Angel L. De Blas

Collybistin (CB) is a rho guanine exchange factor found at GABAergic and glycinergic postsynapses that interacts with the inhibitory scaffold protein, gephyrin, and induces accumulation of gephyrin and GABA_A receptors (GABA_ARs) to the postsynapse. We have previously reported that the isoform without the src homology 3 (SH3) domain, CBSH3-, is particularly active in enhancing the GABAergic postsynapse in both cultured hippocampal neurons as well as in cortical pyramidal neurons after chronic in vivo expression in in utero electroporated (IUE) rats. Deficiency of CB in knockout mice results in absence of gephyrin and gephyrin-dependent GABA_ARs at postsynaptic sites in several brain regions, including hippocampus. In the present study, we have generated an adeno-associated virus (AAV) that expresses CBSH3- in a cre-dependent manner. Using male and female VGLUT1-IRES-cre or VGAT-IRES-cre mice, we explore the effect of overexpression of CBSH3- in hippocampal pyramidal cells or hippocampal interneurons. The results show that: 1) the accumulation of gephyrin and GABA_ARs at inhibitory postsynapses in hippocampal pyramidal neurons or interneurons can be enhanced by CBSH3- overexpression, 2) overexpression of CBSH3- in hippocampal pyramidal cells can enhance the strength of inhibitory neurotransmission, and 3) these enhanced inhibitory synapses provide protection against PTZ-induced seizures. The results indicate that this AAV vector carrying CBSH3- can be used for in vivo enhancement of GABAergic synaptic transmission in selected target neurons in the brain. Significance statement Excessive or imbalanced excitation in the hippocampus can result in acute or chronic pathological conditions, such as seizures, epilepsy, and learning impairments. It is therefore important to uncover target genes that can be manipulated to restore or prevent this imbalance. This study uses a novel adeno-associated virus to express collybistin SH3- in select cells of the hippocampus. We have found that overexpression of this protein enhances GABAergic inhibitory synaptic transmission. The results also bring attention to collybistin as a possible target for therapeutic intervention aimed to restore the balance between excitation and inhibition.

It has been shown that subunit composition is the main determinant of the synaptic or extrasynaptic localization of GABA_A receptors (GABA_ARs). Synaptic and extrasynaptic GABA_ARs are involved in phasic and tonic inhibition, respectively. It has been proposed that synaptic GABA_ARs bind to the postsynaptic gephyrin/collybistin (Geph/CB) lattice, but not the typically extrasynaptic GABA_ARs. Nevertheless, there are no studies of the direct binding of various types of GABA_ARs with the submembranous Geph/CB lattice in the absence of other synaptic proteins, some of which are known to interact with GABA_ARs. We have reconstituted GABA_ARs of various subunit compositions, together with the Geph/CB scaffold, in HEK293 cells, and have investigated the recruitment of surface GABA_ARs by submembranous Geph/CB clusters. Results show that the typically synaptic α1β3γ2 GABA_ARs were trapped by submembranous Geph/CB clusters. The α5β3γ2 GABA_ARs, which are both synaptic and extrasynaptic, were also trapped by Geph/CB clusters. Extrasynaptic α4β3δ GABA_ARs consistently showed little or no trapping by the Geph/CB clusters. However, the extrasynaptic α6β3δ, α1β3, α6β3 (and less α4β3) GABA_ARs were highly trapped by the Geph/CB clusters. AMPA and NMDA glutamate receptors were not trapped. The results suggest: (I) in the absence of other synaptic molecules, the Geph/CB lattice has the capacity to trap not only synaptic but also several typically extrasynaptic GABA_ARs; (II) the Geph/CB lattice is important but does not play a decisive role in the synaptic localization of GABA_ARs; and (III) in neurons there must be mechanisms preventing the trapping of several typically extrasynaptic GABA_ARs by the postsynaptic Geph/CB lattice.

Collybistin (CB) is a guanine nucleotide exchange factor (GEF) selectively localized at GABAergic and glycinergic postsynapses. Analysis of mRNA shows that several isoforms of collybistin are expressed in the brain. Some of the isoforms have a SH3 domain (CBSH3+) and some have no SH3 domain (CBSH3−). The CBSH3+ mRNAs are predominantly expressed over CBSH3−. However, in an immunoblot study of mouse brain homogenates, only CBSH3+ protein isoforms were detected, proposing that CBSH3− protein might not be expressed in the brain. The expression or lack of expression of CBSH3− protein is an important issue because CBSH3− has a strong effect in promoting the postsynaptic clustering of gephyrin and GABA‐A receptors (GABA_ARs). Moreover CBSH3− is constitutively active; therefore lower expression of CBSH3− protein might play a relatively stronger functional role than the more abundant but self‐inhibited CBSH3+ isoforms, which need to be activated. We are now showing that: (a) CBSH3− protein is expressed in the brain; (b) parvalbumin positive (PV+) interneurons show higher expression of CBSH3− protein than other neurons; (c) CBSH3− is associated with GABAergic synapses in various regions of the brain and (d) knocking down CBSH3− in hippocampal neurons decreases the synaptic clustering of gephyrin and GABA_ARs. The results show that CBSH3− protein is expressed in the brain and that it plays a significant role in the size regulation of the GABAergic postsynapse. image

