In Situ Hybridization Evidence of Differential Modulation by Pentobarbital of GABAA Receptor α1- and β3-Subunit mRNAs

Abstract: Tolerance to and withdrawal from pentobarbital were induced in rats by continuous intracerebroventricular infusion via subcutaneously implanted osmotic minipumps. In situ hybridization of GABA_A receptor α₁- and β₃-subunit mRNA was conducted using synthetic 3'- end ³⁵S-dATP-labeled oligodeoxynucleotide probes. Results were quantified by film densitometry. In animals that were tolerant to pentobarbital, levels of α₁-subunit mRNA were decreased in hippocampus, superior colliculus, and inferior colliculus, but levels of β₃-subunit mRNA were not affected. Dramatically increased levels of GABA_A receptor subunit mRNA were observed in animals 24 h after withdrawal from chronic pentobarbital treatment. These increases occurred in cerebral cortex and cerebellum for the α₁ subunit and in cerebral cortex only for the β₃-subunit. These data provide further support to the structural and pharmacological GABA_A receptor heterogeneity in discrete brain areas. The observed changes of subunit expression may underlie, at least in part, the receptor up- and down-regulation observed in receptor ligand binding studies.     

Chronic Ethanol Administration Regulates the Expression of GABAA Receptor α1 and α5 Subunits in the Ventral Tegmental Area and Hippocampus

Abstract: Ethanol dependence and tolerance involve perturbation of GABAergic neurotransmission. Previous studies have demonstrated that ethanol treatment regulates the function and expression of GABA_A receptors throughout the CNS. Conceivably, changes in receptor function may be associated with alterations of subunit composition. In the present study, a comprehensive (1–12 weeks) ethanol treatment paradigm was used to evaluate changes in GABA_A receptor subunit expression in several brain regions including the cerebellum, cerebral cortex, ventral tegmental area (VTA) (a region implicated in drug reward/dependence), and the hippocampus (a region involved in memory/cognition). Expression of α₁ and α₅ subunits was regulated by ethanol in a region-specific and time-dependent manner. Following 2–4 weeks of administration, cortical and cerebellar α₁ and α₅ subunit immunoreactivity was reduced. In the VTA, levels of α₁ subunit immunoreactivity were significantly decreased after 12 weeks but not 1–4 weeks of treatment. Hippocampal α₁ subunit immunoreactivity and mRNA content were also significantly reduced after 12 but not after 4 weeks of treatment. In contrast, α₅ mRNA content was increased in this brain region. These data indicate that chronic ethanol administration alters GABA_A receptor subunit expression in the VTA and hippocampus, effects that may play a role in the abuse potential and detrimental cognitive effects of alcohol.     

Platelet activation by a relapsing fever spirochaete results in enhanced bacterium–platelet interaction via integrin αIIbβ3 activation

Borrelia hermsii , a spirochaete responsible for relapsing fever in humans, grows to high density in the bloodstream and causes thrombocytopenia. We show here that B. hermsii binds to human platelets. Extended culture in bacteriological medium resulted in both diminished infectivity in vivo and diminished platelet binding in vitro . Platelet binding was promoted by the platelet integrin α_IIbβ₃: the bacterium bound to purified integrin α_IIbβ₃, and bacterial binding to platelets was diminished by α_IIbβ₃ antagonists or by a genetic defect in this integrin. Integrin α_IIbβ₃ undergoes a conformational change upon platelet activation, and bacteria bound more efficiently to activated rather than resting platelets. Nevertheless, B. hermsii bound at detectable levels to preparations of resting platelets. The bacterium did not recognize a point mutant of α_IIbβ₃ that cannot acquire an active conformation. Rather, B. hermsii was capable of triggering platelet and integrin α_IIbβ₃ activation, as indicated by the expression of the platelet activation marker P-selectin and integrin α_IIbβ₃ in its active conformation. The degree of platelet activation varied depending upon bacterial strain and growth conditions. Prostacyclin I₂, an inhibitor of platelet activation, diminished bacterial attachment, indicating that activation enhanced bacterial binding. Thus, B. hermsii signals the host cell to activate a critical receptor for the bacterium, thereby promoting high-level bacterial attachment.     

