Mitochondria maintain tight regulation of inner mitochondrial membrane (IMM) permeability to sustain ATP production. Stressful events cause cellular calcium (Ca 2+ ) dysregulation followed by rapid loss of IMM potential known as permeability transition (PT), which produces osmotic shifts, metabolic dysfunction, and cell death. The molecular identity of the mitochondrial PT pore (mPTP) was previously unknown. We show that the purified reconstituted c-subunit ring of the F O of the F 1 F O ATP synthase forms a voltage-sensitive channel, the persistent opening of which leads to rapid and uncontrolled depolarization of the IMM in cells. Prolonged high matrix Ca 2+ enlarges the c-subunit ring and unhooks it from cyclophilin D/cyclosporine A binding sites in the ATP synthase F 1 , providing a mechanism for mPTP opening. In contrast, recombinant F 1 beta-subunit applied exogenously to the purified c-subunit enhances the probability of pore closure. Depletion of the c-subunit attenuates Ca 2+ -induced IMM depolarization and inhibits Ca 2+ and reactive oxygen species-induced cell death whereas increasing the expression or single-channel conductance of the c-subunit sensitizes to death. We conclude that a highly regulated c-subunit leak channel is a candidate for the mPTP. Beyond cell death, these findings also imply that increasing the probability of c-subunit channel closure in a healthy cell will enhance IMM coupling and increase cellular metabolic efficiency.metabolism | necrosis | apoptosis | ion channel | excitotoxicity M itochondria produce ATP by oxidative phosphorylation (OXPHOS). Leak currents in the inner mitochondrial membrane (IMM) reduce the efficiency of this process by uncoupling the electron transport system from ATP synthase activity. Many studies have described the biophysical and pharmacological features of an IMM pore [the mitochondrial permeability transition pore (mPTP)] that is responsible for a rapid IMM uncoupling, causing osmotic shifts within the mitochondrial matrix in the setting of cellular Ca 2+ dysregulation and adenine nucleotide depletion (1-4). Some studies suggest that such uncoupling also functions during physiological events and that the mPTP may transiently operate as a Ca 2+ -release channel (5-7). Although models for the molecular identity of the mPTP have been proposed (8), deletions of putative components, such as adenine nucleotide translocase (ANT) and the voltagedependent anion channel (VDAC), have failed to prevent rapid depolarizations (9). In the meantime, nonpore forming regulatory components of the mPTP, such as cyclophilin D (CypD), have been extensively investigated (10, 11).We recently reported a leak conductance sensitive to ATP/ ADP and the Bcl-2 family member B-cell lymphoma-extra large (Bcl-x L ) within the membrane of isolated submitochondrial vesicles (SMVs) enriched in ATP synthase (12, 13). We demonstrated binding of Bcl-x L within F 1 to the beta-subunit of the ATP synthase, suggesting that the channel responsible for the leak conductance lies within the memb...

Anti-apoptotic Bcl2 family proteins such as Bcl-xL protect cells from death by sequestering apoptotic molecules, but also contribute to normal neuronal function. We find in hippocampal neurons that Bcl-xL enhances the efficiency of energy metabolism. Our evidence suggests that Bcl-xL interacts directly with the beta subunit of the F1FO ATP synthase, decreasing an ion leak within the F1FO ATPase complex and thereby increasing net transport of H+ by F1FO during F1FO ATPase activity. By patch clamping submitochondrial vesicles enriched in F1FO ATP synthase complexes, we find that, in the presence of ATP, pharmacological or genetic inhibition of Bcl-xL increases the membrane leak conductance. In addition, recombinant Bcl-xL protein directly increases ATPase activity of purified synthase complexes, while inhibition of endogenous Bcl-xL decreases F1FO enzymatic activity. Our findings suggest that increased mitochondrial efficiency contributes to the enhanced synaptic efficacy found in Bcl-xL expressing neurons.

Transplantation of GABAergic interneurons (INs) can provide long-term functional benefits in animal models of epilepsy and other neurological disorders. Whereas GABAergic INs can be differentiated from embryonic stem cells, alternative sources of GABAergic INs may be more tractable for disease modeling and transplantation. We identified five factors (Foxg1, Sox2, Ascl1, Dlx5, and Lhx6) that convert mouse fibroblasts into induced GABAergic INs (iGABA-INs) possessing molecular signatures of telencephalic INs. Factor overexpression activates transcriptional networks required for GABAergic fate specification. iGABA-INs display progressively maturing firing patterns comparable to cortical INs, form functional synapses, and release GABA. Importantly, iGABA-INs survive and mature upon being grafted into mouse hippocampus. Optogenetic stimulation demonstrated functional integration of grafted iGABA-INs into host circuitry, triggering inhibition of host granule neuron activity. These five factors also converted human cells into functional GABAergic INs. These properties suggest that iGABA-INs have potential for disease modeling and cell-based therapeutic approaches to neurological disorders.

Graphene has the potential to make a very significant impact on society, with important applications in the biomedical field. The possibility to engineer graphene-based medical devices at the neuronal interface is of particular interest, making it imperative to determine the biocompatibility of graphene materials with neuronal cells. Here we conducted a comprehensive analysis of the effects of chronic and acute exposure of rat primary cortical neurons to few-layer pristine graphene (GR) and monolayer graphene oxide (GO) flakes. By combining a range of cell biology, microscopy, electrophysiology, and "omics" approaches we characterized the graphene-neuron interaction from the first steps of membrane contact and internalization to the long-term effects on cell viability, synaptic transmission, and cell metabolism. GR/GO flakes are found in contact with the neuronal membrane, free in the cytoplasm, and internalized through the endolysosomal pathway, with no significant impact on neuron viability. However, GO exposure selectively caused the inhibition of excitatory transmission, paralleled by a reduction in the number of excitatory synaptic contacts, and a concomitant enhancement of the inhibitory activity. This was accompanied by induction of autophagy, altered Ca(2+) dynamics, and a downregulation of some of the main players in the regulation of Ca(2+) homeostasis in both excitatory and inhibitory neurons. Our results show that, although graphene exposure does not impact neuron viability, it does nevertheless have important effects on neuronal transmission and network functionality, thus warranting caution when planning to employ this material for neurobiological applications.