Dephosphorylation by calcineurin regulates translocation of Drp1 to mitochondria.

Changes in mitochondrial morphology that occur during cell cycle, differentiation, and death are tightly regulated by the balance between fusion and fission processes. Excessive fragmentation can be caused by inhibition of the fusion machinery and is a common consequence of dysfunction of the organelle. Here, we show a role for calcineurin-dependent translocation of the profission dynamin related protein 1 (Drp1) to mitochondria in dysfunction-induced fragmentation. When mitochondrial depolarization is associated with sustained cytosolic Ca(2+) rise, it activates the cytosolic phosphatase calcineurin that normally interacts with Drp1. Calcineurin-dependent dephosphorylation of Drp1, and in particular of its conserved serine 637, regulates its translocation to mitochondria as substantiated by site directed mutagenesis. Thus, fragmentation of depolarized mitochondria depends on a loop involving sustained Ca(2+) rise, activation of calcineurin, and dephosphorylation of Drp1 and its translocation to the organelle.

Hereditary spastic paraplegia and amyotrophy associated with a novel locus on chromosome 19.

We identified a family in Mali with two sisters affected by spastic paraplegia. In addition to spasticity and weakness of the lower limbs, the patients had marked atrophy of the distal upper extremities. Homozygosity mapping using single nucleotide polymorphism arrays showed that the sisters shared a region of extended homozygosity at chromosome 19p13.11-q12 that was not shared by controls. These findings indicate a clinically and genetically distinct form of hereditary spastic paraplegia with amyotrophy, designated SPG43.

Functional modulation of GABAA receptors by cAMP-dependent protein phosphorylation.

gamma-Aminobutyric acidA (GABAA) receptors are ligand-gated ion channels that mediate inhibitory synaptic transmission in the central nervous system. The role of protein phosphorylation in the modulation of GABAA receptor function was examined with cells transiently transfected with GABAA receptor subunits. GABAA receptors consisting of the alpha 1 and beta 1 or the alpha 1, beta 1, and gamma 2 subunits were directly phosphorylated on the beta 1 subunit by adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (PKA). The phosphorylation decreased the amplitude of the GABA response of both receptor types and the extent of rapid desensitization of the GABAA receptor that consisted of the alpha 1 and beta 1 subunits. Site-specific mutagenesis of the serine residue phosphorylated by PKA completely eliminated the PKA phosphorylation and modulation of the GABAA receptor. In primary embryonic rat neuronal cell cultures, a similar regulation of GABAA receptors by PKA was observed. These results demonstrate that the GABAA receptor is directly modulated by protein phosphorylation and suggest that neurotransmitters or neuropeptides that regulate intracellular cAMP levels may modulate the responses of neurons to GABA and consequently have profound effects on synaptic excitability.

Cellular localization of a metabotropic glutamate receptor in rat brain.

In rat brain, the cellular localization of a phosphoinositide-linked metabotropic glutamate receptor (mGluR1 alpha) was demonstrated using antibodies that recognize the C-terminus of the receptor. mGluR1 alpha, a 142 kd protein, is enriched within the olfactory bulb, stratum oriens of CA1 and polymorph layer of dentate gyrus in hippocampus, globus pallidus, thalamus, substantia nigra, superior colliculus, and cerebellum. Lower levels of mGluR1 alpha are present within neocortex, striatum, amygdala, hypothalamus, and medulla. Dendrites, spines, and neuronal cell bodies contain mGluR1 alpha. mGluR1 alpha is not detectable in presynaptic terminals. mGluR1 alpha and ionotropic alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunits show differential distributions, but in Purkinje cells, mGluR1 alpha and specific AMPA receptor subunits colocalize. The postsynaptic distribution of mGluR1 alpha is consistent with postulated physiological roles of this subtype of glutamate receptor.

The distribution of glutamate receptors in cultured rat hippocampal neurons: postsynaptic clustering of AMPA-selective subunits.