CaMKII associates with CaV1.2 L-type calcium channels via selected β subunits to enhance regulatory phosphorylation

Calcium/calmodulin-dependent kinase II (CaMKII) facilitates L-type calcium channel (LTCC) activity physiologically, but may exacerbate LTCC-dependent pathophysiology. We previously showed that CaMKII forms stable complexes with voltage-gated calcium channel (VGCC) β_1b or β_2a subunits, but not with the β₃ or β₄ subunits ( Grueter et al. 2008 ). CaMKII-dependent facilitation of Ca_V1.2 LTCCs requires Thr498 phosphorylation in the β_2a subunit ( Grueter et al. 2006 ), but the relationship of this modulation to CaMKII interactions with LTCC subunits is unknown. Here we show that CaMKII co-immunoprecipitates with forebrain LTCCs that contain Ca_V1.2α₁ and β₁ or β₂ subunits, but is not detected in LTCC complexes containing β₄ subunits. CaMKIIα can be specifically tethered to the I/II linker of Ca_V1.2 α₁ subunits in vitro by the β_1b or β_2a subunits. Efficient targeting of CaMKIIα to the full-length Ca_V1.2α₁ subunit in transfected HEK293 cells requires CaMKII binding to the β_2a subunit. Moreover, disruption of CaMKII binding substantially reduced phosphorylation of β_2a at Thr498 within the LTCC complex, without altering overall phosphorylation of Ca_V1.2α₁ and β subunits. These findings demonstrate a biochemical mechanism underlying LTCC facilitation by CaMKII.     

Mechanism of docosahexaenoic acid-induced inhibition of in vitro Aβ1–42 fibrillation and Aβ1–42-induced toxicity in SH-S5Y5 cells

The mechanism of the effect of docosahexaenoic acid (DHA; C22:6, n -3), one of the essential brain nutrients, on in vitro fibrillation of amyloid β (Aβ_1–42), Aβ_1–42-oligomers and its toxicity imparted to SH-S5Y5 cells was studied with the use of thioflavin T fluorospectroscopy, laser confocal microfluorescence, and transmission electron microscopy. The results clearly indicated that DHA inhibited Aβ_1–42-fibrill formation with a concomitant reduction in the levels of soluble Aβ_1–42 oligomers. The polymerization (into fibrils) of preformed oligomers treated with DHA was inhibited, indicating that DHA not only obstructs their formation but also inhibits their transformation into fibrils. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (12.5%), Tris–Tricine gradient(4–20%) gel electrophoresis and western blot analyses revealed that DHA inhibited at least 2 species of Aβ_1–42 oligomers of 15–20 kDa, indicating that it hinders these on-pathway tri/tetrameric intermediates during fibrillation. DHA also reduced the levels of dityrosine and tyrosine intrinsic fluorescence intensity, indicating DHA interrupts the microenvironment of tyrosine in the Aβ_1–42 backbone. Furthermore, DHA protected the tyrosine from acrylamide collisional quenching, as indicated by decreases in Stern–Volmer constants. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide-reduction efficiency and immunohistochemical examination suggested that DHA inhibits Aβ_1–42-induced toxicity in SH-S5Y5 cells. Taken together, these data suggest that by restraining Aβ_1–42 toxic tri/tetrameric oligomers, DHA may limit amyloidogenic neurodegenerative diseases, Alzheimer's disease.     

N-cadherin-based adhesion enhances Aβ release and decreases Aβ42/40 ratio

In neurons, Presenilin 1(PS1)/γ-secretase is located at the synapses, bound to N-cadherin. We have previously reported that N-cadherin-mediated cell–cell contact promotes cell-surface expression of PS1/γ-secretase. We postulated that N-cadherin-mediated trafficking of PS1 might impact synaptic PS1-amyloid precursor protein interactions and Aβ generation. In the present report, we evaluate the effect of N-cadherin-based contacts on Aβ production. We demonstrate that stable expression of N-cadherin in Chinese hamster ovary cells, expressing the Swedish mutant of human amyloid precursor protein leads to enhanced secretion of Aβ in the medium. Moreover, N-cadherin expression decreased Aβ_42/40 ratio. The effect of N-cadherin expression on Aβ production was accompanied by the enhanced accessibility of PS1/γ-secretase to amyloid precursor protein as well as a conformational change of PS1, as demonstrated by the fluorescence lifetime imaging technique. These results indicate that N-cadherin-mediated synaptic adhesion may modulate Aβ secretion as well as the Aβ_42/40 ratio via PS1/N-cadherin interactions.     

Rat Frontal Cortex β1-Adrenoceptors Are Activated by the β3-Adrenoceptor Agonists SR 58611A and SR 58878A but Not by BRL 37344 or ICI 215,001

Abstract: SR 58611A, a selective agonist of gut and brown adipose tissue β₃-adrenoceptors (β₃ARs), has been reported to have antidepressant-like activity in rodents by indicating brain β₃ARs as the sites of this property. SR 58611A and its acid metabolite SR 58878A, as opposed to BRL 37344, ICI 215,001, and CGP 12177, increased cyclic AMP levels in rat frontal cortex. ICI 215,001, differently from BRL 37344, at concentrations in the millimolar range antagonized norepinephrine- or (−)-isoproterenol-stimulated adenylyl cyclase partially. The increase of cyclic AMP levels induced by SR 58878A was blocked selectively by β₁AR antagonist CGP 20712A but not by β₂AR antagonist ICI 118,551. In addition, PCR analysis did not reveal β₃AR mRNA, and no specific β₃AR binding sites were detected by [³H]CGP 12177 in rat frontal cortex. When down-regulation of the β₁AR ligand binding and mRNA levels had been induced in frontal cortex by chronic administration of imipramine, SR 58878A as well as norepinephrine and (−)-isoproterenol increased the cyclic AMP production less markedly. Our findings indicate that β₃ARs are absent in the adult rat frontal cortex, and that various β₃AR agonists differently affect the frontal cortex β₁ARs, indicating that SR 58611A may exert its putative antidepressant effect acting on the frontal cortex β₁ARs.     

Evidence for the Existence of Differential O-Glycosylated α5-Subunits of the γ-Aminobutyric AcidA Receptor in the Rat Brain

Abstract: Polyclonal antibodies were raised to synthetic peptides having amino acid sequences corresponding with the N- or C-terminal part of the γ-aminobutyric acid_A (GABA_A) receptor α₅-subunit. These anti-peptide α₅(2–10) or anti-peptide α₅(427–433) antibodies reacted specifically with GABA_A receptors purified from the brains of 5–10-day-old rats in an enzyme-linked immunosorbent assay and were able to dose-dependently immunoprecipitate up to 6.3 or 13.1% of the GABA_A receptors present in the incubation, respectively. In immunoblots, each of these antibodies reacted with the same two protein bands with apparent molecular mass of 53 or 57 kDa. After exhaustive treatment of purified GABA_A receptors with N -Glycanase, each of these antibodies identified two proteins with apparent molecular masses of 46 and 48 kDa. Additional treatment of GABA_A receptors with neuraminidase and O -Glycanase resulted in an apparently single protein with molecular mass of 47 kDa, which again was identified by both the anti-peptide α₅(2–10) and the anti-peptide α₅(427–433) antibody. These results indicate the existence of at least two different α₅-sub-units of the GABA_A receptor that differ in their carbohydrate content. In contrast to other α- or β-subunits of GABA_A receptors so far investigated, at least one of these two α₅-subunits contains O-linked carbohydrates.     

Cloning and Characterization of a Mouse σ1 Receptor

Abstract: A cDNA clone (S2-1a) isolated from a mouse brain cDNA library, using a guinea pig σ₁ cDNA as probe, has high homology to the predicted protein sequence of the guinea pig (88%) and human (90%) σ₁ receptors. Northern analysis revealed a major mRNA of ∼1.8 kb in a wide range of mouse tissues, with highest levels in brain, liver, kidney, and thymus. Southern analysis and chromosomal mapping in the mouse suggested a single-copy gene in region A5-B2 of chromosome 4. Expression of the clone in MCF-7 and CHO cells led to a pronounced increase in (+)-[³H]pentazocine binding with a selectivity profile consistent with σ₁ receptors. In vitro translation yielded a protein of ∼28 kDa, as did transfection of a probe containing the hemagglutinin (HA) epitope (S2-1a.HA) into CHO cells, as determined by western analysis using an antibody directed against HA. (+)-[³H]-Pentazocine binding to immunopurified HA-tagged receptor demonstrated conclusively that S2-1a.HA encodes a high-affinity (+)-[³H]pentazocine binding site with characteristics of a murine σ₁ receptor. An antisense oligodeoxynucleotide designed from S2-1a potentiated opioid analgesia in vivo.     

Functional Rescue of β1-Adrenoceptor Dimerization and Trafficking by Pharmacological Chaperones

Site-directed mutagenesis guided by evolutionary trace analysis revealed that substitution of V179 and W183 within a cluster of evolutionarily important residues on the surface of the fourth transmembrane domain of the β₁-adrenergic receptor (β₁AR) significantly reduced the propensity of the receptor to self-assemble into homodimers as assessed by bioluminescence resonance energy transfer in living cells. These results suggest that mutation of V179 and W183 result in conformational changes that reduce homodimerization either directly by interfering with the dimerization interface or indirectly by causing local misfolding that result in reduced self-assembly. However, the mutations did not cause a general misfolding of the β₁AR as they did not prevent heterodimerization with the β₂AR. The homodimerization-compromised mutants were significantly retained in the endoplasmic reticulum (ER) and could not be properly matured and trafficked to the cell surface. Lipophilic β-adrenergic ligands acted as pharmacological chaperones by restoring both dimerization and plasma membrane trafficking of the ER-retained dimerization-compromised β₁AR mutants. These results clearly indicate that homodimerization occurs early in the biosynthetic process in the ER and that pharmacological chaperones can promote both dimerization and cell surface targeting, most likely by stabilizing receptor conformations compatible with the two processes.     

The Subunit Composition of a GABAA/Benzodiazepine Receptor from Rat Cerebellum

Abstract: The pentameric subunit composition of a large population (36%) of the cerebellar granule cell GABA_A receptors that show diazepam (or clonazepam)-insensitive [³H]Ro 15-4513 binding has been determined by immunoprecipitation with subunit-specific antibodies. These receptors have α₆, α₁, γ_2S, γ_2L, and β₂ or β₃ subunits colocalizing in the same receptor complex.     

α2-Macroglobulin Complexes with and Mediates the Endocytosis of β-Amyloid Peptide via Cell Surface Low-Density Lipoprotein Receptor-Related Protein

Abstract: A primary histopathological feature of Alzheimer's disease is the accumulation of β-amyloid (Aβ) in the brain of afflicted individuals. However, Aβ is produced continuously as a soluble protein in healthy individuals where it is detected in serum and CSF, suggesting the existence of cellular clearance mechanisms that normally prevent its accumulation and aggregation. Here, we demonstrate that Aβ forms stable complexes with activated α₂-macroglobulin (α₂M^⋆), a physiological ligand for the low-density lipoprotein receptor-related protein (LRP) that is abundantly expressed in the CNS. These α₂M^⋆/¹²⁵I-Aβ complexes are immunoreactive with both anti-Aβ and anti-α₂M IgG and are stable under various pH conditions, sodium dodecyl sulfate, reducing agents, and boiling. We demonstrate that α₂M^⋆/¹²⁵I-Aβ complexes can be degraded by glioblastoma cells and fibroblasts via LRP, because degradation is partially inhibited by receptor-associated protein (RAP), an antagonist of ligand interactions with LRP. In contrast, the degradation of free ¹²⁵I-Aβ is not inhibited by RAP and thus must be mediated via an LRP-independent pathway. These results suggest that LRP can function as a clearance receptor for Aβ via a physiological ligand.     

