HAP1 facilitates effects of mutant huntingtin on inositol 1,4,5-trisphosphate-induced Ca release in primary culture of striatal medium spiny neurons

Huntington's disease is caused by polyglutamine expansion (exp) in huntingtin (Htt). Htt-associated protein-1 (HAP1) was the first identified Htt-binding partner. The type 1 inositol (1,4,5)-trisphosphate receptor (InsP3R1) is an intracellular Ca2+ release channel that plays an important role in neuronal function. Recently, we identified a InsP3R1-HAP1A-Htt ternary complex in the brain and demonstrated that Httexp, but not normal Htt, activates InsP3R1 in bilayers and facilitates InsP3R1-mediated intracellular Ca2+ release in medium spiny striatal neurons [MSN; T.-S. Tang et al. (2003) Neuron, 39, 227-239]. Here we took advantage of mice with targeted disruption of both HAP1 alleles (HAP1 -/-) to investigate the role of HAP1 in functional interactions between Htt and InsP3R1. We determined that: (i) HAP1 is expressed in the MSN; (ii) HAP1A facilitates functional effects of Htt and Htt(exp) on InsP3R1 in planar lipid bilayers; (iii) HAP1 is required for changes in MSN basal Ca2+ levels resulting from Htt or Htt(exp) overexpression; (iv) HAP1 facilitates potentiation of InsP3R1-mediated Ca2+ release by Htt(exp) in mouse MSN. Our present results indicate that HAP1 plays an important role in functional interactions between Htt and InsP3R1.     

Inositol 1,4,5 trisphosphate receptor and chromogranin B are concentrated in different regions of the hippocampus

Calcium (Ca(2+)) release from intracellular stores plays a crucial role in many cellular functions in the brain. These intracellular signals have been shown to be transmitted within and between cells. We report a non-uniform distribution of proteins essential for Ca(2+) signaling in acutely prepared brain slice preparations and organotypic slice cultures, both made from rat hippocampus. The Type I inositol-1,4,5 trisphosphate receptor (InsP(3)R1) is the main InsP(3)R subtype in neurons. Immunohistochemistry experiments showed a prominent expression of InsP(3)R1 in the CA1 region of the hippocampus whereas the CA3 region and dentate gyrus (DG) showed only moderate immunoreactivity. In contrast, chromogranin B (CGB), a protein binding to the InsP(3)R1 on the luminal side of the endoplasmic reticular membrane was enriched in the CA3 region whereas DG and the CA1 region showed only faint CGB signals. The neuronal kinases leading to the formation of inositol-1,4,5 trisphosphate (InsP(3)), phosphatidylinositol-4-kinase (PI4K), and phosphatidylinositol-4-phosphate-5-kinase (PIPK), showed strong immunoreactivity throughout all hippocampal cell fields with differences in the subcellular distribution. Moreover, a distinct band of strong CGB and PIPK immunoreactivity was observed in the CA3 region that coincides with the mossy fiber tract (stratum lucidum). These data show differential expression of the components of the signaling toolkit leading to InsP(3)-mediated Ca(2+) release in cells of the hippocampus. The regulation of these differences may play an important role in various neuropathologic conditions such as Alzheimer's disease, epilepsy, or schizophrenia.     

Normal phototransduction in Drosophila photoreceptors lacking an InsP(3) receptor gene

The Drosophila light-sensitive channels TRP and TRPL are prototypical members of an ion channel family responsible for a variety of receptor-mediated Ca(2+) influx phenomena, including store-operated calcium influx. While phospholipase Cbeta is essential, downstream events leading to TRP and TRPL activation remain unclear. We investigated the role of the InsP(3) receptor (InsP(3)R) by generating mosaic eyes homozygous for a deficiency of the only known InsP(3)R gene in Drosophila. Absence of gene product was confirmed by RT-PCR, Western analysis, and immunocytochemistry. Mutant photoreceptors underwent late onset retinal degeneration; however, whole-cell recordings from young flies demonstrated that phototransduction was unaffected, quantum bumps, macroscopic responses in the presence and absence of external Ca(2+), light adaptation, and Ca(2+) release from internal stores all being normal. Using the specific TRP channel blocker La(3+) we demonstrated that both TRP and TRPL channel functions were unaffected. These results indicate that InsP(3)R-mediated store depletion does not underlie TRP and TRPL activation in Drosophila photoreceptors.     

