Effects of distinct collybistin isoforms on the formation of GABAergic synapses in hippocampal neurons
Introduction
A key step in the assembly and maintenance of synapses is the anchoring of neurotransmitter receptors in the postsynaptic compartment. Therefore, the receptors have to be bound to submembranous scaffold proteins, a process poorly understood for GABAARs which provide the major source of synaptic inhibition in the forebrain (reviewed by Tretter and Moss, 2008). The majority of GABAergic synapses employ the scaffold protein gephyrin (Essrich et al., 1998) that has originally been reported to cluster glycine receptors via direct binding to its β-subunit (Kirsch et al., 1995, Meyer et al., 1995). Moreover, gephyrin interacts directly with the α2 and α3 but not the γ2 GABAAR subunits (Saiepour et al., 2010, Tretter et al., 2008), the latter being essential for the synaptic localization of GABAARs (Essrich et al., 1998). Thus, additional proteins besides gephyrin have to be involved in the anchoring of GABAARs to the postsynaptic compartment.
A number of such gephyrin interaction partners have been described in recent years (Fritschy et al., 2008) one of which is collybistin (Cb) (Kins et al., 2000). Cb and its human homologue hPEM2, which have been linked to hyperekplexia, epilepsy, anxiety, aggression and mental retardation (Harvey et al., 2004, Kalscheuer et al., 2009), belong to the family of dbl-like guanine nucleotide exchange factors and can activate the small GTPase Cdc42 (Reid et al., 1999, Tyagarajan et al., 2011, Xiang et al., 2006). In the rat, four splice variants of Cb have been reported, three of these (Cb1-3) differ in their C-termini, and one (Cb2) exists with and without src-homology-3 (SH3)-domain near the N-terminus (Harvey et al., 2004, Kins et al., 2000). All Cb isoforms possess a central tandem dbl-homology (DH)/pleckstrin-homology (PH)-domain of which a functional PH- but not DH-domain is required for proper function (Reddy-Alla et al., 2010). Cb is required for establishment and maintenance of postsynaptic gephyrin scaffolds and the synaptic localization of major, gephyrin-dependent GABAAR subtypes. The loss of gephyrin and gephyrin-dependent GABAARs from specific brain regions in Cb knock-out mice, leads to increased neuronal excitability resulting in increased anxiety, impaired spatial learning and altered hippocampal plasticity (Jedlicka et al., 2009, Papadopoulos et al., 2007, Papadopoulos et al., 2008).
Despite these insights from Cb knock-out mice, little is known about the properties of the distinct Cb isoforms. Co-expression of Cb2SH3−, but not Cb1, with gephyrin in non-neuronal cells results in a translocation of gephyrin from intracellular aggregates to submembranous compartments (Kins et al., 2000) suggesting an autoinhibition of Cb function by the SH3-domain. Nevertheless, SH3-domain containing isoforms can be “activated” by the binding of neuroligin 2 or 4, present at inhibitory synapses, to the SH3-domain of Cb (Hoon et al., 2011, Poulopoulos et al., 2009). Moreover, it has been shown recently that overexpression of Cb2SH3+ in hippocampal cultured neurons leads to the formation of many, often non-innervated, GABAergic postsynapses, whereas overexpression of Cb2SH3− induces the formation of abnormally large GABAergic synapses (Chiou et al., 2011, Kalscheuer et al., 2009, Tyagarajan et al., 2011). These differences have been proposed to originate from differential interactions of the Cb2 splice variants with Cdc42 and gephyrin (Tyagarajan et al., 2011).
Besides these recent advances in the understanding of inter-splice variant differences, a systematical assessment of the functional role of the individual Cb isoforms in the absence of endogenous Cb, which may affect the results obtained by overexpression, has not yet been performed. Here we set out to close this gap and study the functional roles of different Cb isoforms by performing sequential rescue experiments after knock-down of endogenous Cb.
