound fission in response to nitric oxide (NO) in cortical neurons of primary cultures. Mitochondrial fission by NO occurs long before neurite injury and neuronal cell death. Furthermore, fission is accompanied by ultrastructural damage of mitochondria, autophagy, ATP decline and generation of free radicals. Fission is occasionally asymmetric and can be reversible. Strikingly, mitochondrial fission is also an early event in ischemic stroke in vivo. Mitofusin 1 (Mfn1) or dominant‐negative Dynamin related protein 1 (Drp1K38A) inhibits mitochondrial fission induced by NO, rotenone and Amyloid‐β peptide. Conversely, overexpression of Drp1 or Fis1 elicits fission and increases neuronal loss. Importantly, NO‐induced neuronal cell death was mitigated by Mfn1 and Drp1K38A. Thus, persistent mitochondrial fission may play a causal role in NO‐mediated neurotoxicity.

Complex I-mediated reactive oxygen species generation: modulation by cytochrome c and NAD(P)+ oxidation-reduction state.

Several lines of evidence indicate that mitochondrial reactive oxygen species (ROS) generation is the major source of oxidative stress in the cell. It has been shown that ROS production accompanies cytochrome c release in different apoptotic paradigms, but the site(s) of ROS production remain obscure. In the current study, we demonstrate that loss of cytochrome c by mitochondria oxidizing NAD(+)-linked substrates results in a dramatic increase of ROS production and respiratory inhibition. This increased ROS production can be mimicked by rotenone, a complex I inhibitor, as well as other chemical inhibitors of electron flow that act further downstream in the electron transport chain. The effects of cytochrome c depletion from mitoplasts on ROS production and respiration are reversible upon addition of exogenous cytochrome c. Thus in these models of mitochondrial injury, a primary site of ROS generation in both brain and heart mitochondria is proximal to the rotenone inhibitory site, rather than in complex III. ROS production at complex I is critically dependent upon a highly reduced state of the mitochondrial NAD(P)(+) pool and is achieved upon nearly complete inhibition of the respiratory chain. Redox clamp experiments using the acetoacetate/L-beta-hydroxybutyrate couple in the presence of a maximally inhibitory rotenone concentration suggest that the site is approx. 50 mV more electronegative than the NADH/NAD(+) couple. In the absence of inhibitors, this highly reduced state of mitochondria can be induced by reverse electron flow from succinate to NAD(+), accounting for profound ROS production in the presence of succinate. These results lead us to propose a model of thermodynamic control of mitochondrial ROS production which suggests that the ROS-generating site of complex I is the Fe-S centre N-1a.

Opa1-Mediated Cristae Opening Is Bax/Bak and BH3 Dependent, Required for Apoptosis, and Independent of Bak Oligomerization

Controversy surrounds the role and mechanism of mitochondrial cristae remodeling in apoptosis. Here we show that the proapoptotic BH3-only proteins Bid and Bim induced full cytochrome c release but only a subtle alteration of crista junctions, which involved the disassembly of Opa1 complexes. Both mitochondrial outer membrane permeabilization (MOMP) and crista junction opening (CJO) were caspase independent and required a functional BH3 domain and Bax/Bak. However, MOMP and CJO were experimentally separable. Pharmacological blockade of MOMP did not prevent Opa1 disassembly and CJO; moreover, expression of a disassembly-resistant mutant Opa1 (Q297V) blocked cytochrome c release and apoptosis but not Bax activation. Thus, apoptosis requires a subtle form of Opa1-dependent crista remodeling that is induced by BH3-only proteins and Bax/Bak but independent of MOMP.

Mitochondrial Cyclic AMP Response Element-binding Protein (CREB) Mediates Mitochondrial Gene Expression and Neuronal Survival

Cyclic AMP response element-binding protein (CREB) is a widely expressed transcription factor whose role in neuronal protection is now well established. Here we report that CREB is present in the mitochondrial matrix of neurons and that it binds directly to cyclic AMP response elements (CREs) found within the mitochondrial genome. Disruption of CREB activity in the mitochondria decreases the expression of a subset of mitochondrial genes, including the ND5 subunit of complex I, down-regulates complex I-dependent mitochondrial respiration, and increases susceptibility to 3-nitropropionic acid, a mitochondrial toxin that induces a clinical and pathological phenotype similar to Huntington disease. These results demonstrate that regulation of mitochondrial gene expression by mitochondrial CREB, in part, underlies the protective effects of CREB and raise the possibility that decreased mitochondrial CREB activity contributes to the mitochondrial dysfunction and neuronal loss associated with neurodegenerative disorders.