Basic properties of an inositol 1,4,5-trisphosphate-gated channel in carp olfactory cilia

In addition to the activation of cAMP-dependent pathways, odorant binding to its receptor can lead to inositol 1,4,5-trisphosphate (InsP3) production that may induce the opening of plasma membrane channels. We therefore investigated the presence and nature of such channels in carp olfactory cilia. Functional analysis was performed by reconstitution of the olfactory cilia in planar lipid bilayers (tip-dip method). In the presence of InsP3 (10 microM) and Ca2+ (100 nM), a current of 1.6 +/- 0.1 pA (mean +/- SEM, n = 4) was measured, using Ba2+ as charge carrier. The I/V curve displayed a slope conductance of 45 +/- 5 pS and a reversal potential of -29 mV indicating a higher selectivity for divalent cations. This current was characterized by two mean open times (3.0 +/- 0.4 ms and 42.0 +/- 2.6 ms, n = 4) and was strongly inhibited by ruthenium red (30 microM) or heparin (10 microg/mL). Importantly, the channel activity was closely dependent on the Ca2+ concentration, with the highest open probability (Po) at 100 nM Ca2+ (Po = 0.50 +/- 0.02, n = 4). Po is lower at both higher and lower Ca2+ concentrations. A structural identification of the channel was attempted by using a large panel of antibodies, raised against several InsP3 receptor (InsP3R)/Ca2+ release channel isoforms. The type 1 InsP3R was detected in carp cerebellum and whole brain, while a lower molecular mass InsP3R, which may correspond to type 2 or 3, was detected in heart, whole brain and the soma of the olfactory neurons. None of the antibodies, however, cross-reacted with olfactory cilia. Taken together, these results indicate that in carp olfactory cilia an InsP3-dependent channel is present, distinct from the classical InsP3Rs localized on intracellular membranes.     

Type II brain 4.1 (4.1B/KIAA0987), a member of the protein 4.1 family, is localized to neuronal paranodes

Histochemical analyses of type II brain 4.1/4.1B/KIAA0987, a member of the protein 4.1 family, were carried out in rat brain. In situ hybridization (ISH) showed that type II brain 4.1 mRNA is expressed in a variety of neuronal cells. In particular, type II brain 4.1 mRNA was actively transcribed in the cells of the mesencephalon and the brainstem, which have large myelinated nerve fibers. Expression of type II brain 4.1 mRNA was not observed at least in glial cells distributed in nerve fiber tracts. In immunohistochemical studies using anti-type II brain 4.1-specific antibody, the major immunosignals appeared as brilliant pairs of dots along nerve fibers. Such immunosignals were detected throughout the brain, but were highly concentrated in nerve fiber tracts. These data suggested that type II brain 4.1 is predominantly localized to neuronal paranodes. Detailed analysis concentrating on the nodal region indicated that type II brain 4.1 is present at the paranodal membrane but not in the axoplasm. Weaker type II brain 4.1-specific immunosignals were observed along the internodal membrane of myelinated axons and in the cytoplasm of some neuronal cells. Finally, comparative immunohistochemical studies using antibodies against the other three protein 4.1 family members, type I brain 4.1/4.1N/KIAA0338, erythroid type 4.1 (4.1R) and 4.1G, demonstrated that each of these proteins is distributed in a unique pattern in the cerebellum. Our results are the first to show that type II brain 4.1 is the only member of the protein 4.1 family localized to neuronal paranodes.     

Astrocytes in adult rat brain express type 2 inositol 1,4,5-trisphosphate receptors

