Antisense suppression of GABA(A) receptor beta subunit levels in cultured cerebellar granule neurons demonstrates their importance in receptor expression

GABA(A) receptors in the CNS are pentameric molecules composed of alpha, beta, gamma, delta, epsilon and theta subunits. Studies on transfected cells have shown that GABA(A) receptor beta subunit isoforms can direct alpha1 subunit localization within the cell. To examine the role of selected subunits in governing GABA(A) receptor expression in neurons, cultures of rat cerebellar granule cells were grown with antisense or sense oligodeoxynucleotides (ODNs) specific for the alpha 1, beta 2 or gamma 2 subunits. These subunits are all expressed in granule neurons where they are thought to contribute to an abundant receptor type. Following ODN treatment, subunit expression and distribution were examined by western blotting, immunocytochemistry and RT-PCR. Treatment of the cultures with the antisense, but not the corresponding sense, ODNs reduced the levels of the targeted subunit polypeptides. In addition, the beta 2 antisense ODN reduced the level of the alpha1 subunit polypeptide without altering the level of its mRNA. In contrast, treatment with the beta 2 subunit antisense ODN did not alter gamma 2 subunit polypeptide expression, distribution or mRNA level. These findings suggest that the alpha1 subunit requires a beta subunit for assembly into GABA(A) receptors in cerebellar granule neurons.     

Cell surface stability of gamma-aminobutyric acid type A receptors. Dependence on protein kinase C activity and subunit composition

Type A gamma-aminobutyric acid receptors (GABA(A)), the major sites of fast synaptic inhibition in the brain, are believed to be composed predominantly of alpha, beta, and gamma subunits. Although cell surface expression is essential for GABA(A) receptor function, little is known regarding its regulation. To address this issue, the membrane stability of recombinant alpha(1)beta(2) or alpha(1)beta(2)gamma(2) receptors was analyzed in human embryonic kidney cells. Alpha(1)beta(2)gamma(2) but not alpha(1)beta(2) receptors were found to recycle constitutively between the cell surface and a microtubule-dependent, perinuclear endosomal compartment. Similar GABA(A) receptor endocytosis was also seen in cultured hippocampal and cortical neurons. GABA(A) receptor surface levels were reduced upon protein kinase C (PKC) activation. Like basal endocytosis, this response required the gamma(2) subunit but not receptor phosphorylation. Although inhibiting PKC activity did not block alpha(1)beta(2)gamma(2) receptor endocytosis, it did prevent receptor down-regulation, suggesting that PKC activity may block alpha(1)beta(2)gamma(2) receptor recycling to the cell surface. In agreement with this observation, blocking recycling from endosomes with wortmannin selectively reduced surface levels of gamma(2)-containing receptors. Together, our results demonstrate that the surface stability of GABA(A) receptors can be dynamically and specifically regulated, enabling neurons to modulate cell surface receptor number upon the appropriate cues.     

GABAA alpha6-containing receptors are selectively compromised in cerebellar granule cells of the ataxic mouse, stargazer

Stargazer mice fail to express the gamma2 isoform of transmembrane alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) receptor regulatory proteins that has been shown to be absolutely required for the trafficking and synaptic targeting of excitatory AMPA receptors in adult murine cerebellar granule cells. Here we show that 30 +/- 6% fewer inhibitory gamma-aminobutyric acid, type A (GABA(A)), receptors were expressed in adult stargazer cerebellum compared with controls because of a specific loss of GABA(A) receptor expression in the cerebellar granule cell layer. Radioligand binding assays allied to in situ immunogold-EM analysis and furosemide-sensitive tonic current estimates revealed that expression of the extrasynaptic (alpha6betaxdelta) alpha6-containing GABA(A) receptor were markedly and selectively reduced in stargazer. These observations were compatible with a marked reduction in expression of GABA(A) receptor alpha6, delta (mature cerebellar granule cell-specific proteins), and beta3 subunit expression in stargazer. The subunit composition of the residual alpha6-containing GABA(A) receptors was unaffected by the stargazer mutation. However, we did find evidence of an approximately 4-fold up-regulation of alpha1betadelta receptors that may compensate for the loss of alpha6-containing GABA(A) receptors. PCR analysis identified a dramatic reduction in the steady-state level of alpha6 mRNA, compatible with alpha6 being the primary target of the stargazer mutation-mediated GABA(A) receptor abnormalities. We propose that some aspects of assembly, trafficking, targeting, and/or expression of extrasynaptic alpha6-containing GABA(A) receptors in cerebellar granule cells are selectively regulated by AMPA receptor-mediated signaling.     

