mRNA at the synapse: analysis of a preparation enriched in hippocampal dendritic spine mRNA (original) (raw)
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Journal of Neurochemistry, 1993
Synaptodendrosomes" are subcellular fractions that contain pinched-off axon terminals and dendrites. These fractions are a potential source of RNAs that are localized in dendrites. However, these fractions may also contain RNAs that are seen in situ in neuronal cell bodies and glia. To evaluate whether synaptodendrosomes could be used as a source of dendritic RNA, we studied the RNA content of this fraction as compared with RNA isolated from total forebrain and a cell body-enriched fraction. RNA was analyzed by gel electrophoresis, oligo-dT chromatography, and northern blot hybridization. RNA from synaptodendrosomes contained a greater proportion of low-molecular-weight nonpolyadenylated RNAs. RNAs known to be present in dendrites (mRNA for the a subunit of the calcium/calmodulin-dependent protein kinase II and the polymerase 111 transcript BC1) were detected in synaptodendrosomes; RNAs that are restricted to neuronal and glial cell bodies (mRNAs for the 68-kDa neurofilament protein, 43-kDa growth-associated protein, p-tubulin, and pactin) were present only at low levels. However, the mRNA for glial fibrillary acidic protein (seen in situ in glial cell bodies and processes) was present at high levels in the synaptodendrosomes. These results support and extend previous studies indicating that a limited subset of mRNAs is present in neuronal and astrocyte processes and reveal that both of these types of mRNAs are present in synaptodendrosomes. Thus, synaptodendrosomes may be useful as a source of dendritic RNAs, but it will be necessary to develop strategies to subtract mRNAs present in astroglial processes.
Identification of Process-Localized mRNAs from Cultured Rodent Hippocampal Neurons
Journal of Neuroscience, 2006
The regulated translation of localized mRNAs in neurons provides a mechanism for spatially restricting gene expression in a synapsespecific manner. To identify the population of mRNAs present in distal neuronal processes of rodent hippocampal neurons, we grew neurons on polycarbonate filters etched with 3 m pores. Although the neuronal cell bodies remained on the top surface of the filters, dendrites, axons, and glial processes penetrated through the pores to grow along the bottom surface of the membrane where they could be mechanically separated from cell bodies. Quantitative PCR and immunochemical analyses of the process preparation revealed that it was remarkably free of somatic contamination. Microarray analysis of RNA isolated from the processes identified over 100 potentially localized mRNAs. In situ hybridization studies of 19 of these transcripts confirmed that all 19 were present in dendrites, validating the utility of this approach for identifying dendritically localized transcripts. Many of the identified mRNAs encoded components of the translational machinery and several were associated with the RNA-binding protein Staufen. These findings indicate that there is a rich repertoire of mRNAs whose translation can be locally regulated and support the emerging idea that local protein synthesis serves to boost the translational capacity of synapses.
RNA Trafficking and Local Protein Synthesis in Dendrites: An Overview
Journal of Neuroscience, 2006
It is now widely accepted that mRNAs localize to dendrites and that translation of these mRNAs is regulated in response to neuronal activity. Recent studies have begun to reveal the underpinnings of these processes and to underscore the importance of local protein synthesis to synaptic remodeling and plasticity. When Steward and Levy (1982) first reported their observation of polyribosomes at the base of spines, the prevailing view was that all proteins were synthesized in the cell body and then transported to distal compartments of neurons. Steward and Levy's discovery, however, raised the intriguing possibility that mRNAs could be transported to synapses and locally translated in response to synaptic stimulation. This provided an elegant mechanism for spatially restricting gene expression within the neuron, such that individual synapses could independently regulate their morphology and efficacy, in a persistent, protein synthesis-dependent manner, in response to specific stimuli. It is now widely accepted that mRNAs do localize to dendrites and that translation of these mRNAs contributes to synaptic plasticity. As is evident from the collection of Mini-Reviews on dendritic protein synthesis in this issue of The Journal of Neuroscience, the field has evolved to focus on a series of key questions, including the following: (1) what mRNAs are present in dendrites? (2) How are these mRNAs transported from the nucleus into the dendrite? (3) How is translation of these mRNAs regulated by neuronal activity? and (4) What is the function of local translation of specific transcripts? In this brief introductory overview, we will consider each of these questions in turn.