Astrocytes respond to neuronal activity by propagating Ca(2+) waves elicited through the inositol 1,4,5-trisphosphate pathway. We have previously shown that wave propagation is supported by specialized Ca(2+) release sites, where a number of proteins, including inositol 1,4,5-trisphosphate receptors (IP(3)R), occur together in patches. The specific IP(3)R isoform expressed by astrocytes in situ in rat brain is unknown. In the present report, we use isoform-specific antibodies to localize immunohistochemically the IP(3)R subtype expressed in astrocytes in rat brain sections. Astrocytes were identified using antibodies against the astrocyte-specific markers, S-100 beta, or GFAP. Dual indirect immunohistochemistry showed that astrocytes in all regions of adult rat brain express only IP(3)R2. High-resolution analysis showed that hippocampal astrocytes are endowed with a highly branched network of processes that bear fine hair-like extensions containing punctate patches of IP(3)R2 staining in intimate contact with synapses. Such an organization is reminiscent of signaling microdomains found in cultured glial cells. Similarly, Bergmann glial cell processes in the cerebellum also contained fine hair-like processes containing IP(3)R2 staining. The IP(3)R2-containing fine terminal branches of astrocyte processes in both brain regions were found juxtaposed to presynaptic terminals containing synaptophysin as well as PSD 95-containing postsynaptic densities. Corpus callosum astrocytes had an elongated morphology with IP(3)R2 studded processes extending along fiber tracts. Our data suggest that PLC-mediated Ca(2+) signaling in astrocytes in rat brain occurs predominantly through IP(3)R2 ion channels. Furthermore, the anatomical arrangement of the terminal astrocytic branches containing IP(3)R2 ensheathing synapses is ideal for supporting glial monitoring of neuronal activity.     

Inositol 1,4,5-trisphosphate-gated channels in cerebellum: presence of multiple conductance states.

The mechanism by which inositol 1,4,5-triphosphate (InsP3) induces calcium (Ca) release from the reticulum of canine cerebellum was examined. Reticular membrane vesicles used in these experiments accumulated Ca in the presence of ATP and then released approximately 30% of the accumulated Ca upon addition of micromolar concentrations of InsP3. When these membrane vesicles were incorporated into planar lipid bilayers, InsP3-gated Ca channels were observed. Up to four current amplitudes were observed at a given voltage, yielding conductances of 20, 40, 60, and 80 pS with 50 mM Ca as the current carrier. Thus, the cerebellar InsP3-gated Ca channel exhibits four conductance levels that are multiples of a unit conductance step. Moreover, examination of the single-channel records showed both openings directly to each of the current levels and rapid transitions between current levels. These four conductance steps may reflect the interaction among the four InsP3 receptors thought to comprise the InsP3-gated Ca channel in these tissues. Examination of the InsP3 dependence of channel openings and Ca release from vesicles, however, yielded Hill coefficients of 1-1.3. Thus, we hypothesize that it takes only one molecule of InsP3 to open the channel. The observation that the conductance of the InsP3-gated Ca channel assumes four levels that are multiples of a unit conductance suggests that the number of interacting InsP3 receptors in one complex can vary from one to four and supports the hypothesis that the channel is a tetramer.     

A novel neuron-enriched homolog of the erythrocyte membrane cytoskeletal protein 4.1.

We report the molecular cloning and characterization of 4.1N, a novel neuronal homolog of the erythrocyte membrane cytoskeletal protein 4.1 (4.1R). The 879 amino acid protein shares 70, 36, and 46% identity with 4.1R in the defined membrane-binding, spectrin-actin-binding, and C-terminal domains, respectively. 4.1N is expressed in almost all central and peripheral neurons of the body and is detected in embryonic neurons at the earliest stage of postmitotic differentiation. Like 4.1R, 4.1N has multiple splice forms as evidenced by PCR and Western analysis. Whereas the predominant 4.1N isoform identified in brain is approximately 135 kDa, a smaller 100 kDa isoform is enriched in peripheral tissues. Immunohistochemical studies using a polyclonal 4.1N antibody revealed several patterns of neuronal staining, with localizations in the neuronal cell body, dendrites, and axons. In certain neuronal locations, including the granule cell layers of the cerebellum and dentate gyrus, a distinct punctate-staining pattern was observed consistent with a synaptic localization. In primary hippocampal cultures, mouse 4.1N is enriched at the discrete sites of synaptic contact, colocalizing with the postsynaptic density protein of 95 kDa (a postsynaptic marker) and glutamate receptor type 1 (an excitatory postsynaptic marker). By analogy with the roles of 4.1R in red blood cells, 4.1N may function to confer stability and plasticity to the neuronal membrane via interactions with multiple binding partners, including the spectrin-actin-based cytoskeleton, integral membrane channels and receptors, and membrane-associated guanylate kinases.     

