Synapse Specific Protein Synthesis Gene Induction and Synaptic Plasticity

3.10.1. Gene Transcription

Because soma-soma paired neurons failed to extend neurites as compared to their single unpaired counterparts, a search began to identify genes in both single and paired neurons that might be differentially regulated under these two different experimental conditions. Quantitative polymerase chain reaction (QPCR) techniques were utilized and this approach resulted in the identification of a molluscan homolog of the multiple endocrine neoplasia type 1 (MEN1) tumor suppressor gene, which encodes the transcription factor menin. This specific gene was found to be upregulated in soma-soma paired neurons during synapse formation35. To demonstrate the significance of menin in synapse formation, MEN1 mRNA was selectively knocked down with antisense in paired neurons in vitro. MEN1 perturbations completely blocked both excitatory and inhibitory synapse formation. Interestingly, cell-specific knock-down of MEN1 mRNA revealed that menin expression was required only in the postsynaptic neuron35. The MEN1 gene is the first such gene that has been shown to be essential for synapse formation between Lymnaea neurons. Consistent with the notion that neurite outgrowth and synapse formation are mutually exclusive, the BERP gene that is thought to be involved in neurite outgrowth in vertebrates36 was found to be upregulated in single, regenerating Lymnaea neurons and downregulated in soma-soma paired cells. Therefore, the BERP gene may regulate neurite outgrowth from Lymnaea neurons37, though its inhibitory effects on the synaptogenic program remain to be determined.

3.10.2. Protein Synthesis

In addition to transcription of specific genes, synapse formation between soma-soma pairs also requires de novo protein synthesis24,28,29. However, the precise site where this protein synthesis is required (pre- versus postsynaptic cell) remained unknown. A step toward resolving this issue was taken by Meems and colleagues32 who used soma-axon pairs, consisting of a presynaptic cell body paired with a postsynaptic axon severed from its respective cell body. The removal of postsynaptic cell body alone did not affect synapse formation between the pairs. However, when presynaptic somata were removed, the synapses failed to develop in cell culture32. These data suggest that during early synapse formation between soma-axon pairs, the gene transcription and protein synthesis is required in presynaptic soma but not the postsynaptic axon. All that is needed of the postsynaptic axon is a redistribution of neurotransmitter receptors32. Specifically, presynaptic target cell contact facilitates the mobilization of postsynaptic cholinergic receptors from extrasynaptic to synaptic sites, independent of gene transcription or protein synthesis, although it does require trophic factors (Figure 2.2C; Colorplate 3). Taken together these studies underscore the importance of trophic factors and site-specific gene transcription and protein synthesis in synapse formation.

3.10.3. Synaptic Plasticity and Synapse Formation

In Lymnaea, the synaptic plasticity-induced formation of long-term memory (LTM) in the intact animals is both transcription and translation dependent38. Interestingly, the locus for this transcription and translation-dependent process is confined to one single neuron termed RPeD1. Ablation of its cell body in the intact animals completely blocks memory formation39. Similarly, long-term facilitation (LTF), which is thought to be the underlying mechanism for LTM in Aplysia, has also been extensively studied and a variety of molecules and underlying mechanisms identified. One of the key issues is to define the precise locus for new protein synthesis during plasticity. For instance, how is a single synaptic connection subjected to modification in a neuron that has multiple synaptic connections? To address this question, a single bifurcated sensory neuron with two branches was allowed to make synapses in vitro with two spatially separated motor neurons. In this configuration individual synapses could be selectively exposed to plasticity-inducing stimuli. LTF was induced by repeated stimulation with the neuro-transmitter serotonin (5-HT) at one of the synaptic sites, whereas the other synapses were left untreated. This paradigm revealed modification only of the treated synapse and indicated that a single neuron can undergo branch-specific LTF. Furthermore, this branch-specific, synaptic modification is dependent upon gene transcription and local (at the synapse) protein synthesis. Interestingly, protein synthesis was only necessary in the presynaptic but not the postsynaptic terminals40. While altered gene transcription likely serves all synaptic connections formed by any given neuron, the local protein synthesis occurs only at synapses that are subjected to plasticity-specific stimuli. The products of altered gene expression and local protein synthesis thus collectively account for the changes that underlie LTF. Consistent with this idea are studies in which cell bodies from Aplysia neurons were removed after synapse formation to determine the contributions of local protein synthesis in synaptic plasticity. The synapses were exposed to LTF-inducing stimuli and changes in synaptic efficacy at synapses either with or without presynaptic somata were compared, showing that somaless axons exhibit LTF similar to their intact counterparts41. However, the LTF induced in the somaless configuration appeared to be transient. It was thus suggested that local protein synthesis in axons accounts only for initial changes in synaptic efficacy, whereas gene transcription was required for the maintenance of LTF41. These results underscore the importance of gene transcription and protein synthesis in synapse formation as well as in long-term changes in synaptic efficacy.

The search for the molecular machinery that contributes to gene transcription and protein synthesis-dependent LTF has led to the identification of a variety of well-known kinases. In Helix for instance, synapsin (a synaptic vesicle-associated phosphoprotein) was found important for increased efficiency of neurotransmitter release in neurons that were otherwise cultured under low-release conditions42. Furthermore, a Helix synapsin ortholog cloned from Aplysia (ApSyn) was mutated on its phosphorylation sites in search of ApSyn substrates that are involved in synaptic plasticity. ApSyn appeared to be an excellent substrate for cAMP-dependent protein kinase. Injection of wild-type ApSyn in identified Helix neurons cultured under low-release conditions resulted in increased neurotransmitter release, whereas injection of mutant ApSyn failed to do so43. These data indicated that cAMP-dependent protein kinase may be an essential player involved in the induction of synaptic plasticity. Earlier, Aplysia protein kinases A and C were shown to be essential for the formation of 5-HT-induced LTF44-47, whereas more recently mitogen-activated protein kinase (MAPK) has also been implicated in the induction of increased synaptic efficacy48,49. Moreover, MAP kinase was shown to translocate to the nucleus50, suggesting that the site for MAPK action could reside within the nucleus, where it may be involved in transcribing various mRNAs and the translation of their encoded proteins. Taken together, a variety of kinases appear to be involved in the cellular and molecular changes underlying synaptic efficacy, although the exact order of these events remains to be elucidated.

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