Isolation of type I and type II GABAA-benzodiazepine receptors by immunoaffinity chromatography.

Anti-peptide alpha 1 (1-9) and anti-peptide alpha 3 (459-467) antibodies coupled to Affigel-10 were used for the isolation of GABAA receptors containing the alpha 1- or alpha 3-subunit, respectively. Both types of GABAA receptors exhibited a high affinity for [3H]flunitrazepam, and binding of [3H]flunitrazepam was stimulated in the presence of GABA. GABAA receptors eluted from the anti-peptide alpha 1 (1-9) immunoaffinity column exhibited a high affinity and those from the anti-peptide alpha 3 (459-467) columns a low affinity for the type I benzodiazepine receptor-selective ligand Cl 218872, indicating the enrichment of type I and type II GABAA-benzodiazepine receptors, respectively.     

GABA influences the development of the ventromedial nucleus of the hypothalamus

The region that becomes the ventromedial nucleus of the hypothalamus (VMH) is surrounded by cells and fibers containing immunoreactive gamma‐aminobutyric acid (GABA) by embryonic day 13 (E13), several days before the nucleus emerges in Nissl stains. As GABA plays many roles during neural development, we hypothesized that it influences VMH development, perhaps by providing boundary information for migrating neurons. To test this hypothesis we examined the VMH in embryonic mice in which the β3 subunit of the GABA_A‐receptor, a receptor subunit that is normally highly expressed in this nucleus, was disrupted by gene targeting. In β3 ?/? embryos the VMH was significantly larger, and the distribution of cells containing immunoreactive estrogen receptor‐α was expanded compared to controls. Using in vitro brain slices from wild‐type C57BL/6J mice killed at E15 we found that treatment with the GABA_A antagonist bicuculline increased the number of cells migrating per video field analyzed in the VMH. In addition, treatment with either bicuculline or the GABA_A agonist muscimol altered the orientation of cell migration in particular regions of this nucleus. These data suggest that GABA is important for the organization of cells during VMH formation. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 264–276, 2001     

A novel site on gamma 3 subunits important for assembly of GABA(A) receptors

Gamma-aminobutyric acid, type A (GABA(A)) receptors are ligand-gated chloride channels and are the major inhibitory transmitter receptors in the central nervous system. The majority of these receptors is composed of two alpha, two beta, and one gamma subunits. To identify sequences important for subunit assembly, we generated C-terminally truncated and chimeric gamma(3) constructs. From their ability to associate with full-length alpha(1) and beta(3) subunits, we concluded that amino acid sequence gamma(3)(70-84) either directly interacts with alpha(1) or beta(3) subunits or stabilizes a contact site elsewhere in the protein. The observation that this sequence contains amino acid residues homologous to gamma(2) residues contributing to the benzodiazepine-binding site at the alpha(1)/gamma(2) interface suggested that in alpha(1)beta(3)gamma(3) receptors the sequence gamma(3)(70-84) is located at the alpha(1)/gamma(3) interface. In the absence of alpha(1) subunits this sequence might allow assembly of beta(3) with gamma(3) subunits. Other experiments indicated that sequences gamma(3)(86-95) and gamma(3)(94-107), which are homologous to previously identified sequences important for assembly of gamma(2) subunits, are also important for assembly of gamma(3) subunits. This indicates that during assembly of the GABA(A) receptor, more than one N-terminal sequence is important for binding to the same neighboring subunit. Whether the three sequences investigated are involved in direct interaction or stabilize other regions involved in intersubunit contacts has to be further studied.     

Caspase inhibition activates HIV in latently infected cells. Role of tumor necrosis factor receptor 1 and CD95