The distribution of several glutamate receptor subunits was investigated in cultured rat hippocampal neurons by in situ hybridization and immunocytochemistry. The AMPA/kainate-selective receptors GluR1-6 exhibited two patterns of mRNA expression: most neurons expressed GluR1, R2, and R6, whereas only about 20% expressed significant levels of GluR3, R4, and R5. By immunocytochemistry, the metabotropic glutamate receptor mGluR1 alpha was detectable only in a subpopulation of GABAergic interneurons. GluR1 and GluR2/3 segregated to the somatodendritic domain within the first week in culture, even in the absence of synaptogenesis. Glutamate receptor-enriched spines developed later and were present only on presumptive pyramidal cells, not on GABAergic interneurons. Clusters of GluR1 and GluR2/3 completely colocalized and were restricted to a subset of postsynaptic sites. Thus, glutamate receptor subunits exhibit both a cell type-specific expression and a selective subcellular localization.

Regulation of GABAA receptor function by protein kinase C phosphorylation.

GABAA receptors possess consensus sequences for phosphorylation by PKC that are located on the presumed intracellular domains of beta and gamma 2 subunits. PKC phosphorylation sites were analyzed using purified receptor subunits and were located on up to 3 serine residues in beta 1 and gamma 2 subunits. The role of phosphorylation in receptor function was studied using recombinant receptors expressed in kidney cells and Xenopus oocytes and was compared with native neuronal GABAA receptors. For recombinant and native GABAA receptors, PKC phosphorylation caused a reduction in the amplitudes of GABA-activated currents without affecting the time constants for current decay. Selective site-directed mutagenesis of the serine residues reduced the effects of phorbol esters and revealed that serine 343 in the gamma 2 subunit exerted the largest effect on the GABA-activated response. These results indicate that PKC phosphorylation can differentially modulate GABAA receptor function.

Phosphorylation of amino acid neurotransmitter receptors in synaptic plasticity.

The precise regulation of synaptic efficacy in the mammalian central nervous system is fundamental for learning, memory, motor control and sensory processing, as well as synaptogenesis. Currently, the molecular mechanisms underlying synaptic plasticity involved in these crucial processes are topics of intense investigation. The modulation of neurotransmitter receptors has received considerable attention, since these receptors mediate signal transduction at the postsynaptic membranes of chemical synapses. Over the past several years, evidence has suggested that protein phosphorylation of neurotransmitter receptors is a common mechanism for the regulation of receptor function. In this reaction, protein kinases catalyse the transfer of a highly charged phosphate moiety from ATP to serine, threonine or tyrosine residues of a neurotransmitter receptor, thereby altering the charge and/or conformation of the receptor and regulating its function. Phosphorylation of neurotransmitter receptors is reversible, can occur rapidly, and might result in prolonged changes in receptor function. Thus, this modification might play an important role in both short- and long-term changes in synaptic transmission.

Protein targeting and calcium signaling microdomains in neuronal cells.

Over the last several years, a number of optical imaging, physiological, and molecular studies have clarified the mechanisms underlying differential calcium signaling in the postsynaptic neuron. These studies have revealed the existence of membrane-associated calcium microdomains, which are often specifically coupled to distinct protein signaling pathways. In this review, we discuss how these signaling microdomains are organized and regulated, emphasizing the structural and molecular features of synaptic protein complexes containing the metabotropic and N-methyl-D-aspartate (NMDA) glutamate receptors and the L-type voltage-dependent calcium channels (VDCCs). We conclude with a discussion of how these different signaling complexes may interact with one another, relationships which may be important in orchestrating the complex calcium signaling underlying developmental and activity-dependent changes in synaptic function.

Immunocytochemical localization of the mGluR1 alpha metabotropic glutamate receptor in the dorsal cochlear nucleus.

