Dendrites as compartments for macromolecular synthesis

In the past decade it has become clear that an important aspect of gene expression involves the targeting of mRNAs to different intracellular locations. This process occurs in cell types ranging from oocytes to the most spatially complex and highly differentiated cells of the body, neurons of the central nervous system. The differential subcellular localization of mRNAs determines where in the cell particular proteins can be made and with what degree of local regulation. The story regarding neurons began with the discovery that polyribosomes are selectively localized beneath postsynaptic sites on the dendrites of a variety of central nervous system neurons (1, 2). The selectivity of the localization and the fact that synapseassociated polyribosomes are particularly prominent during periods of synapse growth (3-5) gave rise to the hypothesis that key molecular constituents of the postsynaptic junction might be synthesized on site. Obvious candidates for locally synthesized macromolecules include the molecules that make synapses operate the way that they do (neurotransmitter receptors, ion channels, and components of second messenger systems). Local synthesis of the molecular components of synapses would provide a mechanism for adjusting the macromolecular composition of individual synaptic sites based on local conditions-an appealing mechanism for synaptic plasticity. From the beginning it was obvious that, to test these hypotheses, it would be important to identify what macromolecules were actually synthesized at synaptic sites. One obvious initial question was what mRNAs are present in dendrites. But when these hypotheses were first advanced, the question could not be addressed for technical reasons. In the first place, none of the candidate molecules had been cloned. The explosion of information over the past decade solved this problem, so that many of the genes that encode neurotransmitter receptors and other important signaling molecules of the synapse have now been cloned. But the in situ hybridization analyses that were done in conjunction with the cloning studies revealed that the mRNAs for neurotransmitter receptors appeared to be localized exclusively in neuronal cell bodies (see, for example, refs. 6-9). There was no significant labeling of neuropil layers in laminated structures (the cerebral cortex, cerebellum, and hippocampus), which contain dendrites but few cell bodies, so it appeared that the mRNAs for glutamate receptors were not among the ones that were present in dendrites. Over the same period, a host of other neuronal mRNAs were cloned and their subcellular distribution was characterized. The vast majority were found to have a distribution that suggested a cell body localization, but a few mRNAs were found that had a tissue distribution that suggested a dendritic localization (i.e., labeling was present at high levels in neuropil layers in laminated structures). So far, the list includes the mRNAs encoding the high molecular weight microtubule-associated protein 2 (10), the mRNA encoding the a subunit of calcium/calmodulin-dependent protein kinase II (11), and the mRNA encoding the inositol trisphosphate type 1 receptor (12). In addition, a nonmessenger RNA of unknown function that is a polymerase III transcript (termed BC1) was also found to be present in dendritic laminae (13). The dendritic localization of some of these RNAs was subsequently confirmed by studies of neurons in culture where the degree of labeling of individual dendrites could be directly visualized (14-16). In addition, there has been one report (so far unconfirmed) that the mRNA encoding amyloid precursor protein is present in the dendrites of neurons in culture (17). Thus the story stood-that a few mRNAs were present in dendrites but that the vast majority of neuronal mRNAs were present only in the cell body. But obviously what was needed was a systematic way to identify the full complement of mRNAs that are present in dendrites and determine their relative abundance. Addressing the question requires that several difficult technical problems be solved. First, there is the problem of selectivity-how to obtain mRNA from dendrites that is not contaminated by mRNA from neuronal cell bodies or supporting cells. Second, there is the problem of obtaining sufficient quantities of mRNA to work with. Finally, there is the problem of quantitative analysis, made more difficult by the first two problems. The report by Miyashiro et al. (18) uses a collection of ingenious approaches that together provide a breakthrough to solve these problems. The problem of selectivity was solved in a simple but technically daunting way, by using patch pipettes to aspirate the cytoplasmic contents of individual dendrites of neurons grown in culture. The problems of obtaining sufficient quantities of RNA and analyzing what mRNAs were present were solved by using newly developed techniques of RNA amplification in conjunction with reverse Northern blotting. By using this array of techniques, the report provides evidence that mRNAs for glutamate receptor subunits can be detected in dendrites. If the mRNAs for glutamate receptors are associated with the polyribosomes that are selectively positioned beneath synaptic sites, then this localization would allow the synthesis of the key functional molecules of a synapse to be regulated by events at that individual synapse. It is easy to imagine how this type of mechanism might underlie activity-dependent synaptic plasticity. The study goes on to use the technique of differential display to compare the total population of mRNAs in dendrites and cell bodies and in different dendrites from the same neuron and to clone and obtain sequence information about some of the mRNAs from dendrites. This part of the study yielded two important conclusions about the nature of the mRNAs in dendrites: (i) that there appears to be a substantial number of different mRNAs in dendrites, and (ii) that there appears to be a different blend of mRNAs in different dendrites and even different parts of the same dendrite. The nature of most of these RNAs remains unknown. Some may be messages that have not yet been cloned; others may derive from the 3'untranslated region of mRNAs that are known but whose 3' sequences have not been characterized. In any case, the evidence suggests that a larger number of different RNAs are present in neurites than had previously been suspected. Taken at face value, this study provides a host of important new clues about the nature of macromolecular synthesis in dendrites and raises a number of new questions. But there are some important