Stimulation of tumor necrosis factor receptor 1 (TNF-R1) triggers both caspase-dependent and caspase-independent signaling activities. The caspase-dependent signaling pathway induces apoptotic cell death in susceptible cells, whereas the caspase-independent signaling cascade leads to activation of nuclear factor kappa B and induces antiapoptotic signaling activities. Stimulation of nuclear factor kappa B via TNF-R1 is known to activate human immunodeficiency virus (HIV) replication in infected cells. Here we show that the broad range caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (ZVAD) activates HIV replication in the chronically infected T-cell line ACH-2. Virus activation was caused by a sensitization of TNF-R1 toward endogenously produced tumor necrosis factor alpha (TNF-alpha). Neutralizing anti-TNF-alpha antibodies completely abolished the virus-inducing activity of ZVAD. Treatment of cells with TNF-alpha in the presence of ZVAD caused increased expression of TNF-alpha and induced enhanced virus replication. Activation of CD95, another member of the TNF receptor family, similarly triggered HIV replication, which was further enhanced in the presence of ZVAD. Our data show that caspase inhibitors sensitize both CD95 and TNF-R1 to mediate activation of HIV in latently infected cells. Activation of HIV replication in latent virus reservoirs is currently discussed as a therapeutic strategy to achieve eradication of HIV in patients treated with antiretroviral therapy. Our results point to a novel role for caspase inhibitors as activators of virus replication in vivo.     

Binding of γ-Aminobutyric AcidA Receptors to Tubulin

Abstract: The rate of axonal transport of tubulin, actin, and the neurofilament proteins was measured in the peripheral and central projections of the rat L5 dorsal root ganglion (DRG). [³⁵S]Methionine was injected into the DRG, and the "front" of the radiolabeled protein was located 7, 14, and 20 days postinjection. Transport rates calculated for the neurofilament triplet proteins, tubulin, and actin in the peripheral nerve were ∼ 1.5-fold faster than those in the dorsal root. A progressive decrease in the rate of transport was observed from 7 to 20 days after radiolabeling in both the central and peripheral directions (neurofilaments, ∼ 1.7-fold; tubulin/actin, 2.1-fold). A surgical preparation, leaving the peripheral sciatic nerve with predominantly sensory fibers, was the basis for ELISAs for phosphorylation-dependent immunoreactivity of the high-molecular-weight neurofilament protein. In both dorsal roots and peripheral sensory axons the degree of phosphorylation was greater in nerve segments further away from the cell bodies. The degree of phosphorylation-related immunoreactivity correlates with the slowing of transport of radiolabeled cytoskeletal protein.     

Sedimentation and release properties of P2 fractions derived from rat cerebral cortex slices incubated with radiolabeled GABA for a short or long time period

On homogenization of rat cerebral cortex slices previously incubated with [³H] GABA or [¹⁴C]GABA for 5 or 30 min, respectively, particles were recovered in P₂ fractions which exhibited similar buoyant density, but different sedimentation velocity on linear sucrose density gradient centrifugation. The K⁺-evoked release of [³H]GABA from particles isolated from slices previously incubated for 5 min with [³H]GABA was increased in the presence of exogenous Ca²⁺. In contrast, the K⁺-evoked release from particles isolated from slices previously incubated for 30 min with [³H]GABA, was not influenced by the presence of exogenous Ca²⁺.These results suggest that, depending on the incubation time of slices, exogenously applied GABA can be detected in differnnt pools. These pools not only seem to differ in their Ca²⁺ dependency of K⁺-evoked release but also in their subcellular localization.     

GABAA receptor phosphorylation and functional modulation in cortical neurons by a protein kinase C-dependent pathway

GABA(A) receptors are critical mediators of fast synaptic inhibition in the brain, and the predominant receptor subtype in the central nervous system is believed to be a pentamer composed of alpha, beta, and gamma subunits. Previous studies on recombinant receptors have shown that protein kinase C (PKC) and PKA directly phosphorylate intracellular serine residues within the receptor beta subunit and modulate receptor function. However, the relevance of this regulation for neuronal receptors remains poorly characterized. To address this critical issue, we have studied phosphorylation and functional modulation of GABA(A) receptors in cultured cortical neurons. Here we show that the neuronal beta3 subunit is basally phosphorylated on serine residues by a PKC-dependent pathway. PKC inhibitors abolish basal phosphorylation, increasing receptor activity, whereas activators of PKC enhance beta3 phosphorylation with a concomitant decrease in receptor activity. PKA activators were shown to increase the phosphorylation of the beta3 subunit only in the presence of PKC inhibitors. We also show that the main sites of phosphorylation within the neuronal beta3 subunit are likely to include Ser-408 and Ser-409, residues that are important for the functional modulation of beta3-containing recombinant receptors. Furthermore, PKC activation did not change the total number of GABA(A) receptors in the plasma membrane, suggesting that the effects of PKC activation are on the gating or conductance of the channel. Together, these results illustrate that cell-signaling pathways that activate PKC may have profound effects on the efficacy of synaptic inhibition by directly modulating GABA(A) receptor function.     