We demonstrate that the metabotropic glutamate receptor mGluR1 alpha is enriched in two interneuron cell populations in the dorsal division of the cochlear nucleus. Electron microscopic analysis confirms that mGluR1 alpha immunoreactivity is concentrated in the dendritic spines of cartwheel cells and in dendrites of the recently described unipolar brush cells. The cartwheel cells, which have many similarities to the Purkinje cells of the cerebellum, participate in a local neuronal circuit that modulates the output of the dorsal cochlear nucleus. Immunostained unipolar brush cells were observed in granule cell regions of the cochlear nucleus and the vestibulocerebellum. The presence of analogous cell types with similar patterns of immunolabeling in the cerebellum and in the dorsal cochlear nucleus suggests that a shared but as yet unknown mode of processing may occur in both structures.

Non-NMDA glutamate receptors are present throughout the primate hypothalamus.

To determine the distributions of glutamate receptors throughout the macaque hypothalamus, we utilized highly specific antipeptide antibodies to visualize alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunits (GluR1, GluR2 and GluR3 [designated as GluR2/3], and GluR4); kainate receptor subunits (GluR6 and GluR7, [designated as GluR6/7]), and a metabotropic receptor (mGluR1 alpha). The results indicate that these glutamate receptors are distributed differentially throughout the monkey hypothalamus. alpha-Amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors are the dominant non-N-methyl-D-aspartate glutamate receptors within the monkey hypothalamus, and the GluR2 subunit is most abundant. GluR1-immunoreactive neurons and neuropil are observed predominantly in the tuberal and mammillary nuclei. GluR2/3-immunoreactive neurons and neuropil have a broader distribution within preoptic, anterior, tuberal, and caudal regions. Separate (but partially overlapping) distributions of GluR1- and GluR2/3-immunoreactive neurons were found, suggesting that the GluR1, GluR2, and/or GluR3 subunits may be coexpressed in subsets of hypothalamic neurons. In contrast, GluR4 immunoreactivity was expressed minimally within monkey hypothalamus. GluR6/7 immunoreactivity was enriched selectively within the suprachiasmatic nucleus. mGluR1 alpha immunoreactivity was present in the mammillary complex. The localization of non-N-methyl-D-aspartate glutamate receptor subunits to neurons throughout the macaque hypothalamus provides further evidence for the glutamatergic regulation of neuroendocrine, autonomic, and limbic circuits. Differential distributions of glutamate receptor subunits may increase the dynamic range of the effects of presynaptic glutamate, allowing for the regulation of several distinct functions subserved by hypothalamic neurons.

AMPA receptor protein in developing rat brain: glutamate receptor-1 expression and localization change at regional, cellular, and subcellular levels with maturation.

We tested the hypothesis that the regional, cellular, and synaptic localizations of the glutamate receptor 1 (GluR 1) subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor are regulated developmentally in rat brain. By immunoblotting, GluR1 was first detected in whole brain at embryonic day E15.5, and levels increased progressively during late embryonic (E20) and early postnatal (P2-P11) days. Regionally, GluR1 increased in cerebral cortex but decreased in striatum with postnatal maturation. These changes occurred in the presence of increased presynaptic maturation, as determined by synaptophysin detection. By immunocytochemistry, distinct cellular populations showed different temporal profiles of GluR1 expression during postnatal maturation. The neocortex and hippocampus showed a progressive maturation-related enrichment of GluR1, whereas the striatum showed a gradual reduction in GluR1 during maturation. In cerebellum, GluR1 protein was expressed transiently at restricted times postnatally by granule cells (P0-P11) and Purkinje cells (P13-P19), but by P21 and thereafter these neurons had sparse GluR1 immunoreactivity. By immunoelectron microscopy. GluR1 was found in neurites, specifically in both dendritic and axon terminal components of developing synapses. GluR1 was clustered at the plasma membrane of apparent growth cone appositions, neuronal cell bodies, and dendrites of developing neurons. The presence of GluR1 at presynaptic sites dissipated with synaptic maturation, as GluR1 became confined to the somatodendritic compartment as maturation progressed. We conclude that the regional expression as well as the cellular and synaptic localizations of the GluR1 are developmentally regulated and are different in immature and mature brain. Differences in glutamate receptor expression and synaptic localization in immature and mature brain may be relevant to the phenomenon that the perinatal and adult brain differ in their regional vulnerability to hypoxia-ischemia and excitotoxicity.