It has been proposed that the combinatorial expression of γ‐protocadherins (Pcdh‐γs) and other clustered protocadherins (Pcdhs) provides a code of molecular identity and individuality to neurons, which plays a major role in the establishment of specific synaptic connectivity and formation of neuronal circuits. Particular attention has been directed to the Pcdh‐γ family, for which experimental evidence derived from Pcdh‐γ‐deficient mice shows that they are involved in dendrite self‐avoidance, synapse development, dendritic arborization, spine maturation, and prevention of apoptosis of some neurons. Moreover, a triple‐mutant mouse deficient in the three C‐type members of the Pcdh‐γ family (Pcdh‐γC3, Pcdh‐γC4, and Pcdh‐γC5) shows a phenotype similar to the mouse deficient in whole Pcdh‐γ family, indicating that the latter is largely due to the absence of C‐type Pcdh‐γs. The role of each individual C‐type Pcdh‐γ is not known. We have developed a specific antibody to Pcdh‐γC4 to reveal the expression of this protein in the rat brain. The results show that although Pcdh‐γC4 is expressed at higher levels in the embryo and earlier postnatal weeks, it is also expressed in the adult rat brain. Pcdh‐γC4 is expressed in both neurons and astrocytes. In the adult brain, the regional distribution of Pcdh‐γC4 immunoreactivity is similar to that of Pcdh‐γC4 mRNA, being highest in the olfactory bulb, dentate gyrus, and cerebellum. Pcdh‐γC4 forms puncta that are frequently apposed to glutamatergic and GABAergic synapses. They are also frequently associated with neuron‐astrocyte contacts. The results provide new insights into the cell recognition function of Pcdh‐γC4 in neurons and astrocytes.

Collybistin (CB) is a guanine nucleotide exchange factor selectively localized to γ‐aminobutyric acid (GABA)ergic and glycinergic postsynapses. Active CB interacts with gephyrin, inducing the submembranous clustering and the postsynaptic accumulation of gephyrin, which is a scaffold protein that recruits GABA_A receptors (GABA_ARs) at the postsynapse. CB is expressed with or without a src homology 3 (SH3) domain. We have previously reported the effects on GABAergic synapses of the acute overexpression of CB_SH3− or CB_SH3+ in cultured hippocampal (HP) neurons. In the present communication, we are studying the effects on GABAergic synapses after chronic in vivo transgenic expression of CB2_SH3− or CB2_SH3+ in neurons of the adult rat cerebral cortex. The embryonic precursors of these cortical neurons were in utero electroporated with CB_SH3− or CB_SH3+ DNAs, migrated to the appropriate cortical layer, and became integrated in cortical circuits. The results show that: 1) the strength of inhibitory synapses in vivo can be enhanced by increasing the expression of CB in neurons; and 2) there are significant differences in the results between in vivo and in culture studies. J. Comp. Neurol. 525:1291–1311, 2017.

We studied the effect of clonal overexpression of neuroligin 3 (NL3) or neuroligin 2 (NL2) in the adult rat cerebral cortex following in utero electroporation (IUEP) at embryonic stage E14. Overexpression of NL3 leads to a large increase in vesicular gamma‐aminobutyric acid (GABA) transporter (vGAT) and glutamic acid decarboxylase (GAD)65 in the GABAergic contacts that the overexpressing neurons receive. Overexpression of NL2 produced a similar effect but to a lesser extent. In contrast, overexpression of NL3 or NL2 after IUEP does not affect vesicular glutamate transporter 1 (vGlut1) in the glutamatergic contacts that the NL3 or NL2‐overexpressing neurons receive. The NL3 or NL2‐overexpressing neurons do not show increased innervation by parvalbumin‐containing GABAergic terminals or increased parvalbumin in the same terminals that show increased vGAT. These results indicate that the observed increase in vGAT and GAD65 is not due to increased GABAergic innervation but to increased expression of vGAT and GAD65 in the GABAergic contacts that NL3 or NL2‐overexpressing neurons receive. The majority of bright vGAT puncta contacting the NL3‐overexpressing neurons have no gephyrin juxtaposed to them, indicating that many of these contacts are nonsynaptic. This contrasts with the majority of the NL2‐overexpressing neurons, which show plenty of synaptic gephyrin clusters juxtaposed to vGAT. Besides having an effect on GABAergic contacts, overexpression of NL3 interferes with the neuronal radial migration, in the cerebral cortex, of the neurons overexpressing NL3. J. Comp. Neurol. 523:1359–1378, 2015.

We have found that the γ2 subunit of the GABA_Areceptor (γ2-GABA_AR) specifically interacts with protocadherin-γC5 (Pcdh-γC5) in the rat brain. The interaction occurs between the large intracellular loop of the γ2-GABA_AR and the cytoplasmic domain of Pcdh-γC5. In brain extracts, Pcdh-γC5 coimmunoprecipitates with GABA_ARs. In cotransfected HEK293 cells, Pcdh-γC5 promotes the transfer of γ2-GABA_AR to the cell surface. We have previously shown that, in cultured hippocampal neurons, endogenous Pcdh-γC5 forms clusters, some of which associate with GABAergic synapses. Overexpression of Pcdh-γC5 in hippocampal neurons increases the density of γ2-GABA_AR clusters but has no significant effect on the number of GABAergic contacts that these neurons receive, indicating that Pcdh-γC5 is not synaptogenic. Deletion of the cytoplasmic domain of Pcdh-γC5 enhanced its surface expression but decreased the association with both γ2-GABA_AR clusters and presynaptic GABAergic contacts. Cultured hippocampal neurons from the Pcdh-γ triple C-type isoform knock-out (TCKO) mouse (Pcdhg^tcko/tcko) showed plenty of GABAergic synaptic contacts, although their density was reduced compared with sister cultures from wild-type and heterozygous mice. Knocking down Pcdh-γC5 expression with shRNA decreased γ2-GABA_AR cluster density and GABAergic innervation. The results indicate that, although Pcdh-γC5 is not essential for GABAergic synapse formation or GABA_AR clustering, (1) Pcdh-γC5 regulates the surface expression of GABA_ARs viacis-cytoplasmic interaction with γ2-GABA_AR, and (2) Pcdh-γC5 plays a role in the stabilization and maintenance of some GABAergic synapses.