The olivocerebellar projection to lobules I and II.

The olivocerebellar projection to lobules I and II was studied by means of retrograde transport from implants of the crystalline WGA-HRP complex. Retrogradely labelled neurons were found in the medial and dorsal accessory olives. Judged from the distribution of labelled cells, we conclude that parasagittally the olivocerebellar terminal zones A and B (i.e., the cerebellar cortical strips receiving axons from the olivary A and B regions) extend anteriorly into lobule Ia, whereas the fused olivocerebellar terminal C1/C3 zones reach lobule IIa. The olivocerebellar terminal C2 zone extends into lobule IIIa but not into lobule IIa. In lobule I the medial border of the B zone lies about 1 mm from the midline, in lobule II the B zone extends somewhat more medially. The lateral border of this zone is 1.9-2 mm from the midline. Compared to previous results, it appears that most of the Purkinje cells in lobule I projecting to the vestibular nuclei lie medially to the olivocerebellar terminal B zone.     

Development and expression of cytoplasmic antigens in Purkinje cells recognized by monoclonal antibodies

Summary Five monoclonal antibodies reacting with intracellular constituents of Purkinje cells were investigated by means of indirect immunofluorescence on fresh-frozen sections of the cerebellum and retina from developing and adult normal and mutant mice. Antibodies PC1, PC2 and PC3, which recognize Purkinje cells, but no other cerebellar neuron type, label these cells from day 4 onward. PC4 antigen is expressed in addition to Purkinje cells also in granule cells and neurons of deep cerebellar nuclei and appears in Purkinje cells at day 4. M1 antigen (Lagenaur et al. 1980) is first detectable in Purkinje cell bodies by day 5; it is also detectable in deep cerebellar neurons. In the adult retina, only PC4 antigen is detectably expressed and is localized in the inner segments of photoreceptor cells.The neurological mutants weaver, reeler,jimpy and wobbler show detectable levels of these antigens in Purkinje cells. However, the mutants staggerer and Purkinje cell degeneration are abnormal in expression PC1, PC2, PC3, and M1 antigens. Staggerer never starts to express the antigens during development, whereas Purkinje cell degeneration first expresses the antigens, but then loses antigen expression after day 23. PC4 antigen is detectable in the remaining Purkinje cells in staggerer and Purkinje cell degeneration mice at all ages tested in this study. Deep cerebellar neurons are positive for both antigens, PC4 and M1, in all mutants and at all ages studied. In retinas of staggerer and Purkinje cell degeneration mutants, PC4 antigen is normally detectable in the inner segments of photoreceptor cells, even when these have started to degenerate in the case of Purkinje cell degeneration.     

Identification of the sites for CaMK-II-dependent phosphorylation of GABA(A) receptors

