Maturation of neuronal synapses is thought to involve mitochondria. Bcl-xL protein inhibits mitochondria-mediated apoptosis but may have other functions in healthy adult neurons in which Bcl-xL is abundant. Here, we report that overexpression of Bcl-xL postsynaptically increases frequency and amplitude of spontaneous miniature synaptic currents in rat hippocampal neurons in culture. Bcl-xL, overexpressed either pre or postsynaptically, increases synapse number, the number and size of synaptic vesicle clusters, and mitochondrial localization to vesicle clusters and synapses, likely accounting for the changes in miniature synaptic currents. Conversely, knockdown of Bcl-xL or inhibiting it with ABT-737 decreases these morphological parameters. The mitochondrial fission protein, dynamin-related protein 1 (Drp1), is a GTPase known to localize to synapses and affect synaptic function and structure. The effects of Bcl-xL appear mediated through Drp1 because overexpression of Drp1 increases synaptic markers, and overexpression of the dominant-negative dnDrp1-K38A decreases them. Furthermore, Bcl-xL coimmunoprecipitates with Drp1 in tissue lysates, and in a recombinant system, Bcl-xL protein stimulates GTPase activity of Drp1. These findings suggest that Bcl-xL positively regulates Drp1 to alter mitochondrial function in a manner that stimulates synapse formation.Bcl-2 ͉ synaptic transmission ͉ mitochondria ͉ cell death ͉ ABT-737

Transient global ischemia is a neuronal insult that induces delayed cell death. A hallmark event in the early. These findings reveal a novel mechanism by which ischemic insults disrupt the functional integrity of the outer mitochondrial membrane and implicate ⌬N-BCL-xL and VDAC in the large, Zn 2ϩ -dependent mitochondrial channels observed in post-ischemic hippocampal mitochondria.

Transient forebrain or global ischemia induces delayed neuronal death in vulnerable CA1 pyramidal cells with many features of apoptosis. A brief period of ischemia, i.e., ischemic preconditioning, affords robust protection of CA1 neurons against a subsequent more prolonged ischemic challenge. Here we show that preconditioning acts via PI3K/Akt signaling to block the ischemia-induced cascade involving mitochondrial translocation of Bad, assembly of Bad with Bcl-x L , cleavage of Bcl-x L to form its prodeath fragment, ΔN-Bcl-x L , activation of large-conductance channels in the mitochondrial outer membrane, mitochondrial release of cytochrome c and Smac/DIABLO (second mitochondria-derived activator of caspases/direct IAP-binding protein with low pI), caspase activation, and neuronal death. These findings show how preconditioning acts to prevent the release of cytochrome c and Smac/DIABLO from mitochondria and to preserve the integrity of the mitochondrial membrane. The specific PI3K inhibitor LY294002 administered in vivo 1 h before or immediately after ischemia or up to 120 h later significantly reverses preconditioning-induced protection, indicating a requirement for sustained PI3K signaling in ischemic tolerance. These findings implicate PI3K/Akt signaling in maintenance of the integrity of the mitochondrial outer membrane.

Transient global ischemia in rats induces delayed death of hippocampal CA1 neurons. Early events include caspase activation, cleavage of anti-death Bcl-2 family proteins and large mitochondrial channel activity. However, a causal role of these events in ischemia-induced neuronal death is unclear. Unexpectedly, we found that the Bcl-2/Bcl-xL inhibitor ABT-737, which enhances death of tumor cells, protects rats against neuronal death in a clinically relevant model of brain ischemia. Bcl-xL is prominently expressed in adult neurons and can be cleaved by caspases to generate a pro-death fragment ΔN-Bcl-xL. We found that ABT-737 administered before or after ischemia inhibited ΔN-Bcl-xL-induced mitochondrial channel activity and neuronal death. To establish a causal role for ΔN-Bcl-xL, we generated knockin mice expressing caspase-resistant Bcl-xL. The knockin mice exhibit strikingly reduced mitochondrial channel activity and reduced vulnerability to ischemia-induced neuronal death. These findings point to truncated Bcl-xL as a potentially important therapeutic target in ischemic brain injury.

