Translation of an integral membrane protein in distal dendrites of hippocampal neurons Jeffrey C. Grigston, Hendrika M. A. VanDongen, James O. McNamara II and Antonius M. J. VanDongen Department of Pharmacology and Cancer Biology, Duke University Medical Center, PO Box 3813, Durham, NC 27710, USA Keywords: mRNA, protein synthesis, Rattus norvegicus, synaptic plasticity, TGN-38, zip code Abstract Maintenance of synaptic plasticity requires protein translation. Because changes in synaptic strength are regulated at the level of individual synapses, a mechanism is required for newly translated proteins to specifically and persistently modify only a subset of synapses. Evidence suggests this may be accomplished through local translation of proteins at or near synapses in response to plasticity-inducing patterns of activity. A number of proteins important for synaptic function are integral membrane proteins, which require a specialized group of organelles, proteins and enzymatic activities for proper synthesis. Dendrites appear to contain machinery necessary for the proper production of these proteins, and mRNAs for integral membrane proteins have been found localized to dendrites. Experiments are described that investigate the local translation of membrane proteins in the dendrites of cultured rat hippocampal neurons, using fluorescence recovery after photobleaching. Neurons were transfected with cDNAs encoding a fluorescently labeled transmembrane protein, TGN-38. Under conditions where the transport of this reporter construct was inhibited, the appearance of newly synthesized protein was observed via fluorescent microscopy. The dendritic translation of this protein required activation of glutamate receptors. The results demonstrate a functional capacity for activity-dependent synthesis of integral membrane proteins for distal dendrites in hippocampal neurons. Introduction Synaptic transmission between neurons is responsible for mediating information processing in the mammalian CNS. Lasting modification of synaptic strength underlies processes such as learning, memory, and synapse development and maturation. While changes in synaptic strength may initially result from the modification and redistribution of existing proteins, the persistence of synaptic plasticity requires synthesis of new proteins (Nguyen et al., 1994; Lisman & Fallon, 1999). Evidence for the necessity of new protein synthesis for the expression of lasting synaptic modification has come from experi- ments involving translational inhibitors. Application of pharmacolo- gical antagonists of protein synthesis blocks long-lasting forms of synaptic plasticity in hippocampal slices and other brain-derived preparations, while the initial stages of plasticity remain intact (Stanton & Sarvey, 1984; Montarolo et al., 1986; Frey et al., 1988). An individual neuron can have thousands of synaptic inputs distributed throughout a complex network of dendrites. The require- ment of protein synthesis for maintenance of synaptic plasticity and the independent regulation of the efficacy of individual synapses raises the issue of how newly synthesized proteins are incorporated only at the particular synapses undergoing changes in strength. A number of findings suggest local mRNA translation as a potential mechanism for delivering newly synthesized proteins specifically to activated synapses (Tang & Schuman, 2002). Dendritic shafts contain protein synthetic machinery and polyribosome complexes, which are frequently found localized at the base of synaptic spines (Steward & Levy, 1982; Steward & Reeves, 1988; Tiedge & Brosius, 1996; Pierce et al., 2001). Furthermore, while most mRNA species are confined to the neuronal cell body, certain mRNAs are selectively localized to the dendritic compartment (Wells et al., 2000; Steward & Schuman, 2003). Dendritic targeting elements have been identified for a number of dendritically localized mRNAs (Mori et al., 2000; Rook et al., 2000), and disruption of these elements impairs persistent plasticity and memory consolidation (Miller et al., 2002). Dendritic localization of specific mRNAs can be stimulated by synaptic activity or depolarization (Muslimov et al., 1998; Steward et al., 1998; Zhang et al., 1999; Mori et al., 2000; Havik et al., 2003). Additional support for the involvement of dendritic translation in the maintenance of synaptic potentiation is provided by findings that localized protein synthesis can be activated in dendrites by protocols shown to induce lasting plasticity (Kang & Schuman, 1996; Weiler et al., 1997; Ouyang et al., 1999; Scheetz et al., 2000). Experiments using a green fluorescent protein (GFP) construct expressed in cultured neurons have demonstrated that protein synthesis occurs preferentially at distinct foci along the dendrites coinciding with sites of presynaptic innervation (Aakalu et al., 2001). While substantial data have been provided for the local synthesis of a number of soluble proteins, evidence for the dendritic translation of integral membrane proteins is more limited. The synthesis and processing of this class of proteins requires a set of organelles, including rough endoplasmic reticulum (RER) and Golgi, which allow for their proper membrane insertion and glycosylation. Membranous cisternae immunoreactive for markers of RER and Golgi can be found in the dendrites (Gardiol et al., 1999; Pierce et al., 2000). Many of the polyribosomes within the dendrites are associated with membranous organelles in a pattern that resembles RER (Steward & Reeves, 1988). Enzymatic activities associated with these organelles are seen in synaptoneurosomes and in isolated neuronal dendrites. A number of Correspondence: Dr A. M. J. VanDongen, as above. E-mail: vando005@mc.duke.edu Received 7 January 2004, revised 12 January 2004, accepted 13 January 2004 European Journal of Neuroscience, Vol. 21, pp. 1457–1468, 2005 ª Federation of European Neuroscience Societies doi:10.1111/j.1460-9568.2005.03999.x