The AMPA glutamate receptor GluR3 is enriched in oxytocinergic magnocellular neurons and is localized at synapses.

The cellular localization of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate glutamate receptor, GluR3, was identified using antibodies that recognize the N-terminus of the predicted polypeptide sequence of GluR3. Regional immunoblot analysis of monkey brain homogenates identified a protein of approximately 102,000 mol. wt that was enriched in hypothalamus. Immunocytochemistry demonstrated that GluR3 was enriched within the hypothalamic magnocellular neurosecretory nuclei and axons of the hypothalamo-neurohypophysial tract in rat and monkey. GluR3 immunoreactivity co-localized to oxytocin-containing, but not vasopressin-containing, neurons of the hypothalamic paraventricular nucleus, supraoptic nucleus and accessory magnocellular nuclei. Ultrastructurally, GluR3 immunoreactivity was enriched throughout cytoplasm of the somatodendritic compartment and was associated with postsynaptic and presynaptic structures. GluR3 immunoreactivity was frequently observed to be clustered at the plasma membrane of the somatodendritic compartment, consistent with the predicted localization of a membrane-bound ion channel. Additionally, GluR3-immunoreactive axon terminals in synaptic contact with unlabeled dendrites within the retrochiasmatic area and bed nucleus of the stria terminalis were observed, providing morphological evidence for a presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor. By immunoblot analysis and immunocytochemistry using antibodies directed against a specific alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor in rat and monkey brain, our findings suggest a highly selective hypothalamic distribution of the GluR3 subunit that may have functional significance in the glutamatergic regulation of oxytocinergic neurons.

Distribution of glutamate receptor subtypes in the vertebrate retina.

The distribution of glutamate receptor subunit/subtypes in the vertebrate retina was investigated by immunocytochemistry using anti-peptide antibodies against AMPA (GluR1-4), kainate (GluR6/7) and metabotropic (mGluR1 alpha) receptors. All receptor subtypes examined are present in the mammalian retina, but they are distributed differentially. GluR1 is present in the inner plexiform layer as well as amacrine and ganglion cell bodies. GluR2 is present mainly in the outer plexiform layer and bipolar cells. An anti-GluR2/3 antibody labels both plexiform layers and various cell bodies in the inner nuclear layer and the ganglion cell layer. GluR4 is present on Müller glial cells. In the goldfish retina, GluR2 immunoreactivity is prominent in the Mb type of ON-bipolar cells, including the dendrites and the large synaptic terminal. The putative dendritic localization is surprising, because no depolarizing conductance increase induced by glutamate is thought to be present in these cells. An AMPA receptor at a presynaptic terminal is also unusual, and probably provides feedback control of glutamate release. GluR6/7 is most widespread in the retina, being present in horizontal, bipolar, amacrine and ganglion cells. Ion channels composed of GluR6 are now known to be phosphorylated by protein kinase A, resulting in current potentiation. This property and our present observation together suggest that the glutamate receptors previously studied electrophysiologically by others in horizontal cells may contain GluR6. mGluR1 alpha is found mostly in the inner plexiform layer; its localization partially overlaps with that of the inositol trisphosphate receptor in the retina. Our results suggest that, in the retina, glutamate receptor subtypes may be expressed in selective cell types according to their specific functions.

AMPA glutamate receptor subunits are differentially distributed in rat brain.

