Egr3, a synaptic activity regulated transcription factor that is essential for learning and memory

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Abstract

Learning and memory depend upon poorly defined synaptic and intracellular modifications that occur in activated neurons. Mitogen activated protein kinase-extracellular regulated kinase (MAPK-ERK) signaling and de novo protein synthesis are essential aspects of enduring memory formation, but the precise effector molecules of MAPK-ERK signaling in neurons are not well defined. Early growth response (Egr) transcriptional regulators are examples of MAPK-ERK regulated genes and Egr1 (zif268) has been widely recognized as essential for some aspects of learning and memory. Here we show that Egr3, a transcriptional regulator closely related to Egr1, is essential for normal hippocampal long-term potentiation (LTP) and for hippocampal and amygdala dependent learning and memory. In the absence of Egr3, the defects in learning and memory appear to be independent of Egr1 since Egr1 protein levels are not altered in amygdala, hippocampus or cortex. Moreover, unlike Egr1-deficient mice which have impairments in late phase hippocampal LTP and consolidation of some forms of long-term hippocampus- and amygdala-dependent memory, Egr3-deficient mice have profound defects in early- and late-phase hippocampal LTP, as well as short-term and long-term hippocampus- and amygdala-dependent learning and memory. Thus, Egr3 has an essential role in learning and memory processing that appears to be partly distinct from the role of Egr1.

Introduction

Neurons process and retain information by forming synaptic connections that are modified by the intensity and frequency of their historical activity. This remarkable capacity to regulate the efficacy of synaptic transmission is essential for continual remodeling of neural networks required for cognitive processes such as learning and memory. Long lasting synaptic enhancement of excitatory neurotransmission, known as long-term potentiation (LTP), is one of the best studied cellular forms of synaptic “plasticity.” Many types of synapses throughout the brain undergo LTP after appropriate patterned stimulation but the phenomenon has been best studied in the hippocampus, a brain structure that is essential for memory acquisition and consolidation (Milner et al., 1998). Enduring memory-related synaptic changes depend upon new gene transcription and translation (protein synthesis), but the specific molecular pathways involved have only been partially characterized. For example, activity dependent calcium flux into neurons through excitatory N-methyl d-aspartate (NMDA) glutamate receptors and/or voltage-gated calcium channels engages the mitogen-activated protein kinase (MAPK)-extracellular regulated kinase (ERK) signaling cascade which is essential for NMDA-dependent induction of LTP in area CA1 (English and Sweatt, 1996, English and Sweatt, 1997) and the dentate gyrus of the hippocampus in vitro (Coogan et al., 1999), and for regulation of LTP in vivo (Davis et al., 2000, Rosenblum et al., 2000). Moreover, pharmacologic inhibitors of ERK signaling impair contextual and spatial memory (Atkins et al., 1998, Blum et al., 1999). A major deficit in our understanding of MAPK-ERK signaling related to the regulation of LTP and neuronal information storage at the synaptic (LTP) and network (behavioral) level remains because the genes regulated by MAPK-ERK signaling are still poorly characterized (Thomas and Huganir, 2004).

Early growth response (Egr) transcription factors are among a relatively small number of regulatory immediate early genes (IEGs) that are expressed in response to neuronal activity and coupled to MAPK-ERK signaling. There are four structurally related Egr genes (Egr1–4) that encode proteins with highly conserved zinc finger DNA-binding domains. As MAPK-ERK effector molecules, they may regulate target gene expression required for long-term structural and/or physiologic synaptic changes associated with learning and memory. Egr1 (also known as zif268, NGFI-A, TIS8, Krox24 or ZENK) and Egr3 are the most abundant Egr proteins upregulated by synaptic activity in the brain (Li et al., 2005, O'Donovan and Baraban, 1999). Early studies correlated patterned synaptic activity required to induce LTP with de novo induction of Egr1 and Egr3 expression (Cole et al., 1989, Yamagata et al., 1994). Recently, Egr1 has been shown to be required for the maintenance of late phase (but not early phase) LTP, long-term memory consolidation and reconsolidation of previously formed memories (Bozon et al., 2003, Jones et al., 2001, Lee et al., 2004).

Although a role of Egr1 in learning and memory is now well established, it is not clear what role, if any, Egr3 may have in learning and memory processing. In recent studies, we showed that Egr1 and Egr3 can regulate target genes such as the plasticity associated Arc gene (Activity Regulated Cytoskeletal associated gene; also known as Arg3.1 (Link et al., 1995, Lyford et al., 1995)), suggesting that Egr1 and Egr3 may have some overlapping roles in regulating gene expression in the brain (Li et al., 2005). Similarly, other plasticity associated genes encoding the GABA receptor subunit 4 gene, GABRA4 (Roberts et al., 2005) and synaptic vesicle associated proteins, Syn1 (Thiel et al., 1994) and Syn2 (Petersohn et al., 1995) may be directly regulated by either Egr1 or Egr3. Thus, if Egr3 modulates the expression of important plasticity-associated genes in a physiologically relevant manner, it may be essential for some aspects of learning and/or memory. Here, we report that despite the fact that Egr3-deficient (Egr3−/−) mice appear to have normally developed brains and normal basal synaptic transmission in CA3–CA1 hippocampal neurons where Egr1 and Egr3 are highly expressed, they have abnormal LTP in CA1 neurons, they have profound impairments in context and cued-associative learning/memory, and they have profoundly impaired short-term and long-term object recognition memory. Thus, Egr3 has an essential role in learning and memory presumably by regulating effector target genes required for memory acquisition, consolidation and/or retrieval.

Section snippets

Normal brain development and basal synaptic transmission in Egr3−/− mice

Egr3−/− mice were previously generated but brain development was not systematically studied (Tourtellotte et al., 2001, Tourtellotte and Milbrandt, 1998). Egr3−/− brains were compared to littermate WT to determine whether there were any detectable morphologic, pharmacologic and/or physiologic differences. Detailed examination of littermate WT and Egr3 −/− embryonic, perinatal and adult brains showed no gross or microscopic differences (data not shown). The hippocampus in particular, a brain

Discussion

Egr3 is a poorly studied member of the Egr family of transcriptional regulators that is structurally related to Egr1 (zif268). Like Egr1, Egr3 is regulated by synaptic activity in vivo and in excitatory cortical and hippocampal neurons by an NMDA receptor/MAPK-ERK signaling dependent mechanism (Li et al., 2005, Li et al., 2004, O'Donovan and Baraban, 1999, Yamagata et al., 1994). Several studies have indicated that Egr1 is essential for maintenance of late phase LTP, long-term memory in

Animals

Egr3 heterozygous mice from a BL/6-129Sv/J hybrid strain were backcrossed 4 generations to BL/6 isogenic mice and maintained as an inbred strain. The mice were generated and progeny were genotyped as previously described (Tourtellotte and Milbrandt, 1998, Whitehead et al., 2005). All experimental procedures complied with protocols approved by the Northwestern University and University of Nebraska Institutional Animal Care and Use Committees.

Histology and immunohistochemistry

Deeply anesthetized adult WT and Egr3−/− mice were

Acknowledgments

We thank P. Penzes and R. Huganir for antibody reagents. We thank J. Disterhoft and R. Miller for comments on the manuscript, and members of the Tourtellotte lab for helpful discussion and advice. Technical assistance was provided by J. Whitehead. This study was supported by The National Institutes of Health (NS046468 and NS040748) and a Howard Hughes Faculty Scholar Award to W.G.T.

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