Endogenous inhibitors of InsP3-induced Ca2+ release in neuroblastoma cells

Cerebellar Purkinje neurons and neuroblastoma N1E-115 cells require 10-50 times more InsP3 to induce Ca2+ release than do a variety of non-neuronal cells (including astrocytes, hepatocytes, endothelial cells, or smooth muscle cells). Given the importance of InsP3-induced Ca2+ release for the development of synaptic plasticity in Purkinje neurons, a low InsP3 sensitivity may facilitate the integration of numerous synaptic inputs before initiating a change in synaptic strength. In the present study, attention is directed at the mechanism underlying this low InsP3 sensitivity of Ca2+ release. We show that permeabilization of neuroblastoma cells with saponin increased InsP3 sensitivity of Ca2+ release, indicating the presence of a diffusible, cytosolic inhibitor(s) of Ca2+ release. Consistent with this hypothesis, gel filtration of the neuroblastoma cytosol yielded three peaks that inhibited InsP3-induced Ca2+ release from permeabilized cells. The prominent inhibitory peak decreased the InsP3 sensitivity of Ca2+ release from permeabilized cells, did not bind 3H-InsP3, and was present in sufficient levels to account for the low InsP3 sensitivity of Ca2+ release in intact neuroblastoma cells. Purification of this prominent inhibitory fraction yielded a protein band that was identified by mass spectrometry as stress-induced phosphoprotein 1 (mSTI1). Furthermore, immunoprecipitation of mSTI1 decreased the inhibitory activity of N1E-115 cytosol, indicating that mSTI1 contributes to the inhibition of InsP3-induced Ca2+ release. Thus, the low InsP3 sensitivity of Ca2+ release in neuroblastoma cells can be explained by the presence of cytosolic inhibitors of Ca2+ release and include stress-induced phosphoprotein 1.     

Effect of intracellular injection of inositol trisphosphate on cytosolic calcium and membrane currents in Aplysia neurons.

Pacemaker cells of Aplysia californica display a regular bursting that results from a complex interplay of Ca(2+)-mediated conductances and a continuous influx and extrusion of Ca2+. The effect of the second messenger 1,4,5-inositol trisphosphate (InsP3) on intracellular free Ca2+ concentration (Cai) regulation and electrical properties was investigated in identified neurons of the abdominal ganglion (R15, L2-L4, L6). Double-barreled Ca-selective microelectrodes were used to pressure inject InsP3 and measure Cai at the same point. Brief injection of InsP3 resulted in an average increase of Cai of 9.2 +/- 10.0 microM (+/- SE; n = 14) that decayed in about 1 min. The InsP3-induced elevation of Cai increased in a dose-dependent manner and saturated when large amounts of InsP3 were injected. The InsP3-induced Cai increase was the result of mobilization from intracellular stores; Cai could be repeatedly mobilized by InsP3 in cells superfused with 0 Ca artificial seawater for more than 60 min. Following multiple injections of InsP3, there was no evidence of immediate inhibition or facilitation. the spatial nature of the InsP3-induced Cai increase was investigated by moving the double-barreled Ca-selective microelectrode tip in a stepwise manner relative to the membrane surface. The largest InsP3-induced Cai increases were measured in an area 0-80 microns from the membrane surface; some cells had their largest InsP3-induced Cai increase some 120-160 microns away from the membrane. Injection of InsP3 in a bursting neuron induced an immediate train of action potentials followed by membrane hyperpolarization and a decrease in the burst frequency. Injection of InsP3 in voltage-clamped cells induced a biphasic response: a rapid inward current followed by a more prolonged outward current; the temporal overlap of the currents was depth dependent. Injection of InsP3 or Ca2+ from a double-barreled injecting electrode induced currents that were different in waveform and time course, indicating that part of the conductance change induced by InsP3 is direct and not mediated by the mobilized Ca2+. In BAPTA [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'tetra-acetic acid]-loaded cells, the InsP3-induced inward current was mostly unaffected while the Ca-induced outward current was largely attenuated. The results suggest that InsP3 mobilizes Ca2+ from discrete intracellular compartments and induces distinct changes in membrane currents that seem to be independent of the Cai increase.     

Inositol 1,4,5-trisphosphate receptors and protein kinase C in olivopontocerebellar atrophy.