Phosphorylation can affect both the function and trafficking of GABA(A) receptors with significant consequences for neuronal excitability. Serine/threonine kinases can phosphorylate the intracellular loops between M3-4 of GABA(A) receptor beta and gamma subunits thereby modulating receptor function in heterologous expression systems and in neurons (1, 2). Specifically, CaMK-II has been demonstrated to phosphorylate the M3-4 loop of GABA(A) receptor subunits expressed as GST fusion proteins (3, 4). It also increases the amplitude of GABA(A) receptor-mediated currents in a number of neuronal cell types (5-7). To identify which substrate sites CaMK-II might phosphorylate and the consequent functional effects, we expressed recombinant GABA(A) receptors in NG108-15 cells, which have previously been shown to support CaMK-II modulation of GABA(A) receptors containing the beta3 subunit (8). We now demonstrate that CaMK-II mediates its effects on alpha1beta3 receptors via phosphorylation of Ser(383) within the M3-4 domain of the beta subunit. Ablation of beta3 subunit phosphorylation sites for CaMK-II revealed that for alphabetagamma receptors, CaMK-II has a residual effect on GABA currents that is not mediated by previously identified sites of CaMK-II phosphorylation. This residual effect is abolished by mutation of tyrosine phosphorylation sites, Tyr(365) and Tyr(367), on the gamma2S subunit, and by the tyrosine kinase inhibitor genistein. These results suggested that CaMK-II is capable of directly phosphorylating GABA(A) receptors and activating endogenous tyrosine kinases to phosphorylate the gamma2 subunit in NG108-15 cells. These findings were confirmed in a neuronal environment by expressing recombinant GABA(A) receptors in cerebellar granule neurons.     

gamma-Aminobutyric acid(A) receptor subunit expression predicts functional changes in hippocampal dentate granule cells during postnatal development

Profound alterations in the function of GABA occur over the course of postnatal development. Changes in GABA(A) receptor expression are thought to contribute to these differences in GABAergic function, but how subunit changes correlate with receptor function in individual developing neurons has not been defined precisely. In the current study, we correlate expression of 14 different GABA(A) receptor subunit mRNAs with changes in the pharmacological properties of the receptor in individual hippocampal dentate granule cells over the course of postnatal development in rat. We demonstrate significant developmental differences in GABA(A) receptor subunit mRNA expression, including greater than two-fold lower expression of alpha1-, alpha4- and gamma2-subunit mRNAs and 10-fold higher expression of alpha5-mRNA in immature compared with adult neurons. These differences correlate both with regional changes in subunit protein level and with alterations in GABA(A) receptor function in immature dentate granule cells, including two-fold higher blockade by zinc and three-fold lower augmentation by type-I benzodiazepine site modulators. Further, we find an inverse correlation between changes in GABA(A) receptor zinc sensitivity and abundance of vesicular zinc in dentate gyrus during postnatal development. These findings suggest that developmental differences in subunit expression contribute to alterations in GABA(A) receptor function during postnatal development.     

ATP-Dependent Glutamate Uptake into Synaptic Vesicles from Cerebellar Mutant Mice

The ATP-dependent glutamate uptake system in synaptic vesicles prepared from mouse cerebellum was characterized, and the levels of glutamate uptake were investigated in the cerebellar mutant mice, staggerer and weaver, whose main defect is the loss of cerebellar granule cells, and the nervous mutant, whose main defect is the loss of Purkinje cells. The ATP-dependent glutamate uptake is stimulated by low concentrations of chloride, is insensitive to aspartate, and is inhibited by agents known to dissipate the electrochemical proton gradient. These properties are similar to those of the glutamate uptake system observed in the highly purified synaptic vesicles prepared from bovine cortex. The ATP-dependent glutamate uptake system is reduced by 68% in the staggerer and 57-67% in the weaver mutant; these reductions parallel the substantial loss of granule cells in those mutants. In contrast, the cerebellar levels of glutamate uptake are not altered significantly in the nervous mutant, which has lost Purkinje cells, but not granule cells. In view of evidence that granule cells are glutamatergic neurons and Purkinje cells are GABAergic neurons, these observations support the notion that the ATP-dependent glutamate uptake system is present in synaptic vesicles of glutamatergic neurons.     

