Michael Forte

Properties of the Permeability Transition Pore in Mitochondria Devoid of Cyclophilin D

We have studied the properties of the permeability transition pore (PTP) in mitochondria from the liver of mice where the Ppif gene encoding for mitochondrial Cyclophilin D (CyP-D) had been inactivated. Mitochondria from Ppif–/– mice had no CyP-D and displayed a striking desensitization of the PTP to Ca2+, in that pore opening required about twice the Ca2+ load necessary to open the pore in strain-matched, wild-type mitochondria. Mitochondria lacking CyP-D were insensitive to Cyclosporin A (CsA), which increased the Ca2+ retention capacity only in mitochondria from wild-type mice. The PTP response to ubiquinone 0, depolarization, pH, adenine nucleotides, and thiol oxidants was similar in mitochondria from wild-type and Ppif–/– mice. These experiments demonstrate that (i) the PTP can form and open in the absence of CyP-D, (ii) that CyP-D represents the target for PTP inhibition by CsA, and (iii) that CyP-D modulates the sensitivity of the PTP to Ca2+ but not its regulation by the proton electrochemical gradient, adenine nucleotides, and oxidative stress. These results have major implications for our current understanding of the PTP and its modulation in vitro and in vivo.

Dimers of mitochondrial ATP synthase form the permeability transition pore

Here we define the molecular nature of the mitochondrial permeability transition pore (PTP), a key effector of cell death. The PTP is regulated by matrix cyclophilin D (CyPD), which also binds the lateral stalk of the FOF1 ATP synthase. We show that CyPD binds the oligomycin sensitivity-conferring protein subunit of the enzyme at the same site as the ATP synthase inhibitor benzodiazepine 423 (Bz-423), that Bz-423 sensitizes the PTP to Ca2+ like CyPD itself, and that decreasing oligomycin sensitivity-conferring protein expression by RNAi increases the sensitivity of the PTP to Ca2+. Purified dimers of the ATP synthase, which did not contain voltage-dependent anion channel or adenine nucleotide translocator, were reconstituted into lipid bilayers. In the presence of Ca2+, addition of Bz-423 triggered opening of a channel with currents that were typical of the mitochondrial megachannel, which is the PTP electrophysiological equivalent. Channel openings were inhibited by the ATP synthase inhibitor AMP-PNP (γ-imino ATP, a nonhydrolyzable ATP analog) and Mg2+/ADP. These results indicate that the PTP forms from dimers of the ATP synthase.

The mitochondrial permeability transition from in vitro artifact to disease target

The mitochondrial permeability transition pore is a high conductance channel whose opening leads to an increase of mitochondrial inner membrane permeability to solutes with molecular masses up to ≈ 1500 Da. In this review we trace the rise of the permeability transition pore from the status of in vitro artifact to that of effector mechanism of cell death. We then cover recent results based on genetic inactivation of putative permeability transition pore components, and discuss their meaning for our understanding of pore structure. Finally, we discuss evidence indicating that the permeability transition pore plays a role in pathophysiology, with specific emphasis on in vivo models of disease.

Associative Learning Disrupted by Impaired Gs Signaling in Drosophila Mushroom Bodies

Disruptions in mushroom body (MB) or central complex (CC) brain structures impair Drosophila associative olfactory learning. Perturbations in adenosine 3′,5′ monophosphate signaling also disrupt learning. To integrate these observations, expression of a constitutively activated stimulatory heterotrimeric guanosine triphosphate-binding protein α subunit (Gαs*) was targeted to these brain structures. The ability to associate odors with electroshock was abolished when Gαs* was targeted to MB, but not CC, structures, whereas sensorimotor responses to these stimuli remained normal. Expression of Gαs* did not affect gross MB morphology, and wild-type Gαs expression did not affect learning. Thus, olfactory learning depends on regulated Gs signaling in Drosophila MBs.

A mitochondrial perspective on cell death

The role of mitochondria as crucial participants in cell death programs is well established, yet the mechanisms responsible for the release of mitochondrial activators and the role of BCL2 family proteins in this process remain controversial. Here, we point out the limitations of current approaches used to monitor the physiological responses of mitochondria during cell death, the implications arising from modern views of mitochondrial structure, and briefly assess two proposed mechanisms for the release of mitochondrial proteins during apoptosis.

Heterotrimeric G Proteins Direct Two Modes of Asymmetric Cell Division in the Drosophila Nervous System

In Drosophila, distinct mechanisms orient asymmetric cell division along the apical-basal axis in neuroblasts and along the anterior-posterior axis in sensory organ precursor (SOP) cells. Here, we show that heterotrimeric G proteins are essential for asymmetric cell division in both cell types. The G protein subunit G(alpha)i localizes apically in neuroblasts and anteriorly in SOP cells before and during mitosis. Interfering with G protein function by G(alpha)i overexpression or depletion of heterotrimeric G protein complexes causes defects in spindle orientation and asymmetric localization of determinants. G(alpha)i is colocalized and associated with Pins, a protein that induces the release of the betagamma subunit and might act as a receptor-independent G protein activator. Thus, asymmetric activation of heterotrimeric G proteins by a receptor-independent mechanism may orient asymmetric cell divisions in different cell types.