To demonstrate the regional, cellular and subcellular distributions of non-N-methyl-D-aspartate glutamate receptors in rat brain, we generated antipeptide antibodies that recognize the C-terminal domains of individual subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-preferring glutamate receptors (i.e. GluR1, GluR4, and a region highly conserved in GluR2, GluR3 and GluR4c). On immunoblots, antibodies detect distinct proteins with mol. wts ranging from 102,000 to 108,000 in homogenates of rat brain. Immunocytochemistry shows that glutamate receptor subunits are distributed abundantly and differentially within neuronal cell bodies and processes in cerebral cortex, basal ganglia, limbic system, thalamus, cerebellum and brainstem. The precise patterns and cellular localizations of glutamate receptor subunit immunoreactivities are unique for each antibody. In neocortex and hippocampus, pyramidal neurons express GluR1 and GluR2/3/4c immunoreactivities; many non-pyramidal, calcium-binding, protein-enriched neurons in cerebral cortex are selectively immunoreactive for GluR1. In striatum, the cellular localizations of GluR1, GluR2/3/4c and GluR4 immunoreactivities are different; in this region, GluR1 co-localizes with many cholinergic neurons but is only present in a minor proportion of nicotinamide adenine dinucleotide phosphate diaphorase-positive striatal neurons. GluR1 co-localizes with most dopaminergic neurons within the substantia nigra. In several brain regions, astrocytes show GluR4 immunoreactivity. Within the cerebellar cortex, cell bodies and processes of Bergmann glia express intense GluR4 and GluR1 immunoreactivities; perikarya and dendrites of Purkinje cells show GluR2/3/4c immunoreactivity but no evidence of GluR1 or GluR4. Ultrastructurally, GluR subunit immunoreactivities are localized within cell bodies, dendrites and dendritic spines of specific subsets of neurons and, in the case of GluR1 and GluR4, in some populations of astrocytes. This investigation demonstrates that individual AMPA-preferring glutamate receptor subunits are distributed differentially in the brain and suggests that specific neurons and glial cells selectively express glutamate receptors composed of different subunit combinations. Thus, the co-expression of all AMPA receptor subunits within individual cells may not be obligatory for the functions of this glutamate receptor in vivo.

Glutamate receptor modulation by protein phosphorylation.

Glutamate-gated ion channels mediate most excitatory synaptic transmission in the mammalian central nervous system and play major roles in synaptic plasticity, neuronal development, and in some neuropathological conditions. Recent studies have suggested that protein phosphorylation of neuronal glutamate receptors by cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC) may regulate their function and play a role in some forms of synaptic plasticity. To test whether these protein kinase effects are due to direct phosphorylation of the receptors and to further examine the sites and mechanisms by which the receptors are modulated, we transiently expressed recombinant glutamate receptors in HEK-293 cells and studied their biochemical and biophysical properties. Our results indicate that the kainate-preferring receptor GluR6 is phosphorylated by PKA, primarily on a single serine in the proposed major intracellular loop. Moreover, using the whole cell patch clamp recording technique, we have shown that phosphorylation at this site increases the amplitude of the GluR6-mediated glutamate current without significantly altering its dose-response, current-voltage relation or desensitization kinetics. In other experiments, we have demonstrated that the NMDA receptor subunit NR1 is phosphorylated by PKC on several distinct sites, and most of these sites are located within a single alternatively spliced exon in the C-terminal domain. These findings suggest that RNA splicing can regulate NMDA receptor phosphorylation and that, contrary to the previously proposed membrane topology model, the NR1 C-terminus is intracellular. Furthermore, in HEK-293 cells co-transfected with NR2A and NR1 subunits containing the C-terminal exon with the PKC phosphorylation sites, our preliminary studies indicate that the NMDA-evoked current is potentiated by intracellular PKC. We are currently examining PKC effects on the NMDA-evoked current responses of mutant NR1 receptors that lack the C-terminal phosphorylation sites. These studies provide evidence that glutamate receptors are directly phosphorylated and functionally modulated by protein kinases. Moreover, by identifying phosphorylation sites within the receptor proteins, our results provide information about the structure and membrane topology of these receptors.

Inositolphospholipid-linked glutamate receptors mediate cerebellar parallel-fiber-Purkinje-cell synaptic transmission.

In slices of adult rat cerebellum inositolphospholipid turnover is stimulated markedly by glutamate and its rigid analogues quisqualate and ibotenate. The drug and amino acid specificity of the response reflects a quisqualate-preferring excitatory amino acid receptor. The absence of glutamate-enhanced inositolphospholipid turnover in mice with Purkinje-cell degeneration indicates that the inositolphospholipid-linked quisqualate receptor mediates parallel fiber-Purkinje cell synaptic transmission. The quantitative prominence of this synapse accounts for the massive enrichment of elements of the inositolphospholipid system in cerebellar Purkinje cells.