Section snippets
Knock-down of collybistin leads to loss of functional GABAergic synapses
To investigate the functional role of distinct Cb isoforms in the formation and maintenance of GABAergic synapses, we first established a simultaneous knock-down of all known endogenous rat Cb isoforms (Harvey et al., 2004). Examination of neurons expressing Cb knock-down shRNA by immunofluorescence microscopy revealed that the number of dendritic gephyrin clusters was strongly reduced when compared to untransduced or scrambled control shRNA-expressing neurons (knock-down (kd): 1.1 ± 0.4 puncta
Discussion
In the present study, we systematically investigated the functional role of all four known rat Cb isoforms. Therefore, we first established the knock-down of endogenous Cb in hippocampal cultured neurons that was then sequentially rescued by expression of a single shRNA insensitive Cb isoform. Our results show that all four isoforms are able to restore Cb function as assessed by the re-establishment of functional GABAergic synapses. Distinct Cb isoforms differed only slightly in their
Constructs
Sequences coding for either a shRNA directed against the DH-domain common to all Cb isoforms (AATCCGGAGAGACATCCTATA) or a scrambled control (TTGGTCTTGTGGCATTACA) were cloned into pFSGW, a modified version of the lentiviral vector pFUGW (Lois et al., 2002), in which the expression of the reporter EGFP is under the control of the human synapsin promoter instead of the originally published ubiquitin promoter. The shRNA expression is driven by the U6 promoter.
Coding sequences of Cb2SH3+ and Cb3
Acknowledgments
We thank Rita Rosner and Claudia Kocksch for excellent technical assistance.
References (32)
- et al.
Differential regulation of the postsynaptic clustering of gamma-aminobutyric acid type A (GABAA) receptors by collybistin isoforms
J. Biol. Chem.
(2011) - et al.
Functional regions of the presynaptic cytomatrix protein bassoon: significance for synaptic targeting and cytomatrix anchoring
Mol. Cell. Neurosci.
(2003) - et al.
Gephyrin: where do we stand, where do we go?
Trends Neurosci.
(2008) - et al.
Increased network excitability and impaired induction of long-term potentiation in the dentate gyrus of collybistin-deficient mice in vivo
Mol. Cell. Neurosci.
(2009) - et al.
Targeting of glycine receptor subunits to gephyrin-rich domains in transfected human embryonic kidney cells
Mol. Cell. Neurosci.
(1995) - et al.
Gephyrin-independent clustering of postsynaptic GABA(A) receptor subtypes
Mol. Cell. Neurosci.
(2001) - et al.
Monitoring synaptic transmission in primary neuronal cultures using local extracellular stimulation
J. Neurosci. Methods
(2007) - et al.
Identification of a gephyrin binding motif on the glycine receptor beta subunit
Neuron
(1995) - et al.
Collybistin is required for both the formation and maintenance of GABAergic postsynapses in the hippocampus
Mol. Cell. Neurosci.
(2008) - et al.
Neuroligin 2 Drives Postsynaptic Assembly at Perisomatic Inhibitory Synapses through Gephyrin and Collybistin
Neuron
(2009)
Identification and characterization of hPEM-2, a guanine nucleotide exchange factor specific for Cdc42
J. Biol. Chem.
Complex role of collybistin and gephyrin in GABAA receptor clustering
J. Biol. Chem.
The crystal structure of Cdc42 in complex with collybistin II, a gephyrin-interacting guanine nucleotide exchange factor
J. Mol. Biol.
Postsynaptic clustering of major GABAA receptor subtypes requires the gamma 2 subunit and gephyrin
Nat. Neurosci.
Reduced synaptic clustering of GABA and glycine receptors in the retina of the gephyrin null mutant mouse
J. Comp. Neurol.
The GDP-GTP exchange factor collybistin: an essential determinant of neuronal gephyrin clustering
J. Neurosci.
Cited by (0)
- 1
Present addres: Edinger Institute/Institute of Neurology, Frankfurt University Medical School, Frankfurt, Germany.