Summary Mitochondria are dynamic organelles, remodeling and exchanging contents during cyclic fusion and fission. Genetic mutations of mitofusin (Mfn) 2 interrupt mitochondrial fusion and cause the untreatable neurodegenerative condition, Charcot Marie Tooth disease type 2A (CMT2A). It has not been possible to directly modulate mitochondrial fusion, in part because the structural basis of mitofusin function is incompletely understood. Here we show that mitofusins adopt either a fusion-constrained or fusion-permissive molecular conformation directed by specific intramolecular binding interactions, and demonstrate that mitofusin-dependent mitochondrial fusion can be regulated by targeting these conformational transitions. Based on this model we engineered a cell-permeant minipeptide to destabilize fusion-constrained mitofusin and promote the fusion-permissive conformation, reversing mitochondrial abnormalities in cultured fibroblasts and neurons harboring CMT2A gene defects. The relationship between mitofusin conformational plasticity and mitochondrial dynamism uncovers a central mechanism regulating mitochondrial fusion whose manipulation can correct mitochondrial pathology triggered by defective or imbalanced mitochondrial dynamics.

Endogenous neurosteroids have rapid actions on ion channels, particularly GABA(A) receptors, which are potentiated by nanomolar concentrations of 3alpha-hydroxypregnane neurosteroids. Previous evidence suggests that 3beta-hydroxypregnane steroids may competitively antagonize potentiation induced by their 3alpha diastereomers. Because of the potential importance of antagonists as experimental and clinical tools, we characterized the functional effect of 3beta-hydroxysteroids. Although 3beta-hydroxysteroids reduced the potentiation induced by 3alpha-hydroxysteroids, 3beta-hydroxysteroids acted noncompetitively with respect to potentiating steroids and inhibited the largest degrees of potentiation most effectively. Potentiation by high concentrations of barbiturates was also reduced by 3beta-hydroxysteroids. 3beta-Hydroxysteroids are also direct, noncompetitive GABA(A) receptor antagonists. 3beta-Hydroxysteroids coapplied with GABA significantly inhibited responses to > or =15 microm GABA. The profile of block was similar to that exhibited by sulfated steroids, known blockers of GABA(A) receptors. This direct, noncompetitive effect of 3beta-hydroxysteroids was sufficient to account for the apparent antagonism of potentiating steroids. Mutated receptors exhibiting decreased sensitivity to sulfated steroid block were insensitive to both the direct effects of 3beta-hydroxysteroids on GABA(A) responses and the reduction of potentiating steroid effects. At concentrations that had little effect on GABAergic synaptic currents, 3beta-hydroxysteroids and low concentrations of sulfated steroids significantly reversed the potentiation of synaptic currents induced by 3alpha-hydroxysteroids. We conclude that 3beta-hydroxypregnane steroids are not direct antagonists of potentiating steroids but rather are noncompetitive, likely state-dependent, blockers of GABA(A) receptors. Nevertheless, these steroids may be useful functional blockers of potentiating steroids when used at concentrations that do not affect baseline neurotransmission.

The ability to fuel neurons via glycogenolysis is believed to be an important function of glia. Indeed, the slow, rather than immediate, depression of synaptic transmission in hippocampal slices during exogenous glucose deprivation suggests that intrinsic energy reservoirs help to sustain neurotransmission. It is believed that glia fuel neighboring neurons via diffusible monocarboxylates such as pyruvate and lactate, although a role for glucose has been proposed also. Using alpha-cyano-4-hydroxycinnamate (4-CIN) to inhibit monocarboxylate transport and cytochalasin B (CCB) to inhibit glucose transport, we examined the role of glucose and monocarboxylates in supporting the functional and morphological integrity of hippocampal neurons during glucose deprivation. Although 200 microM 4-CIN failed to depress EPSPs supported by 10 mM glucose, pretreatment with 4-CIN accelerated the depression of EPSPs during glucose deprivation. 4-CIN also accelerated the decline in glucose-supported EPSPs after administration of 50 microM CCB, whereas CCB failed to alter the slow decay of pyruvate-supported EPSPs during pyruvate deprivation. 4-CIN did not alter the morphology of pyramidal neurons in the presence of 10 mM glucose but produced significant damage during glucose deprivation or CCB administration. These results suggest that endogenous monocarboxylates rather than glucose maintain neuronal integrity during energy deprivation. Furthermore, EPSPs supported by 2-3.3 mM glucose were sensitive to 4-CIN, suggesting that endogenous monocarboxylates are involved in maintaining neuronal function even under conditions of mild glucose deprivation.

1. We used whole cell recordings to compare passive membrane properties and synaptic properties of postnatal rat hippocampal neurons grown for 7-15 days in either conventional mass cultures or on physically restricted microisland cultures. Despite matching microisland and mass culture cell across several variables, there were significant differences between neurons in the two groups regarding passive membrane characteristics and synaptic properties. 2. Microisland neurons displayed significantly faster charging of the membrane capacitance than mass culture counterparts matched with microisland neurons for age, somal diameter, and transmitter phenotype. When we used a two-compartment equivalent circuit model to quantify this result, microisland neurons displayed approximately half the distal capacitance of mass culture neurons. These data suggest that microisland neurons elaborate less extensive neuritic arborizations than mass culture neurons. 3. Evoked synaptic responses were enhanced on microislands compared with mass cultures. Excitatory and inhibitory autaptic currents were more frequent and displayed larger amplitudes on single-neuron microislands than in matched mass culture neurons. 4. In recordings from pairs of neurons in the two environments, we observed a significantly higher probability of obtaining a monosynaptic response on two-neuron microislands than in matched mass culture pairs (85% vs. 42%). Evoked excitatory postsynaptic currents were also significantly larger in the microisland environment, with evoked excitatory synaptic currents from two-neuron microislands exhibiting a mean amplitude 20-fold larger than mass culture monosynaptic responses. 5. The differences in evoked synaptic responses were not reflected in differences in the amplitude or frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs). Analysis of mEPSC rise times, decay times, and peak amplitudes within individual cells suggests that electrotonic filtering is not an important contributor to the variability of peak amplitudes and decay times of synaptic currents in cells of either culture environment. However, composite data across neurons in both cultures reveal a significant correlation between mEPSC rise and decay times. 6. Out results suggest that the microisland preparation may be a useful tool for exploring factors that influence synapse formation and development. Additionally, the preparation is a particularly convenient model for the study of single-neuron-mediated synaptic events.

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