Messenger RNAs in synaptosomal fractions from rat brain
Brain Research, 2001
Synaptosomal fractions from rat brain have been analyzed with semi-quantitative RT-PCR methods to determine their content of mRNAs coding for presynaptic, postsynaptic, glial, and neuronal proteins. Each mRNA was determined with reference to the standard HPRT mRNA. In our analyses, mRNAs were considered to be associated with synaptosomes only if their relative amounts were higher 11 1 than in microsomes prepared in a polysome stabilizing medium, rich in Mg and K ions, or in the homogenate. According to this stringent criterion, the following synaptosomal mRNAs could not be attributed to microsomal contamination and were assumed to derive from the subcellular structures known to harbor their translation products, i.e. GAT-1 mRNAs from presynaptic terminals and glial processes, MAP2 mRNA from dendrites, GFAP mRNA from glial processes, and TAU mRNA from neuronal fragments. This interpretation is in agreement with the involvement of extrasomatic mRNAs in local translation processes.
Synaptic Regulation of Translation of Dendritic mRNAs
Journal of Neuroscience, 2006
The selective localization of protein synthetic machinery at postsynaptic sites makes it possible for the synthesis of particular proteins to be regulated by synaptic signals. Here we consider how the structure of the machinery constrains synthetic capacity and the evidence that mRNA translation is locally controlled by synaptic signals. Since the discovery of protein synthetic machinery at synaptic sites on dendrites (Steward and Levy, 1982), substantial progress has been made in identifying dendritic mRNAs and in showing that dendritic protein synthesis is critical for persistent synaptic modifications like long-term potentiation (LTP) and long-term depression (LTD). Although many pieces of the puzzle have been identified, major questions remain. Here we focus on one of the unknowns: how translational activity at synapses is regulated and whether regulation involves upregulation or downregulation of overall translation or differential regulation of the translation of particular transcripts. It is useful to begin by considering constraints imposed by the nature of the protein synthetic machinery at synapses.
Internal initiation of translation of five dendritically localized neuronal mRNAs
Proceedings of the National Academy of Sciences, 2001
In neurons, translation of dendritically localized mRNAs is thought to play a role in affecting synaptic efficacy. Inasmuch as components of the translation machinery may be limiting in dendrites, we investigated the mechanisms by which translation of five dendritically localized mRNAs is initiated. The 5′ leader sequences of mRNAs encoding the activity-regulated cytoskeletal protein, the α subunit of calcium–calmodulin-dependent kinase II, dendrin, the microtubule-associated protein 2, and neurogranin (RC3) were evaluated for their ability to affect translation in the 5′ untranslated region of a monocistronic reporter mRNA. In both neural and nonneural cell lines, the activity-regulated cytoskeletal protein, microtubule-associated protein 2, and α-CaM Kinase II leader sequences enhanced translation, whereas the dendrin and RC3 5′ untranslated regions slightly inhibited translation as compared with controls. When cap-dependent translation of these constructs was suppressed by overex...
Dendritic mRNA: transport, translation and function
Nature Reviews Neuroscience, 2007
Lasting activity-dependent changes in synaptic strength depend on new protein synthesis and the growth or remodelling of excitatory synapses. The dendritic tree of a typical projection neuron in the adult mammalian brain contains approximately 10,000 dendritic spines, onto each of which is formed a single excitatory synapse. As a discrete structural, physiological and biochemical compartment, dendritic spines afford a necessary degree of autonomy during information processing and storage. The discovery of mRNA, ribosomes and translation factors in dendrites, and even in the dendritic spines themselves, suggested that synapses could be modified directly -and perhaps individually -through regulation of local protein synthesis 1,2 .