Faculty Bibliography

The Ca(V)2 family of voltage-gated calcium channels, present in presynaptic nerve terminals, regulates exocytosis and synaptic transmission. Cumulative inactivation of these channels occurs during trains of action potentials, and this may control short-term dynamics at the synapse. Inactivation during brief, repetitive stimulation is primarily attributed to closed-state inactivation, and several factors modulate the susceptibility of voltage-gated calcium channels to this form of inactivation. We show that alternative splicing of an exon in a cytoplasmic region of the Ca(V)2.2 channel modulates its sensitivity to inactivation during trains of action potential waveforms. The presence of this exon, exon 18a, protects the Ca(V)2.2 channel from entry into closed-state inactivation specifically during short (10 ms to 3 s) and small depolarizations of the membrane potential (-60 mV to -50 mV). The reduced sensitivity to closed-state inactivation within this dynamic range likely underlies the differential responsiveness of Ca(V)2.2 splice isoforms to trains of action potential waveforms. Regulated alternative splicing of Ca(V)2.2 represents a possible mechanism for modulating short-term dynamics of synaptic efficacy in different regions of the nervous system.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional enzyme that is very critical for synaptic plasticity and memory formation. Although significant progress has been made in understanding the role of postsynaptic CaMKII in synaptic plasticity, very little is known about its presynaptic function during plasticity changes. Here we report that KN-93, a membrane-permeable CaMKII inhibitor, blocked glutamate-induced increases in the frequency of miniature excitatory postsynaptic currents (mEPSCs) and the number of presynaptic functional boutons in cultured hippocampal pyramidal neurons. In addition, presynaptic injection of the membrane-impermeable CaMKII inhibitor peptide 281-309 blocked synaptic plasticity induced by tetanus, glutamate, or NO/cGMP pathway activation as expressed by long-lasting increases in EPSC amplitude and functional presynaptic boutons. Presynaptic injection of CaMKII itself coupled with weak tetanus produced an immediate and long-lasting enhancement of EPSC amplitude. Thus, the present results conclusively prove that presynaptic CaMKII is essential for synaptic plasticity in cultured hippocampal neurons

Mutations in presenilins are the major cause of familial Alzheimer's disease, but the pathogenic mechanism by which presenilin mutations cause memory loss and neurodegeneration remains unclear. Here we demonstrate that conditional double knockout mice lacking both presenilins in the postnatal forebrain exhibit impairments in hippocampal memory and synaptic plasticity. These deficits are associated with specific reductions in NMDA receptor-mediated responses and synaptic levels of NMDA receptors and alphaCaMKII. Furthermore, loss of presenilins causes reduced expression of CBP and CREB/CBP target genes, such as c-fos and BDNF. With increasing age, mutant mice develop striking neurodegeneration of the cerebral cortex and worsening impairments of memory and synaptic function. Neurodegeneration is accompanied by increased levels of the Cdk5 activator p25 and hyperphosphorylated tau. These results define essential roles and molecular targets of presenilins in synaptic plasticity, learning and memory, and neuronal survival in the adult cerebral cortex

Although synapsins are abundant synaptic vesicle proteins that are widely used as markers of presynaptic terminals, the mechanisms that target synapsins to presynaptic terminals have not been elucidated. We have addressed this question by imaging the targeting of green fluorescent protein-tagged synapsins in cultured hippocampal neurons. Whereas all synapsin isoforms targeted robustly to presynaptic terminals in wild-type neurons, synapsin Ib scarcely targeted in neurons in which all synapsins were knocked-out. Coexpression of other synapsin isoforms significantly strengthened the targeting of synapsin Ib in knock-out neurons, indicating that heterodimerization is required for synapsin Ib to target. Truncation mutagenesis revealed that synapsin Ia targets via distributed binding sites that include domains B, C, and E. Although domain A was not necessary for targeting, its presence enhanced targeting. Domain D inhibited targeting, but this inhibition was overcome by domain E. Thus, multiple intermolecular and intramolecular interactions are required for synapsins to target to presynaptic terminals

