The regulation of synaptic glutamate receptor and GABAAR (γ-aminobutyric acid subtype A receptor) levels is a key component of synaptic plasticity. Most forms of neuronal plasticity are associated with the induction of the transcription factor zif268 (egr1). Hence, it is predicted that zif268 may regulate transcription of genes associated with glutamate receptors and/or GABAARs. It turns out that receptor regulation by zif268 tends to be indirect. Induction of zif268 in neurons leads to altered expression of proteasome subunit and proteasome-regulatory genes, thereby changing the capacity of the neuron to degrade synaptic proteins, including receptors and receptor subunits. In addition, zif268 alters the transcription of genes associated with GABAAR expression and trafficking, such as ubiquilin and gephyrin. This indirect regulation of receptor turnover is likely to contribute to the delayed, but long-lasting, phases of synaptic plasticity and also to the synaptic dysfunction associated with diseases such as schizophrenia and Alzheimer's disease, where zif268 expression is reduced.

Introduction

Neurons are remarkable in their ability to modify the sensitivity with which they communicate with their neighbours. This synaptic plasticity is fundamental to physiological CNS (central nervous system) function and also to pathological change in the CNS. Many forms of plasticity are triggered by the stimulation of glutamate receptors such as the NMDA (N-methyl-D-aspartate) receptor. Plasticity is dependent on protein synthesis, yet recent reports indicate that plasticity is also dependent on protein degradation by the proteasome (a large multi-subunit protein complex that degrades ubiquitinated proteins). Proteasome inhibitors attenuate learning and memory in both Aplysia [13] and rats [4]. Hence, it seems that synaptic plasticity is dependent on a precise balance between the synthesis of some proteins and the degradation of others. A large number of proteasome-related genes were identified in a recent unbiased screen for genes affecting synapse structure and function [5], and the proteasome is implicated in regulating the turnover of a subset of synaptic proteins, including NMDA receptor subunits and postsynaptic density scaffolding molecules [68]. Hence, the proteasome is emerging as an important mediator of glutamatergic plasticity in the CNS.

Zif268 and neuronal plasticity

There is considerable evidence that the transcription factor zif268 (egr1), is involved in the late phase of synaptic plasticity [9]. Zif268 is induced in many varied models of plasticity in the hippocampus [i.e. exposure to learning tasks, or induction of LTP (long-term potentiation), which is one of the best understood models of physiological plasticity], cerebral cortex, amygdala and spinal cord, in diverse species from primates to songbirds [9]. Hippocampal LTP duration correlates with the degree of zif268 induction and mice with targeted disruption of the zif268 gene show deficits in a range of memory tests [10]. We have recently identified candidate target genes of zif268 in neurons [11]. The proportion of these genes encoding receptors or receptor subunits was small. Remarkably, however, approx. 20% of the genes identified were either components of the proteasome or proteins with functions closely allied to the proteasome [12,13]. The challenge is now to ascertain how altered expression of these genes modulates synaptic function.

GABAARs (γ-aminobutyric acid subtype A receptors) and neuronal plasticity

GABAARs are ligand-gated chloride channels that exist in numerous distinct subunit combinations, but the α1β2γ2 combination is the most predominantly expressed receptor multimers, followed by α2β3γ2 and α3β3γ2 [14]. GABAARs containing α and β subunits can reach the cell surface [15]; however, there is a necessity for the γ subunit for synaptic localization [16]. Gephyrin anchors glycine and GABAAR to the subsynaptic cytoskeleton [17] and has been linked with the α1–3, β3 and γ2 subunits of the GABAARs at postsynaptic sites [16,18]. Other proteins are also involved in co-ordinating synaptic GABAAR levels: the ubiquitin-related protein ubiquilin binds to the α1–3, α6 and β1–3 subunits of the GABAAR [19]. Reduced gephyrin expression is predicted to decrease the number of GABAARs clustered at synaptic sites, whereas decreased ubiquilin expression is predicted to reduce GABAAR number via enhanced proteasomal degradation.

Since both gephyrin and ubiquilin are associated with GABAARs at inhibitory synapses, it is of interest that both genes were identified as possible transcriptional targets of zif268 [11]. Indeed, we found that gephyrin mRNA and protein expression levels were down-regulated in response to increased levels of zif268 [via transient transfection in PC12 cells] and also by low level (non-excitotoxic) NMDA stimulation in primary cultured cortical neurons. In addition, ubiquilin mRNA and protein levels were also down-regulated within the same experimental paradigms, implying that both gephyrin and ubiquilin are downstream transcriptional targets of this plasticity-related gene.