Intracellular trafficking of GABA(A) receptors

Some of the mechanisms that control the intracellular trafficking of GABA(A) receptors have recently been described. Following the synthesis of alpha, beta, and gamma subunits in the endoplasmic reticulum, ternary receptor complexes assemble slowly and are inefficiently inserted into surface membranes of heterologous cells. While beta3, beta4, and gamma2S subunits appear to contain polypeptide sequences that alone are sufficient for surface targeting, these sequences are neither conserved nor essential for surface expression of heteromeric GABA(A) receptors formed from alpha1beta or alpha1betagamma subunits. At the neuronal surface, native GABA(A) receptor clustering and synaptic targeting require a gamma2 subunit and the participation of gephyrin, a clustering protein for glycine receptors. A linker protein, such as the GABA(A) receptor associated protein (GABARAP), may be necessary for the formation of GABA(A) receptor aggregates containing gephyrin. A substantial fraction of surface receptors are sequestered by endocytosis, another process which apparently requires a GABA(A) receptor gamma2 subunit. In heterologous cells, constitutive endocytosis seems to predominate while, in cortical neurons, internalization is evoked when receptors are occupied by GABA(A) agonists. After constitutive endocytosis, receptors are relatively stable and can be rapidly recycled to the cell surface, a process that may be regulated by protein kinase C. On the other hand, a portion of the intracellular GABA(A) receptors derived from ligand-dependent endocytosis is apparently degraded. The clustering of GABA(A) receptors at synapses and at coated pits are two mechanisms that may compete for a pool of diffusable receptors, providing a model for plasticity at inhibitory synapses.     

A gamma 2(R43Q) mutation, linked to epilepsy in humans, alters GABAA receptor assembly and modifies subunit composition on the cell surface

Genetic defects leading to epilepsy have been identified in gamma2 GABA(A) receptor subunit. A gamma2(R43Q) substitution is linked to childhood absence epilepsy and febrile seizure, and a gamma2(K289M) mutation is associated with generalized epilepsy with febrile seizures plus. To understand the effect of these mutations, surface targeting of GABA(A) receptors was analyzed by subunit-specific immunofluorescent labeling of living cells. We first transfected hippocampal neurons in culture with recombinant gamma2 constructs and showed that the gamma 2(R43Q) mutation prevented surface expression of the subunit, unlike gamma2(K289M) substitution. Several gamma2-subunit constructs, bearing point mutations within the Arg-43 domain, were expressed in COS-7 cells with alpha3- and beta3-subunits. R43Q and R43A substitutions dramatically reduced surface expression of the gamma2-subunit, whereas R43K, P44A, and D39A substitutions had a lesser, but still significant, impact and K289M substitution had no effect. Whereas the mutant gamma2(R43Q) was retained within intracellular compartments, alphabeta complexes were still targeted at the cell membrane. Coimmunoprecipitation experiments showed that gamma2(R43Q) was able to associate with alpha3- or beta3-subunits, although the stoichiometry of the complex with alpha3 was altered. Our data show that gamma2(R43Q) is not a dominant negative and that the mutation leads to a modification of GABA(A) receptor subunit composition on the cell surface that impairs the synaptic targeting in neurons. This study reveals an involvement of the gamma2-Arg-43 domain in the control of receptor assembly that may be relevant to the effect of the heterozygous gamma2(R43Q) mutation leading to childhood absence epilepsy and febrile seizure.     

The mRNAs encoding GABAA/benzodiazepine receptor subunits are localized in different cell populations of the bovine cerebellum

The expression of the mRNAs encoding the alpha and beta subunits of the GABAA/benzodiazepine receptor was examined in the bovine cerebellum by in situ hybridization histochemistry. The alpha subunit mRNA, which encodes the benzodiazepine binding site, was localized in all Purkinje and granule cells and in some cells of the molecular layer. The distribution of the beta subunit mRNA, which encodes the GABA binding site, only partially overlapped with that of the alpha subunit mRNA. While cells in the granule cell layer expressed the beta subunit mRNA, no message could be detected in other cell populations. These findings suggest that the subunit composition of the GABAA/benzodiazepine receptor is heterogeneous and that additional, as yet unidentified, beta subunits exist.     