Evidence for recombination of live, attenuated immunodeficiency virus vaccine with challenge virus to a more virulent strain

Live, attenuated immunodeficiency virus vaccines, such as nef deletion mutants, are the most effective vaccines tested in the simian immunodeficiency virus (SIV) macaque model. In two independent studies designed to determine the breadth of protection induced by live, attenuated SIV vaccines, we noticed that three of the vaccinated macaques developed higher set point viral load levels than unvaccinated control monkeys. Two of these vaccinated monkeys developed AIDS, while the control monkeys infected in parallel remained asymptomatic. Concomitant with an increase in viral load, a recombinant of the vaccine virus and the challenge virus could be detected. Therefore, the emergence of more-virulent recombinants of live, attenuated immunodeficiency viruses and less-aggressive wild-type viruses seems to be an additional risk of live, attenuated immunodeficiency virus vaccines.     

Phosphorylation state of the native high-molecular-weight neurofilament subunit protein from cervical spinal cord in sporadic amyotrophic lateral sclerosis

The intraneuronal aggregation of phosphorylated high-molecular-weight neurofilament protein (NFH) in spinal cord motor neurons is considered to be a key pathological marker of amyotrophic lateral sclerosis (ALS). In order to determine whether this observation is due to the aberrant or hyper-phosphorylation of NFH, we have purified and characterized NFH from the cervical spinal cords of ALS patients and controls. We observed no differences between ALS and normal controls in the physicochemical properties of NFH in Triton X-100 insoluble protein fractions, with respect to migration patterns on 2D-iso electrofocusing (IEF) gels, the rate of Escherichia coli alkaline phosphatase mediated dephosphorylation, or the rate of calpain-mediated proteolysis. The rate of calpain-mediated proteolysis was unaffected by either exhaustive NFH dephosphorylation or by the addition of calmodulin to the reaction. Phosphopeptides and the phosphorylated motifs characterized by liquid chromatography tandem mass spectroscopy (LC/MS/MS) analysis demonstrated that all the phosphorylated residues found in ALS NFH were also found to be phosphorylated in normal human NFH samples. Hence, we have observed no difference in the physicochemical properties of normal and ALS NFH extracted from cervical spinal cords, suggesting that the perikaryal aggregation of highly phosphorylated NF in ALS neurons reflects the aberrant somatotopic localization of normally phosphorylated NFH.     

Targeted disruption of the GABA(A) receptor delta subunit gene leads to an up-regulation of gamma 2 subunit-containing receptors in cerebellar granule cells

GABA(A) receptors are chloride channels composed of five subunits. Cerebellar granule cells express abundantly six subunits belonging to four subunit classes. These are assembled into a number of distinct receptors, but the regulation of their relative proportions is yet unknown. Here, we studied the composition of cerebellar GABA(A) receptors after targeted disruption of the delta subunit gene. In membranes and extracts of delta-/- cerebellum, [(3)H]muscimol binding was not significantly changed, whereas [(3)H]Ro15-4513 binding was increased by 52% due to an increase in diazepam-insensitive binding. Immunocytochemical and Western blot analysis revealed no change in alpha(6) subunits but an increased expression of gamma(2) subunits in delta-/- cerebellum. Immunoaffinity chromatography of cerebellar extracts indicated there was an increased coassembly of alpha(6) and gamma(2) subunits and that 24% of all receptors in delta-/- cerebellum did not contain a gamma subunit. Because 97% of delta subunits are coassembled with alpha(6) subunits in the cerebellum of wild-type mice, these results indicated that, in delta-/- mice, alpha(6)betagamma(2) and alphabeta receptors replaced delta subunit-containing receptors. The availability of the delta subunit, thus, influences the level of expression or the extent of assembly of the gamma(2) subunit, although these two subunits do not occur in the same receptor.