The mouse gephyrin promoter has been described by Ramming et al. [20] and was used to identify the rat gephyrin promoter region. Within this promoter region, bioinformatic analysis identified three potential zif268 sites, along with three potential Sp1 (specificity protein 1) sites (a general zinc-finger transcription factor known to enhance promoter activity and functionally linked with zif268 actions), along with an NRSE (neuron-restrictive silencer element; Figure 1). The presence of the putative zif268-binding sites in close proximity to the transcriptional start site (Figure 1) is consistent with a major influence of zif268 on the transcription of this gene [12]. We recently confirmed this using a promoter-reporter construct transfected into PC12 cells along with a zif268 expression vector.

Bioinformatic analysis of the rat gephyrin promoter region

Figure 1
Bioinformatic analysis of the rat gephyrin promoter region

Predicted zif268-binding sites (Egr-response elements), identified using previously described procedures [11,12], are clustered upstream of the transcriptional start site (TSS). As noted with other zif268 target genes [11,12], the zif268-binding site is frequently associated with a predicted Sp1 site. The presence of an NRSE is typical of neuronally expressed genes.

Figure 1
Bioinformatic analysis of the rat gephyrin promoter region

Predicted zif268-binding sites (Egr-response elements), identified using previously described procedures [11,12], are clustered upstream of the transcriptional start site (TSS). As noted with other zif268 target genes [11,12], the zif268-binding site is frequently associated with a predicted Sp1 site. The presence of an NRSE is typical of neuronally expressed genes.

Functional implications

The ability of zif268 to down-regulate the level of expression of both ubiquilin and gephyrin suggests that one of the consequences of elevated zif268 expression may be a functional depletion of synaptic GABAARs. Using primary hippocampal cultures, we observed a loss of dendritic GABAAR-immunoreactive puncta following low-level NMDA receptor stimulation, consistent with this hypothesis. Regulating the number of receptors at the postsynaptic membrane in this way allows alterations in the efficacy of inhibitory synaptic transmission. There is evidence that plasticity of inhibitory transmission frequently occurs alongside plasticity of excitatory transmission. A decrease in inhibition may be required for the potentiated activation of excitatory synapses allowing the encoding of new information. There is evidence to suggest that synaptic GABAARs may be down-regulated during forms of plasticity involving zif268 induction. Hippocampal LTP is associated with the suppression of GABAergic synaptic events [21]. Similarly BDNF (brain-derived neurotrophic factor) treatment or epileptogenesis cause a down-regulation of synaptic GABAARs [22,23]. Enhancing network activity in hippocampal slices leads to increased internalization of GABAARs [23]. It has been reported that interneurons display a reduced level of activity during novel spatial experiences [24,25]. Decreasing GABAARs at the synapse may hence play some part in the strengthening of network activity that occurs during plasticity, implying that changes in excitability could be facilitated by changes in inhibitory synaptic transmission. Activity-dependent modulation of GABAergic transmission may therefore contribute to long-lasting plasticity. The potential suppression by zif268 of gephyrin and/or ubiquilin expression provides a molecular substrate for this phenomenon.

It is of interest that reduced zif268 expression is believed to be an early event in both schizophrenia and Alzheimer's disease. Understanding the molecular consequences of zif268 action may be highly relevant for developing future strategies to ameliorate the neurobiological dysfunction in these diseases.

Neuronal Glutamate and GABAA Receptor Function in Health and Disease: Biochemical Society Focused Meeting held at University of St Andrews, St Andrews, U.K., 21–24 July 2009. Organized and Edited by Chris Connolly and Jenni Harvey (Dundee, U.K.).

Abbreviations

     
  • CNS

    central nervous system

  •  
  • GABAAR

    γ-aminobutyric acid subtype A receptor

  •  
  • LTP

    long-term potentiation

  •  
  • NMDA

    N-methyl-D-aspartate

  •  
  • NRSE

    neuron-restrictive silencer element

  •  
  • Sp1

    specificity protein 1

Funding

This work was partially supported by the Biotechnology and Biological Sciences Research Council.

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Author notes

1

Present address: Lab901, Unit 53, IMEX Business Centre, Loanhead, Midlothian EH20 9LZ, U.K.