猫小脑皮质GABA能神经元年龄相关性变化

为探讨青年猫和老年猫小脑皮质GABA能神经元及其表达的年龄相关性变化,利用Nissl染色显示小脑皮质结构及神经元,免疫组织化学ABC法标记GABA免疫阳性神经元。光镜下观察,采集图像,并利用图像分析软件对分子层、蒲肯野细胞层和颗粒层神经元及GABA免疫阳性神经元及其灰度值进行分析统计。结果显示,GABA免疫阳性神经元、阳性纤维及终末在青年猫和老年猫小脑皮质各层均有分布。与青年猫相比,老年猫分子层、蒲肯野细胞层神经元和GABA免疫阳性神经元密度及其GABA免疫阳性反应强度均显著下降(P<0.01),颗粒层神经元密度和GABA免疫阳性强度也显著下降(P<0.01),但其GABA免疫阳性神经元密度无显著变化(P>0.05);蒲肯野细胞的胞体萎缩,阳性树突分枝减少。因此认为,衰老过程中猫小脑皮质GABA能神经元的丢失和GABA表达的下降,可能是老年个体运动协调、精确调速和运动学习等能力下降的重要原因之一。     

Cerebellar development: afferent organization and Purkinje cell heterogeneity.

Olivo- and spinocerebellar maps in the adult cerebellum of small rodents are discontinuous, with sharp boundaries. Cortical Purkinje cells constitute a heterogeneous population, organized into parasagittal, mutually exclusive compartments. The boundaries of the intrinsic cortical compartments and those of the projectional maps are congruent. During development; (i) The incoming olivary fibres, once they penetrate in the cerebellar parenchyma, are attracted toward their ultimate terminal fields, without passing through a stage of random dispersion. (ii) Migrating Purkinje cells and inferior olivary neurons begin, asynchronously, to express cellular markers in an independent manner, giving rise to a transient compartmentation of the cerebellar cortex and the inferior olivary complex respectively. In both instances, the biochemical heterogeneity disappears during the first postnatal week, simultaneously with the acquisition of adult-like cerebellar maps. (iii) The formation of the maps is an early event, prior to the establishment of the synaptology of the cerebellar cortical circuitry. Moreover, the organization of the spinocerebellar projection in adult mutant mice does not depend on the presence of granule cells (staggerer) but on the presence of normal Purkinje cells (weaver), indicating that synaptogenesis with their target neurons is not involved in the process of map formation. The matching of region specific chemical labels between incoming afferent fibres and heterogeneous sets of Purkinje cells is the most appealing mechanism for the formation of cerebellar maps.     

A GABAA receptor mutation linked to human epilepsy (gamma2R43Q) impairs cell surface expression of alphabetagamma receptors

A mutation in the gamma2 subunit of the gamma-aminobutyric acid (GABA) type A receptor (GABAR), which changes an arginine to a glutamine at position 43 (R43Q), is linked to familial idiopathic epilepsies. We used radioligand binding, immunoblotting, and immunofluorescence techniques to examine the properties of wild-type alpha1beta2gamma2 and mutant alpha1beta2gamma2R43Q GABARs expressed in HEK 293 cells. The gamma2R43Q mutation had no affect on the binding affinity of the benzodiazepine flunitrazepam. However, in cells expressing alpha1beta2gamma2R43Q GABARs, the number of binding sites for [3H]flunitrazepam relative to wild-type receptors was decreased 75%. Using surface protein biotinylation, affinity purification, and immunoblotting, we demonstrated that expression of cell surface alpha1beta2gamma2R43Q GABARs was decreased. Surface immunostaining of HEK 293 cells expressing alpha1beta2gamma2R43Q GABARs confirmed that surface expression of the gamma2R43Q subunit was reduced. These data demonstrate that the gamma2R43Q mutation impairs expression of cell surface GABARs. A deficit in surface GABAR expression would reduce synaptic inhibition and result in neuronal hyperexcitability, which could explain why families possessing the gamma2R43Q subunit have epilepsy.