We examined protein kinase C (PKC) activity and inositol 1,4,5-trisphosphate (InsP3) binding in frontal cortex (FC) and cerebellar cortex (CC) of normal humans, patients with dominant ataxia ("C" kindred) and in Lurcher mutant mouse brain (LMB), a suggested animal model for olivopontocerebellar atrophy (OPCA). PKC activity and [3H]InsP3 binding were decreased in CC of human OPCA by 70% and 90% respectively. The decreases were specific to CC as there were no changes in FC. PKC activity and [3H]InsP3 binding in cerebellum (CB) of LMB were similarly decreased as compared to normal littermate controls. The LMB decrease of PKC and [3H]InsP3 binding was evident on the 15th day of age, the day of onset of ataxia. InsP3-mediated calcium release was also decreased significantly in the cerebellar microsomes of 25-day-old LMB and human OPCA when compared with their respective controls. These data indicate that the decrease of second messenger linked PKC activity and InsP3 receptor binding in CB may be a biochemical marker that reflects neuronal degeneration in dominant cerebellar ataxia.     

Neuronal cytotoxicity of inositol hexakisphosphate (phytate) in the rat hippocampus

d-myo-Inositol hexakisphosphate (InsP6, phytate), a normal cellular constituent, was found to be toxic to neuronal perikarya when injected into the rat hippocampus. However, the extrinsic cholinergic innervation of the hippocampus (as estimated by staining for acetylcholinesterase) was unaffected. Its potency as a toxin was approximately equal to that of the excitotoxin quinolinate. Other highly charged derivatives of Inositol (Inositol hexakissulphate, Inositol monophosphate) were not toxic. The cytotoxicity of InsP6 was not due to a high osmolality, or to seizure-induced lesions, but was reduced by calcium. Nevertheless, the toxicity was not due to chelation of brain calcium by InsP6, as another calcium chelator with a higher affinity for calcium, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (BAPTA), produced only a very mild lesion. Thus, abnormal metabolism of InsP6 might possibly contribute to neuronal death in neurodegenerative diseases.     

Chronic suppression of inositol 1,4,5-triphosphate receptor-mediated calcium signaling in cerebellar purkinje cells alleviates pathological phenotype in spinocerebellar ataxia 2 mice

Spinocerebellar ataxia 2 (SCA2) is a neurodegenerative disorder characterized by progressive ataxia. SCA2 results from a poly(Q) (polyglutamine) expansion in the cytosolic protein ataxin-2 (Atx2). Cerebellar Purkinje cells (PCs) are primarily affected in SCA2, but the cause of PC dysfunction and death in SCA2 is poorly understood. In previous studies, we reported that mutant but not wild-type Atx2 specifically binds the inositol 1,4,5-trisphosphate receptor (InsP(3)R) and increases its sensitivity to activation by InsP(3). We further proposed that the resulting supranormal calcium (Ca(2+)) release from the PC endoplasmic reticulum plays a key role in the development of SCA2 pathology. To test this hypothesis, we achieved a chronic suppression of InsP(3)R-mediated Ca(2+) signaling by adenoassociated virus-mediated expression of the inositol 1,4,5-phosphatase (Inpp5a) enzyme (5PP) in PCs of a SCA2 transgenic mouse model. We determined that recombinant 5PP overexpression alleviated age-dependent dysfunction in the firing pattern of SCA2 PCs. We further discovered that chronic 5PP overexpression also rescued age-dependent motor incoordination and PC death in SCA2 mice. Our findings further support the important role of supranormal Ca(2+) signaling in SCA2 pathogenesis and suggest that partial inhibition of InsP(3)-mediated Ca(2+) signaling could provide therapeutic benefit for the patients afflicted with SCA2 and possibly other SCAs.     