Mitochondrial Shape Governs BAX-Induced Membrane Permeabilization and Apoptosis

Proapoptotic BCL-2 proteins converge upon the outer mitochondrial membrane (OMM) to promote mitochondrial outer membrane permeabilization (MOMP) and apoptosis. Here we investigated the mechanistic relationship between mitochondrial shape and MOMP and provide evidence that BAX requires a distinct mitochondrial size to induce MOMP. We utilized the terminal unfolded protein response pathway to systematically define proapoptotic BCL-2 protein composition after stress and then directly interrogated their requirement for a productive mitochondrial size. Complementary biochemical, cellular, in vivo, and ex vivo studies reveal that Mfn1, a GTPase involved in mitochondrial fusion, establishes a mitochondrial size that is permissive for proapoptotic BCL-2 family function. Cells with hyperfragmented mitochondria, along with size-restricted OMM model systems, fail to support BAX-dependent membrane association and permeabilization due to an inability to stabilize BAXα9·membrane interactions. This work identifies a mechanistic contribution of mitochondrial size in dictating BAX activation, MOMP, and apoptosis.

Bax Activation Initiates the Assembly of a Multimeric Catalyst that Facilitates Bax Pore Formation in Mitochondrial Outer Membranes

Bax promotes mitochondrial permeabilization during apoptosis via a phase-transition-like event in the membrane and oligomerization of a catalyst molecule that facilitates Bax pore formation.

Excitotoxic injury to mitochondria isolated from cultured neurons

Neuronal death in response to excitotoxic levels of glutamate is dependent upon mitochondrial Ca2+ accumulation and is associated with a drop in ATP levels and a loss in ionic homeostasis. Yet the mapping of temporal events in mitochondria subsequent to Ca2+ sequestration is incomplete. By isolating mitochondria from primary cultures, we discovered that glutamate treatment of cortical neurons for 10 min caused 44% inhibition of ADP-stimulated respiration, whereas the maximal rate of electron transport (uncoupler-stimulated respiration) was inhibited by ∼10%. The Ca2+ load in mitochondria from glutamate-treated neurons was estimated to be 167 ± 19 nmol/mg protein. The glutamate-induced Ca2+ load was less than the maximal Ca2+ uptake capacity of the mitochondria determined in vitro (363 ± 35 nmol/mg protein). Comparatively, mitochondria isolated from cerebellar granule cells demonstrated a higher Ca2+ uptake capacity (686 ± 71 nmol/mg protein) than the cortical mitochondria, and the glutamate-induced load of Ca2+ was a smaller percentage of the maximal Ca2+ uptake capacity. Thus, this study indicated that Ca2+-induced impairment of mitochondrial ATP production is an early event in the excitotoxic cascade that may contribute to decreased cellular ATP and loss of ionic homeostasis that precede commitment to neuronal death.

Bioenergetics and cell death

Mitochondrial bioenergetic function is a key to cell life and death. Cells need energy not only to support their vital functions but also to die gracefully. Execution of an apoptotic program includes energy‐dependent steps, including kinase signaling, formation of the apoptosome, and effector caspase activation. Under conditions of bioenergetic collapse, cells are diverted toward necrotic demise. Mitochondrial outer membrane permeabilization (MOMP) is a decisive event in the execution of apoptosis. It is also causally linked to a decline in bioenergetic function via different mechanisms, not merely due to cytochrome c dispersion. MOMP‐induced bioenergetic deficiency is usually irreversible and commits cells to die, even when caspases are inactive. Here, we discuss the mechanisms by which MOMP impacts bioenergetics in different cell death paradigms.

Caspase-independent Mitochondrial Cell Death Results from Loss of Respiration, Not Cytotoxic Protein Release

In apoptosis, mitochondrial outer membrane permeabilization (MOMP) triggers caspase-dependent death. However, cells undergo clonogenic death even if caspases are blocked. One proposed mechanism involved the release of cytotoxic proteins (e.g., AIF and endoG) from mitochondria. To initiate MOMP directly without side effects, we created a tamoxifen-switchable BimS fusion protein. Surprisingly, even after MOMP, caspase-inhibited cells replicated DNA and divided for approximately 48 h before undergoing proliferation arrest. AIF and endoG remained in mitochondria. However, cells gradually lost mitochondrial membrane potential and ATP content, and DNA synthesis slowed to a halt by 72 h. These defects resulted from a partial loss of respiratory function, occurring 4-8 h after MOMP, that was not merely due to dispersion of cytochrome c. In particular, Complex I activity was completely lost, and Complex IV activity was reduced by approximately 70%, whereas Complex II was unaffected. Later, cells exhibited a more profound loss of mitochondrial protein constituents. Thus, under caspase inhibition, MOMP-induced clonogenic death results from a progressive loss of mitochondrial function, rather than the release of cytotoxic proteins from mitochondria.

Mitochondrial ROS metabolism: 10 Years later

The role of mitochondria in oxidative stress is well recognized, but many questions are still to be answered. This article is intended to update our comprehensive review in 2005 by highlighting the progress in understanding of mitochondrial reactive oxygen species (ROS) metabolism over the past 10 years. We review the recently identified or re-appraised sources of ROS generation in mitochondria, such as p66shc protein, succinate dehydrogenase, and recently discovered properties of the mitochondrial antioxidant system. We also reflect upon some controversies, disputes, and misconceptions that confound the field.