VDAC, a channel in the outer mitochondrial membrane.

Proteins that form aqueous channels in membranes generate conduction pathways with a variety of shapes and sizes. Perhaps the largest channel-forming protein is the 2-MDa ryanodine receptor while the smallest may be gramicidin. However, the size of the conducting pathway is not correlated with the amount of protein mass needed to make up the structure, as demonstrated by the fact that some of the narrowest conducting pathways are produced by very large amounts of protein (e. g., 0.3 MDa for the Na+/ K+/Ca2+ channel family). In contrast, the focus of this review, the voltage-dependent anion channel (VDAC) of the mitochondrial outer membrane, produces one of the largest aqueous pathways from a single 30-kDa protein. VDAC also demonstrates that functional complexity does not seem to correlate well with the amount of protein used to form a channel. VDAC has a small amount of protein mass but displays complex behavior. It has two voltage-gating processes, can be controlled by metabolites and regulatory proteins, is able to form complexes with other proteins and enzymes, and responds to the protein concentration of the cytoplasm (Colombini, 1994). Thus, many functions are packed into a single, relatively small VDAC protein.

Ectopic G-protein expression in dopamine and serotonin neurons blocks cocaine sensitization in Drosophila melanogaster

Sensitization to repeated doses of psychostimulants is thought to be an important component underlying the addictive process in humans [1] [2] [3] [4]. In all vertebrate animal models, including humans [5], and even in fruit flies, sensitization is observed after repeated exposure to volatilized crack cocaine [6]. In vertebrates, sensitization is thought to be initiated by processes occurring in brain regions that contain dopamine cell bodies [2] [7]. Here, we show that modulated cell signaling in the Drosophila dopamine and serotonin neurons plays an essential role in cocaine sensitization. Targeted expression of either a stimulatory (Galpha(s)) or inhibitory (Galpha(i)) Galpha subunit, or tetanus toxin light chain (TNT) in dopamine and serotonin neurons of living flies blocked behavioral sensitization to repeated cocaine exposures. These flies showed alterations in their initial cocaine responsiveness that correlated with compensatory adaptations of postsynaptic receptor sensitivity. Finally, repeated drug stimulation of a nerve cord preparation that is postsynaptic to the brain amine cells failed to induce sensitization, further showing the importance of presynaptic modulation in sensitization.

Hexokinase II detachment from mitochondria triggers apoptosis through the permeability transition pore independent of voltage-dependent anion channels

Type II hexokinase is overexpressed in most neoplastic cells, and it mainly localizes on the outer mitochondrial membrane. Hexokinase II dissociation from mitochondria triggers apoptosis. The prevailing model postulates that hexokinase II release from its mitochondrial interactor, the voltage-dependent anion channel, prompts outer mitochondrial membrane permeabilization and the ensuing release of apoptogenic proteins, and that these events are inhibited by growth factor signalling. Here we show that a hexokinase II N-terminal peptide selectively detaches hexokinase II from mitochondria and activates apoptosis. These events are abrogated by inhibiting two established permeability transition pore modulators, the adenine nucleotide translocator or cyclophilin D, or in cyclophilin D knock-out cells. Conversely, insulin stimulation or genetic ablation of the voltage-dependent anion channel do not affect cell death induction by the hexokinase II peptide. Therefore, hexokinase II detachment from mitochondria transduces a permeability transition pore opening signal that results in cell death and does not require the voltage-dependent anion channel. These findings have profound implications for our understanding of the pathways of outer mitochondrial membrane permeabilization and their inactivation in tumors.

The Mitochondrial Permeability Transition Pore: Channel Formation by F-ATP Synthase, Integration in Signal Transduction, and Role in Pathophysiology

The mitochondrial permeability transition (PT) is a permeability increase of the inner mitochondrial membrane mediated by a channel, the permeability transition pore (PTP). After a brief historical introduction, we cover the key regulatory features of the PTP and provide a critical assessment of putative protein components that have been tested by genetic analysis. The discovery that under conditions of oxidative stress the F-ATP synthases of mammals, yeast, and Drosophila can be turned into Ca(2+)-dependent channels, whose electrophysiological properties match those of the corresponding PTPs, opens new perspectives to the field. We discuss structural and functional features of F-ATP synthases that may provide clues to its transition from an energy-conserving into an energy-dissipating device as well as recent advances on signal transduction to the PTP and on its role in cellular pathophysiology.