Impaired mitochondrial energy metabolism and neuronal apoptotic cell death after chronic dichlorvos (OP) exposure in rat brain

The present study elucidates a possible mechanism by which chronic organophosphate exposure (dichlorvos 6 mg/kg bw, s.c. for 12 weeks) causes neuronal degeneration. Mitochondria, as a primary site of cellular energy generation and oxygen consumption represent itself a likely target for organophosphate poisoning. Therefore, the objective of the current study was planned with an aim to investigate the effect of chronic dichlorvos exposure on mitochondrial calcium uptake, oxidative stress generation and its implication in the induction of neuronal apoptosis in rodent model. Mitochondrial preparation from dichlorvos (DDVP) treated rat brain demonstrated significant increase in mitochondrial Ca(2+) uptake (644.2 nmol/mg protein). Our results indicated decreased mitochondrial electron transfer activities of cytochrome oxidase (complex IV) along with altered mitochondrial complex I, and complex II activity, which might have resulted from elevated mitochondrial calcium uptake. The alterations in the mitochondrial calcium uptake and mitochondrial electron transfer enzyme activities in turn might have caused an increase in malondialdehyde, protein carbonyl and 8-hydroxydeoxyguanosine formation as a result of enhanced lipid peroxidation, and as well as protein and mtDNA oxidation. All this could have been because of enhanced oxidative stress, decreased GSH levels and also decreased Mn-SOD activity in the mitochondria isolated from dichlorvos treated rat brain. Thus, chronic organophosphate exposure has the potential to disrupt cellular antioxidant defense system which in turn triggers the release of cytochrome c from mitochondria to cytosol as well as caspase-3 activation in dichlorvos treated rat brain as revealed by immunoblotting experiments. Low-level long-term organophosphate exposure finally resulted in oligonucleosomal DNA fragmentation, a hallmark of apoptosis. These studies provide an evidence of impaired mitochondrial bioenergetics and apoptotic neuronal degeneration after chronic low-level exposure to dichlorvos.     

Ca1.2 and CaV1.3 neuronal L-type calcium channels: differential targeting and signaling to pCREB

Neurons express multiple types of voltage-gated calcium (Ca2+) channels. Two subtypes of neuronal L-type Ca2+ channels are encoded by CaV1.2 and CaV1.3 pore-forming subunits. To compare targeting of CaV1.2 and CaV1.3 L-type Ca2+ channels, we transfected rat hippocampal neuronal cultures with surface-epitope-tagged sHA-CaV1.2 or sHA-CaV1.3a constructs and found that: (i) both sHA-CaV1.2 and sHA-CaV1.3a form clusters on the neuronal plasma membrane surface; (ii) when compared with sHA-CaV1.2 surface clusters, the sHA-CaV1.3a surface clusters were 10% larger and 25% brighter, but 35% less abundant; (iii) 81% of sHA-CaV1.2 surface clusters, but only 48% of sHA-CaV1.3a surface clusters, co-localized with synapsin clusters; (iv) co-expression with GFP-Shank-1B had no significant effect on sHA-CaV1.2 surface clusters, but promoted formation and synaptic localization of sHA-CaV1.3a surface clusters. In experiments with dihydropyridine-resistant CaV1.2 and CaV1.3a mutants we demonstrated that CaV1.3a L-type Ca2+ channels preferentially mediate nuclear pCREB signaling in hippocampal neurons at low, but not at high, levels of stimulation. In experiments with primary neuronal cultures from CaV1.3 knockout mice we discovered that CaV1.3 channels play a more important role in pCREB signaling in striatal medium spiny neurons than in hippocampal neurons. Our results provide novel insights into the function of CaV1.2 and CaV1.3 L-type Ca2+ channels in the brain.     

Death of motor neurons and molecular change of glutamate receptors in ALS]

AMPA receptor-mediated neuronal death is initiated by exaggerated Ca2+ influx through AMPA receptor channels, and the Ca2+ permeability of the AMPA receptor ion channel depends strongly upon the presence or absence in its composition of an edited GluR2 subunit whose glutamine (Q) residue is substituted by arginine (R) at the Q/R site due to RNA editing. The pivotal role of the RNA editing at the GluR2 Q/R site in neuronal death has been clearly demonstrated in animal experiments and its deficiency is a direct cause of neuronal death. We demonstrated that the editing efficiency at the GluR2 mRNA Q/R site varied greatly, from 0% to 100%, among the single motoneurons of each individual with ALS, whereas it remained 100% among those of normal controls. In addition, the editing efficiency was more than 99% in the cerebellar Purkinje cells of ALS, spinocerebellar degeneration and normal control groups. By contrast, there was no significant difference as to both the amount and the proportion to total GluRs mRNA of GluR2 mRNA between normal and ALS motoneurons. Thus, marked GluR2 underediting in ALS motoneurons occurs in a disease specific and region selective manner, and may be closely relevant to ALS etiology.     