Phosphorylation of recombinant non-NMDA glutamate receptors on serine and tyrosine residues.

Glutamate receptors are the major excitatory neurotransmitter receptors in the central nervous system. A variety of data has recently suggested that protein phosphorylation of glutamate receptors regulates their function. To examine at a molecular level the role of protein phosphorylation in the modification of glutamate receptors, we have transiently expressed the non-NMDA glutamate receptor subunit GluR1 (flop) in human embryonic kidney 293 cells. Using a polyclonal antipeptide antiserum directed specifically against GluR1, we have immunoprecipitated a 106 kDa phosphoprotein corresponding to the GluR1 subunit. Phosphoamino acid analysis and thermolytic peptide mapping demonstrate that this basal phosphorylation occurs exclusively on serine residues in two phosphopeptides. Application of activators of endogenous cAMP-dependent protein kinase or protein kinase C revealed no consistent changes in the phosphorylation of GluR1. However, co-expression of the GluR1 subunit with the well characterized protein tyrosine kinase v-src results in phosphorylation of GluR1 on tyrosine residues, in a single thermolytic phosphopeptide. These results suggest that GluR1 may be a substrate for protein serine/threonine kinases as well as protein tyrosine kinases in the central nervous system.

Phosphorylation and modulation of recombinant GluR6 glutamate receptors by cAMP-dependent protein kinase.

Glutamate-gated ion channels mediate most excitatory synaptic transmission in the central nervous system and play crucial roles in synaptic plasticity, neuronal development and some neuropathological conditions. These ionotropic glutamate receptors have been classified according to their preferred agonists as NMDA (N-methyl-D-aspartate), AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate) and KA (kainate) receptors. On the basis of sequence similarity and pharmacological properties, the recently cloned glutamate receptor subunits have been assigned as components of NMDA (NMDAR1, 2A-D), AMPA (GluR1-4) and KA (GluR5-7, KA1, KA2) receptors. Protein phosphorylation of glutamate receptors by protein kinase C and cyclic AMP-dependent protein kinase (PKA) has been suggested to regulate their function, possibly playing a prominent role in certain forms of synaptic plasticity such as long-term potentiation and long-term depression. Here we report that the GluR6 glutamate receptor, transiently expressed in mammalian cells, is directly phosphorylated by PKA, and that intracellularly applied PKA increases the amplitude of the glutamate response. Site-specific mutagenesis of the serine residue (Ser 684) representing a PKA consensus site completely eliminates PKA-mediated phosphorylation of this site as well as the potentiation of the glutamate response. These results provide evidence that direct phosphorylation of glutamate receptors modulates their function.

Phosphorylation of ligand-gated ion channels: a possible mode of synaptic plasticity.

Most neurotransmitter receptors examined to date have been shown either to be regulated by protein phosphorylation or to contain consensus sequences for phosphorylation by protein kinases. Neurotransmitter receptors that mediate rapid synaptic transmission in the nervous system are the ligand-gated ion channels and include the nicotinic acetylcholine receptors of muscle and nerve and the excitatory and inhibitory amino acid receptors: the glutamate, GABAA, and glycine receptors. These receptors are multimeric proteins composed of homologous subunits which each span the membrane several times and contain a large intracellular loop that is a mosaic of consensus sites for protein phosphorylation. Recent evidence has suggested that extracellular signals released from the presynaptic neuron, such as neurotransmitters and neuropeptides as well as an extracellular matrix protein, regulate the phosphorylation of ligand-gated ion channels. The functional effects of phosphorylation are varied and include the regulation of receptor desensitization rate, subunit assembly, and receptor aggregation at the synapse. These results suggest that phosphorylation of neurotransmitter receptors represents a major mechanism in the regulation of their function and may play an important role in synaptic plasticity.