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...

Previous imaging and postmortem studies have reported a reduction in brain volume and a decrease in the size and density of neurons in the dorsolateral prefrontal cortex (dlPFC, area 9) of subjects with major depressive disorder (MDD).1,2 These findings suggest that synapse number and function are decreased in dlPFC of depressed patients. However, there has been no direct evidence for synapse loss in MDD and the gene expression alterations underlying these effects have not been identified. Here we use microarray gene profiling and electron microscopic stereology to reveal decreased expression of synaptic function-related genes in dlPFC of MDD subjects and a corresponding reduction in the number of synapses. We also identify a transcriptional repressor that is increased in MDD, and that when expressed in PFC neurons is sufficient to decrease expression of synapse-related genes, cause loss of spines and dendrites, and produce depressive behavior in rodent models of depression.

The gene encoding the forkhead box transcription factor, FOXP2, is essential for developing the full articulatory power of human language. Mutations of FOXP2 cause developmental verbal dyspraxia (DVD), a speech and language disorder that compromises the fluent production of words and the correct use and comprehension of grammar. FOXP2 patients have structural and functional abnormalities in the striatum of the basal ganglia, which also express high levels of FOXP2. Since human speech and learned vocalizations in songbirds bear behavioral and neural parallels, songbirds provide a genuine model for investigating the basic principles of speech and its pathologies. In zebra finch Area X, a basal ganglia structure necessary for song learning, FoxP2 expression increases during the time when song learning occurs. Here, we used lentivirus-mediated RNA interference (RNAi) to reduce FoxP2 levels in Area X during song development. Knockdown of FoxP2 resulted in an incomplete and inaccurate imitation of tutor song. Inaccurate vocal imitation was already evident early during song ontogeny and persisted into adulthood. The acoustic structure and the duration of adult song syllables were abnormally variable, similar to word production in children with DVD. Our findings provide the first example of a functional gene analysis in songbirds and suggest that normal auditory-guided vocal motor learning requires FoxP2.

It is becoming increasingly clear that single cortical neurons encode complex and behaviorally relevant signals, but efficient means to study gene functions in small networks and single neurons in vivo are still lacking. Here, we establish a method for genetic manipulation and subsequent phenotypic analysis of individual cortical neurons in vivo. First, lentiviral vectors are used for neuron-specific gene delivery from ␣-calcium͞calmodulin-dependent protein kinase II or Synapsin I promoters, optionally in combination with gene knockdown by means of U6 promoter-driven expression of short-interfering RNAs. Second, the phenotypic analysis at the level of single cortical cells is carried out by using two-photon microscopy-based techniques: high-resolution two-photon timelapse imaging is used to monitor structural dynamics of dendritic spines and axonal projections, whereas cellular response properties are analyzed electrophysiologically by two-photon microscopydirected whole-cell recordings. This approach is ideally suited for analysis of gene functions in individual neurons in the intact brain.patch-clamp recording ͉ two-photon imaging