Expression of recombinant NMDA receptors in hippocampal neurons by adenoviral-mediated gene transfer.

N-methyl-d-aspartate (NMDA) receptors have attracted a great deal of attention because they are intimately involved in brain development, synaptic plasticity and a variety of neurological disorders. The ability to artificially alter the properties of NMDA receptors in central nervous system (CNS) neurons would be useful for elucidating the physiological roles of these receptors. It would also raise the possibility of gene therapy of neurological diseases caused by malfunction of NMDA receptors. In this study, we constructed three recombinant adenoviruses encoding rat NMDA receptor subunit cDNAs, NMDAR1 (NR1), NMDAR2B (NR2B) and mutant NR1(N598R) in which the asparagine (N) site of the wild-type NR1 was replaced with arginine (R) by site-directed mutagenesis. PC12 cells co-infected with recombinant adenoviruses bearing NR1 and NR2B cDNAs expressed conventional NMDA receptors that were permeable to Ca2+ and sensitive to Mg2+, whereas those with viruses bearing NR1(N598R) and NR2B cDNAs expressed Ca2+-impermeable and Mg2+-insensitive receptors. When rat hippocampal neurons in culture were infected with NR1(N598R) and NR2B viruses, both Ca2+ permeability and Mg2+ sensitivity of NMDA receptors were markedly reduced in the infected neurons. Excitatory postsynaptic currents (EPSCs) mediated by NMDA receptors also became much less sensitive to Mg2+. Thus, the NR1(N598R)/NR2B receptors were more dominant than the native NMDA receptors in the infected neurons, and the former receptors introduced by the adenoviral vectors functioned as postsynaptic receptors. These results indicate that the functional properties of postsynaptic NMDA receptors can be manipulated by gene transfer technology using adenoviral vectors.     

Protein kinase C activators reduce the inositol trisphosphate-induced outward current and the Ca2+-activated outward current in identified neurons of Aplysia

Effects of intracellularly injected activators of protein kinase C on the InsP3-induced K+ current and the Ca2+-activated K+ current recorded from identified neurons (R9-R12) of Aplysia kurodai were investigated with conventional voltage-clamp and pressure-injection techniques. Intracellular injection of InsP3 into identified neurons produced a 4-aminopiridine (4-AP)-resistant, tetraethylammonium (TEA)-sensitive, and quinidine-sensitive K+ current similar to the Ca2+ activated K+ current elicited by direct injection of Ca2+ ions into the same neurons. The diacylglycerol analogue 1,2-oleoylacetylglycerol (OAG) at an intracellular concentration of 65 nM produced irreversible decreases in both the InsP3-induced K+ current and the Ca2+-activated K+ current. The phorbol 12,13-dibutyrate (PDBu) at an intracellular concentration of 150 nM also decreased irreversibly both the InsP3-induced K+ current and the Ca2+-activated K+ current. These results suggest that protein kinase C activators reduce both the InsP3-induced K+ current and the Ca2+-activated K+ current recorded from certain identified neurons of Aplysia and that protein kinase C reduces the ability of Ca2+ to open K+ channels rather than affecting the ability of InsP3 to release Ca2+ from intracellular stores.     

In vivo characterization of a smart MRI agent that displays an inverse response to calcium concentration

Contrast agents for magnetic resonance imaging (MRI) that exhibit sensitivity toward specific ions or molecules represent a challenging but attractive direction of research. Here a Gd(3+) complex linked to an aminobis(methylenephosphonate) group for chelating Ca(2+) was synthesized and investigated. The longitudinal relaxivity (r(1)) of this complex decreases during the relaxometric titration with Ca(2+) from 5.76 to 3.57 mM(-1) s(-1) upon saturation. The r(1) is modulated by changes in the hydration number, which was confirmed by determination of the luminescence emission lifetimes of the analogous Eu(3+) complex. The initial in vivo characterization of this responsive contrast agent was performed by means of electrophysiology and MRI experiments. The investigated complex is fully biocompatible, having no observable effect on neuronal function after administration into the brain ventricles or parenchyma. Distribution studies demonstrated that the diffusivity of this agent is significantly lower compared with that of gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA).