Spreading depression-induced preconditioning in the mouse cortex: differential changes in the protein expression of ionotropic nicotinic acetylcholine and glutamate receptors

Preconditioning of the cerebral cortex was induced in mice by repeated cortical spreading depression (CSD), and the major ionotropic glutamate (GluRs) and nicotinic acetylcholine receptor (nAChRs) subunits were compared by quantitative immunoblotting between sham- and preconditioned cortex, 24 h after treatment. A 30% reduction in alpha-amino-3-hydroxy-5-methyl-4-iso- xazolepropionate (AMPA) GluR1 and 2 subunit immunoreactivities was observed in the preconditioned cortex (p < 0.03), but there was no significant change in the NMDA receptor subunits, NR1, NR2A and NR2B. A 12-15-fold increase in alpha7 nAChR subunit expression following in vivo CSD (p < 0.001) was by far the most remarkable change associated with preconditioning. In contrast, the alpha4 nAChR subunit was not altered. These data point to the alpha7 nAChR as a potential new target for neuroprotection because preconditioning increases consistently the tolerance of the brain to acute insults such as ischaemia. These data complement recent studies implicating alpha7 nAChR overexpression in the amelioration of chronic neuropathologies, notably Alzheimer's disease (AD).     

α4 but Not α3 and α7 Nicotinic Acetylcholine Receptor Subunits Are Lost from the Temporal Cortex in Alzheimer''s Disease

Neuronal nicotinic acetylcholine receptors labelled with tritiated agonists are reduced in the cerebral cortex in Alzheimer's disease (AD), but to date it has not been demonstrated which nicotinic receptor subunits contribute to this deficit. In the present study, autopsy tissue from the temporal cortex of 14 AD cases and 15 age-matched control subjects was compared using immunoblotting with antibodies against recombinant peptides specific for alpha3, alpha4, and alpha7 subunits, in conjunction with [3H]epibatidine binding. Antibodies to alpha3, alpha4, and alpha7 produced one major band on western blots at 59, 51, and 57 kDa, respectively. [3H]Epibatidine binding and alpha4-like immunoreactivity (using antibodies against the extracellular domain and cytoplasmic loop of the alpha4 subunit) were reduced in AD cases compared with control subjects (p < 0.02) and with a subgroup of control subjects (n = 9) who did not smoke prior to death (p < 0.05) for the former two parameters. [3H]Epibatidine binding and cytoplasmic alpha4-like immunoreactivity were significantly elevated in a subgroup of control subjects (n = 4) known to have smoked prior to death (p < 0.05). There were no significant changes in alpha3- or alpha7-like immunoreactivity associated with AD or tobacco use. The selective involvement of alpha4 has implications for understanding the role of nicotinic receptors in AD and potential therapeutic targets.     

Identification of the alpha 3-subunit in the GABAA receptor purified from bovine brain

Polyclonal antibodies have been raised to synthetic amino acid sequences of the bovine GABAA receptor alpha 1 and alpha 3 subunits. Anti-alpha 1 subunit antibodies recognise a polypeptide of 53 kDa whereas anti-alpha 3 subunit antibodies recognise a polypeptide of 59-60 kDa, in Western blots of GABAA receptor purified from adult bovine cerebral cortex, cerebellum and 12-day calf cerebral cortex.     

Immunological Identification of Multiple α-Like Subunits of the γ-Aminobutyric AcidA Receptor Complex Purified from Neonatal Rat Cortex

Antibodies were prepared against a synthetic peptide corresponding to amino acid sequences 174-203 of the bovine gamma-aminobutyric acidA (GABAA) receptor alpha 1-subunit. The antibodies recognized this synthetic alpha 1-peptide, but failed to react with the homologous peptide sequence, 170-199, of the bovine beta 1-subunit. On Western blots, anti-alpha 1-subunit antibody recognized a 50-kilodalton (kDa) protein in affinity-purified receptor preparations from adult rat cortex and cerebellum. In receptor purified from neonatal cortex, the anti-alpha 1-antibody reacted with 50-kDa, 53-54-kDa, and 59-kDa proteins. After digestion with endoglycosidase F, these three protein bands retained differing electrophoretic mobilities. The 50-kDa and 59-kDa subunits of affinity-purified neonatal receptor, which were photoaffinity-labeled with [3H]flunitrazepam, were immunoprecipitated to different extents by alpha-subunit antibody. These data suggest the existence in GABAA receptor from neonatal cortex of three proteins (50 kDa, 53 kDa, and 59 kDa) which have immunological homology to alpha 1-subunit of bovine GABAA receptor. The presence of an alpha- and a beta-like subunit with similar mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis may account for the relatively high concentration of protein in the 53-54-kDa band which has been observed in receptor purified from neonatal cortex. The presence of multiple alpha-like subunits may be related to the presence of a relatively high concentration of type II GABA receptor in this tissue.     

Detection of alpha7 nicotinic acetylcholine receptors with the aid of antibodies and toxins

Neuronal nicotinic acetylcholine receptors (nAChRs) containing alpha7 subunit are well represented in the brain and some non-neuronal tissues, and their malfunctioning is associated with diverse pathologies. Therefore, detection and quantification of alpha7 nAChR are important tasks. The affinity-purified antibodies were prepared against the 1-23 and 179-190 fragments of the human and rat alpha7 nAChR extracellular domain. The specificity and selectivity of these alpha7 (1-23) and alpha7 (179-190) antibodies was tested by ELISA in model systems: the E. coli-expressed alpha7 subunit extracellular domain and the pituitary cell line GH(4)C(1) stably expressing human alpha7 nAChR. On the rat brain slices two antibodies and biotinylated alpha-cobratoxin specifically stained the hippocampus region known to be rich in alpha7 nAChR. Western blot analysis revealed that in the human thalamus membranes and in rat brain membranes, antibodies alpha7 (1-23) stained a single band of 62 kDa, while the alpha7 (179-190) antibodies stained a doublet of 53-54 kDa. The results obtained show that utilization of model systems and a combination of several antibodies with appropriately labeled toxins may provide better ways for detection of alpha7 nAChR.     

Expression of nicotinic receptors on primary cultures of rat astrocytes and up-regulation of the alpha7, alpha4 and beta2 subunits in response to nanomolar concentrations of the beta-amyloid peptide(1-42)

Neuronal nicotinic acetylcholine receptors (nAChRs) are thought to be involved in the pathogenesis of Alzheimer's disease (AD). Interestingly, in the brains of patients with this disease, losses of several subtypes of nAChRs on neurons have been reported, while an increase in alpha7 nAChRs was recently detected in the astrocytes. However, little is presently known about the expressions of individual subunits of nAChR on rat astrocytes in primary culture or the possible influence of exposure to beta-amyloid peptide (Abeta), a neuropathological hallmark of AD, on this expression. Thus, in the present investigation the levels of individual nAChR subunits on primary rat astrocytes and the possible direct influence of Abetas on the receptors were examined by RT-PCR, Western blotting, monitoring intracellular free calcium and immunohistochemistry. The alpha4, alpha7, beta2 and beta3 subunits and related calcium channel responses were found in these cells, whereas neither alpha2 nor alpha3 could be detected. Elevation in the levels of alpha7, alpha4 and beta2 mRNAs and proteins were observed in astrocytes exposed to 0.1-100nM Abeta(1-42). In contrast, incubation with 1muM Abeta(1-42) or Abeta(35-25) did not affect these levels. We propose that the enhanced expression of alpha7, alpha4 and beta2 nAChRs by astrocytes stimulated directly by nanomolar concentrations of Abeta(1-42) might be related to ongoing defensive or compensative mechanisms.     

Molecular characterization of Dalpha6 and Dalpha7 nicotinic acetylcholine receptor subunits from Drosophila: formation of a high-affinity alpha-bungarotoxin binding site revealed by expression of subunit chimeras

Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission in the insect brain and are target sites for neonicotinoid insecticides. Seven nAChR subunits (four alpha-type and three beta-type) have been cloned previously from Drosophila melanogaster, the model insect system and characterized by heterologous expression. Recently, three further putative nAChR alpha subunits (Dalpha5, Dalpha6 and Dalpha7) with sequence similarity to the vertebrate alpha7 subunit have been identified from Drosophila genome sequence data but there have been no reports, as yet, of their characterization by heterologous expression. In the present study, we report the first isolation of a full-length Dalpha7 cDNA and the independent molecular cloning of Dalpha6. Binding of nicotinic radioligands was not detected to full-length Dalpha6 or Dalpha7 subunits when expressed alone or when or co-expressed with other nAChR subunits in Drosophila or mammalian cell lines, but specific cell-surface binding of [(125)I]alpha-bungarotoxin (K(d) = 0.68 +/- 0.22 nm) and [(3)H]methyllycaconitine (K(d) = 0.27 +/- 0.06 nm) was detected after expression of a subunit chimera containing the ligand-binding domains of Dalpha6 fused to the C-terminal domain of the 5-hydroxytryptamine receptor 5HT(3A). Although cell-surface binding was not detected with a Dalpha7/5HT(3Alpha) chimera expressed alone, co-expression of the two subunit chimeras resulted in significantly enhanced levels of nicotinic radioligand binding (with no change in affinity). This is the first evidence for the formation of a nAChR binding site by heterologously expressed Drosophila nAChR subunits in the absence of a co-expressed vertebrate nAChR subunit. In addition to the formation of homomeric nAChR complexes, evidence has been obtained from both radioligand binding and co-immunoprecipitation studies for the co-assembly of Dalpha6 and Dalpha7 into heteromeric cell surface complexes.