Olfactory cilia contain the known components of olfactory signal transduction, including a high density of cyclic-nucleotide-gated (CNG) channels. CNG channels play an important role in mediating odor detection. The channels are activated by cAMP, which is formed by a G-protein-coupled transduction cascade. Frog olfactory cilia are 25-200 microm in length, so the spatial distribution of CNG channels along the length should be important in determining the sensitivity of odor detection. We have recorded from excised cilia and modeled diffusion of cAMP into a cilium to determine the spatial distribution of the CNG channels along the ciliary length. The proximal segment, which in frog is the first 20% of the cilium, appears to express a small fraction of the CNG channels, whereas the distal segment contains the majority, mostly clustered in one region.

Defects in primary cilia lead to a variety of human diseases. One of these, polycystic kidney disease, can be caused by defects in a Ca²⁺-gated ion channel (TRPP2) found on the cilium. Other ciliary functions also contribute to cystogenesis, and defects in apical Ca²⁺ homeostasis have been implicated. By recording directly from the native cilia of mIMCD-3 cells, a murine cell line of renal epithelial origin, we have identified a second Ca²⁺-gated channel in the ciliary membrane: the transient receptor potential cation channel, subfamily M, member 4 (TRPM4). In excised primary cilia, TRPM4 was found to have a low sensitivity to Ca²⁺, with an EC₅₀ of 646 μM at +100 mV. It was inhibited by MgATP and by 9-phenanthrol. The channel was not permeable to Ca²⁺ or Cl⁻ and had a permeability ratio PK/PNa of 1.42. Reducing the expression of Trpm4 mRNA with short hairpin (sh) RNA reduced the TRPM4 current by 87% and shortened primary cilia by 43%. When phospholipase C was inhibited, the sensitivity to cytoplasmic Ca²⁺ greatly increased (EC₅₀ = 26 μM at +100 mV), which is consistent with previous reports that phosphatidylinositol 4,5-bisphosphate (PIP2) modulates the channel. MgATP did not restore the channel to a preinactivation state, suggesting that the enzyme or substrate necessary for making PIP2 is not abundant in primary cilia of mIMCD-3 cells. The function of TRPM4 in renal primary cilia is not yet known, but it is likely to influence the apical Ca²⁺ dynamics of the cell, perhaps in tandem with TRPP2.

J. Neurochem. (2010) 114, 430–439. Abstract Proteins that control the excitability of neurons, including voltage‐dependent ion channels and neurotransmitter receptors, reside in a membrane lipid environment that includes sphingomyelin, but the influence of the metabolism of this lipid on excitability is unknown. Sphingomyelin in the plasma membrane can be cleaved by neutral sphingomyelinases (nSMase) to generate ceramides and sphingosine‐1‐phosphate (S1P) which have been shown to play a variety of roles in cellular signaling processes. We found that application of nSMase to hippocampal slices results in a selective enhancement in the population spike amplitude, resulting in fEPSP‐PS potentiation of the CA3‐CA1 schaeffer collateral synapse. Single cell recordings showed that nSMase activity increases action potential frequency in CA1 neurons in a reversible manner. Additional current clamp recordings showed that nSMase reduces the slow after‐hyperpolarization after a burst of action potentials. Mass spectrometry‐based measurements demonstrated that nSMase activity induces a rapid increase in the levels of ceramides and S1P in cells in hippocampal slices. The ability of nSMase to increase CA1 neuron excitability was blocked by an inhibitor of sphingosine kinase, the enzyme that converts ceramide to S1P. Moreover, direct intracellular application of S1P to CA1 neurons increased action potential firing. Our findings suggest roles for sphingomyelin metabolism and S1P in the positive regulation of the excitability of hippocampal neurons.

Identification of detailed features of neuronal systems is an important challenge in the biosciences today. Cilia are long thin structures that extend from the olfactory receptor neurons into the nasal mucus. Transduction of an odor into an electrical signal occurs in the membranes of the cilia. The cyclic-nucleotide-gated (CNG) channels which reside in the ciliary membrane and are activated by adenosine 3',5'-cyclic monophosphate (cAMP) allow a depolarizing influx of Ca(2+) and Na(+) and are thought to initiate the electrical signal.In this paper, a mathematical model consisting of two nonlinear differential equations and a constrained Fredholm integral equation of the first kind is developed to model experiments involving the diffusion of cAMP into cilia and the resulting electrical activity. The unknowns in the problem are the concentration of cAMP, the membrane potential and, the quantity of most interest in this work, the distribution of CNG channels along the length of a cilium. A simple numerical method is derived that can be used to obtain estimates of the spatial distribution of CNG ion channels along the length of a cilium. Certain computations indicate that this mathematical problem is ill-conditioned.