Human and rat Kv10.1a and b cDNAs encode silent K+ channel pore-forming subunits that modify the electrophysiological properties of Kv2.1. These alternatively spliced variants arise by the usage of an alternative site of splicing in exon 1 producing an 11-amino acid insertion in the linker between the first and second transmembrane domains in Kv10.1b. In human, the Kv10s mRNA were detected by Northern blot in brain kidney lung and pancreas. In brain, they were expressed in cortex, hippocampus, caudate, putamen, amygdala and weakly in substantia nigra. In rat, Kv10.1 products were detected in brain and weakly in testes. In situ hybridization in rat brain shows that Kv10.1 mRNAs are expressed in cortex, olfactory cortical structures, basal ganglia/striatal structures, hippocampus and in many nuclei of the amygdala complex. The CA3 and dentate gyrus of the hippocampus present a gradient that show a progression from high level of expression in the caudo-ventro-medial area to a weak level in the dorso-rostral area. The CA1 and CA2 areas had low levels throughout the hippocampus. Several small nuclei were also labeled in the thalamus, hypothalamus, pons, midbrain, and medulla oblongata. Co-injection of Kv2.1 and Kv10.1a or b mRNAs in Xenopus oocytes produced smaller currents that in the Kv2.1 injected oocytes and a moderate reduction of the inactivation rate without any appreciable change in recovery from inactivation or voltage dependence of activation or inactivation. At higher concentration, Kv10.1a also reduces the activation rate and a more important reduction in the inactivation rate. The gene that encodes for Kv10.1 mRNAs maps to chromosome 2p22.1 in human, 6q12 in rat and 17E4 in mouse, locations consistent with the known systeny for human, rat and mouse chromosomes

Neuropathy target esterase (NTE) is a neuronal membrane protein originally identified for its property to be modified by organo-phosphates (OPs), which in humans cause neuropathy characterized by axonal degeneration. Drosophila mutants for the homolog gene of NTE, swisscheese (sws), indicated a possible involvement of sws in the regulation of axon-glial cell interaction during glial wrapping. However, the role of NTE/sws in mammalian brain pathophysiology remains unknown. To investigate NTE function in vivo, we used the cre/loxP site-specific recombination strategy to generate mice with a specific deletion of NTE in neuronal tissues. Here we show that loss of NTE leads to prominent neuronal pathology in the hippocampus and thalamus and also defects in the cerebellum. Absence of NTE resulted in disruption of the endoplasmic reticulum, vacuolation of nerve cell bodies, and abnormal reticular aggregates. Thus, these results identify a physiological role for NTE in the nervous system and indicate that a loss-of-function mechanism may contribute to neurodegenerative diseases characterized by vacuolation and neuronal loss

GABA(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. Whereas heterodimerization between GABA(B) receptor GABA(B)R1 and GABA(B)R2 subunits is essential for functional expression, how neurons coordinate the assembly of these critical receptors remains to be established. Here we have identified Marlin-1, a novel GABA(B) receptor-binding protein that associates specifically with the GABA(B)R1 subunit in yeast, tissue culture cells, and neurons. Marlin-1 is expressed in the brain and exhibits a granular distribution in cultured hippocampal neurons. Marlin-1 binds different RNA species including the 3'-untranslated regions of both the GABA(B)R1 and GABA(B)R2 mRNAs in vitro and also associates with RNA in cultured neurons. Inhibition of Marlin-1 expression via small RNA interference technology results in enhanced intracellular levels of the GABA(B)R2 receptor subunit without affecting the level of GABA(B)R1. Together our results suggest that Marlin-1 functions to regulate the cellular levels of GABA(B) R2 subunits, which may have significant effects on the production of functional GABA(B) receptor heterodimers. Therefore, our observations provide an added level of regulation for the control of GABA(B) receptor expression and for the efficacy of inhibitory synaptic transmission

Pericytes in the central nervous system (CNS) are hypothesized to be involved in important circulatory functions, including local blood flow regulation, angiogenesis, immune reaction, and regulation of blood-brain barrier. Despite these putative functions, functional correlates of pericytes in vivo are scarce. We have labeled CNS pericytes using the dextran-conjugated fluorescent calcium indicator Calcium Green I and imaged them in somatosensory cortex of the mouse in vivo. Intracellular calcium concentration in pericytes showed spontaneous surges lasting for several seconds. Furthermore, population bursts of neuronal activity were associated with increased Ca(2+) signal in a portion of the pericytes. Selective in vivo labeling of pericytes with functional markers may help reveal their physiological function in neuronal activity-associated regulation of local cerebral blood flow