α2-Macroglobulin Attenuates β-Amyloid Peptide 1–40 Fibril Formation and Associated Neurotoxicity of Cultured Fetal Rat Cortical Neurons

Abstract: β-Amyloid peptides (Aβ) are deposited in an aggregated fibrillar form in both diffuse and senile plaques in the brains of patients with Alzheimer's disease. The neurotoxicity of Aβ in cultured neurons is dependent on its aggregation state, but the factors contributing to aggregation and fibril formation are poorly understood. In the present study, we investigated whether α₂-macroglobulin (α₂M), a protein present in neuritic plaques and elevated in Alzheimer's disease brain, is a potential regulatory factor for Aβ fibril formation. Previous studies in our laboratory have shown that α₂M is an Aβ binding protein. We now report that, in contrast to another plaque-associated protein, α₁-antichymotrypsin, α₂M coincubated with Aβ significantly reduces aggregation and fibril formation in vitro. Additionally, cultured fetal rat cortical neurons are less vulnerable to the toxic actions of aged Aβ following pretreatment with α₂M. We postulate that α₂M is able to maintain Aβ in a soluble state, preventing fibril formation and associated neurotoxicity.     

Monoclonal Antibodies to the Human γ2 Subunit of the GABAA/Benzodiazepine Receptors

Abstract: The large intracellular loop (IL) of the γ₂ subunit of the cloned human γ-aminobutyric acid_A (GABA_A) receptor (γ₂IL) was expressed in bacteria as glutathione- S -transferase and staphylococcal protein A fusion proteins. Mice were immunized with the fusion proteins (one protein per animal), and monoclonal antibodies were obtained. Six monoclonal antibodies reacted with the γ₂IL moiety of the fusion proteins. Three of these monoclonal antibodies also immunoprecipitated a high proportion of the GABA_A/benzodiazepine receptors from bovine and rat brain and reacted with a wide 44,000–49,000-M_r peptide band in immunoblots of affinity-purified GABA_A receptors. These monoclonal antibodies are valuable reagents for the molecular characterization of the GABA_A receptors in various brain regions.     

Rapid Degeneration of Cultured Human Brain Pericytes by Amyloid β Protein

Abstract: Amyloid β protein (Aβ) deposition in the cerebral arterial and capillary walls is one of the major characteristics of brains from patients with Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D). Vascular Aβ deposition is accompanied by degeneration of smooth muscle cells and pericytes. In this study we found that Aβ_1–40 carrying the "Dutch" mutation (HCHWA-D Aβ_1–40) as well as wild-type Aβ_1–42 induced degeneration of cultured human brain pericytes and human leptomeningeal smooth muscle cells, whereas wild-type Aβ_1–40 and HCHWA-D Aβ_1–42 were inactive. Cultured brain pericytes appeared to be much more vulnerable to Aβ-induced degeneration than leptomeningeal smooth muscle cells, because in brain pericyte cultures cell viability already decreased after 2 days of exposure to HCHWA-D Aβ_1–40, whereas in leptomeningeal smooth muscle cell cultures cell death was prominent only after 4–5 days. Moreover, leptomeningeal smooth muscle cell cultures were better able to recover than brain pericyte cultures after short-term treatment with HCHWA-D Aβ_1–40. Degeneration of either cell type was preceded by an increased production of cellular amyloid precursor protein. Both cell death and amyloid precursor protein production could be inhibited by the amyloid-binding dye Congo red, suggesting that fibril assembly of Aβ is crucial for initiating its destructive effects. These data imply an important role for Aβ in inducing perivascular cell pathology as observed in the cerebral vasculature of patients with Alzheimer's disease or HCHWA-D.