Selectively enriched mRNAs in rat synaptoneurosomes.

Differential display was used to identify synapse-enriched mRNAs. Of 15 mRNAs initially identified, all were found in multiple synaptoneurosome preparations; 58% were subsequently shown to be enriched in all the preparations by Northern blotting and semiquantitative RT-PCR. RNAs involved in signal transduction, vesicle trafficking, lipid modification and cell shape and remodeling were among these messages. Tip60a mRNA, recently found to associate with the fragile X mental retardation protein, was also identified. These data demonstrate the diversity of the local message pool at synapses.

Synaptic regulation of protein synthesis and the fragile X protein.

Protein synthesis occurs in neuronal dendrites, often near synapses. Polyribosomal aggregates often appear in dendritic spines, particularly during development. Polyribosomal aggregates in spines increase during experience-dependent synaptogenesis, e.g., in rats in a complex environment. Some protein synthesis appears to be regulated directly by synaptic activity. We use "synaptoneurosomes," a preparation highly enriched in pinched-off, resealed presynaptic processes attached to resealed postsynaptic processes that retain normal functions of neurotransmitter release, receptor activation, and various postsynaptic responses including signaling pathways and protein synthesis. We have found that, when synaptoneurosomes are stimulated with glutamate or group I metabotropic glutamate receptor agonists such as dihydroxyphenylglycine, mRNA is rapidly taken up into polyribosomal aggregates, and labeled methionine is incorporated into protein. One of the proteins synthesized is FMRP, the protein that is reduced or absent in fragile X mental retardation syndrome. FMRP has three RNA-binding domains and reportedly binds to a significant number of mRNAs. We have found that dihydroxyphenylglycine-activated protein synthesis in synaptoneurosomes is dramatically reduced in a knockout mouse model of fragile X syndrome, which cannot produce full-length FMRP, suggesting that FMRP is involved in or required for this process. Studies of autopsy samples from patients with fragile X syndrome have indicated that dendritic spines may fail to assume a normal mature size and shape and that there are more spines per unit dendrite length in the patient samples. Similar findings on spine size and shape have come from studies of the knockout mouse. Study of the development of the somatosensory cortical region containing the barrel-like cell arrangements that process whisker information suggests that normal dendritic regression is impaired in the knockout mouse. This finding suggests that FMRP may be required for the normal processes of maturation and elimination to occur in cerebral cortical development.

Abnormal dendritic spine characteristics in the temporal and visual cortices of patients with fragile-X syndrome: a quantitative examination.

Fragile-X syndrome is a common form of mental retardation resulting from the inability to produce the fragile-X mental retardation protein. Qualitative examination of human brain autopsy material has shown that fragile-X patients exhibit abnormal dendritic spine lengths and shapes on parieto-occipital neocortical pyramidal cells. Similar quantitative results have been obtained in fragile-X knockout mice, that have been engineered to lack the fragile-X mental retardation protein. Dendritic spines on layer V pyramidal cells of human temporal and visual cortices stained using the Golgi-Kopsch method were investigated. Quantitative analysis of dendritic spine length, morphology, and number was carried out on patients with fragile-X syndrome and normal age-matched controls. Fragile-X patients exhibited significantly more long dendritic spines and fewer short dendritic spines than did control subjects in both temporal and visual cortical areas. Similarly, fragile-X patients exhibited significantly more dendritic spines with an immature morphology and fewer with a more mature type morphology in both cortical areas. In addition, fragile-X patients had a higher density of dendritic spines than did controls on distal segments of apical and basilar dendrites in both cortical areas. Long dendritic spines with immature morphologies and elevated spine numbers are characteristic of early development or a lack of sensory experience. The fact that these characteristics are found in fragile-X patients throughout multiple cortical areas may suggest a global failure of normal dendritic spine maturation and or pruning during development that persists throughout adulthood.

Evidence for altered Fragile-X mental retardation protein expression in response to behavioral stimulation.

The Fragile-X mental retardation protein, the protein absent in Fragile-X syndrome, is synthesized near synapses upon neurotransmitter activation. Humans and mice lacking this protein exhibit abnormal dendritic spine lengths and numbers. Here we investigated Fragile-X protein levels in animals exposed to behavioral paradigms that induce neuronal morphological change. Fragile-X protein immunoreactivity was examined in visual cortices of rats reared in a complex environment for 10 or 20 days, motor cortices of rats trained on motor-skill tasks for 3 or 7 days, and either visual or motor cortices of inactive controls. Rats exposed to a complex environment for 20 days or trained for 7 days on motor-skill tasks exhibited increased Fragile-X protein immunoreactivity in visual or motor cortices, respectively. These results provide the first evidence for a behaviorally induced alteration of Fragile-X protein expression and are compatible with previous findings suggesting synaptic regulation of its expression. These results also strengthen the association of Fragile-X mental retardation protein expression with the alteration of synaptic structure.

Evidence for altered Fragile-X mental retardation protein expression in response to behavioral stimulation.

The Fragile-X mental retardation protein, the protein absent in Fragile-X syndrome, is synthesized near synapses upon neurotransmitter activation. Humans and mice lacking this protein exhibit abnormal dendritic spine lengths and numbers. Here we investigated Fragile-X protein levels in animals exposed to behavioral paradigms that induce neuronal morphological change. Fragile-X protein immunoreactivity was examined in visual cortices of rats reared in a complex environment for 10 or 20 days, motor cortices of rats trained on motor-skill tasks for 3 or 7 days, and either visual or motor cortices of inactive controls. Rats exposed to a complex environment for 20 days or trained for 7 days on motor-skill tasks exhibited increased Fragile-X protein immunoreactivity in visual or motor cortices, respectively. These results provide the first evidence for a behaviorally induced alteration of Fragile-X protein expression and are compatible with previous findings suggesting synaptic regulation of its expression. These results also strengthen the association of Fragile-X mental retardation protein expression with the alteration of synaptic structure.

Synaptic synthesis of the Fragile X protein: possible involvement in synapse maturation and elimination.

Fragile X mental retardation syndrome results from the absence of or a defect in the protein (FMRP) encoded by the FMR1 gene. FMRP is found in dendrites and synapses as well as in the neuronal cell soma and nucleus, and although it is known to bind to RNA, the function of the protein in neurons is not known. We have studied activity-dependent changes in postsynaptically localized protein translation in central nervous system neurons. We find that FMRP is one of the proteins produced at synapses following stimulation of metabotropic glutamate receptors. We have also observed that Fragile X knockout mice, like human Fragile X patients, have excess numbers of long, thin, immature-appearing dendritic processes. Together, these findings suggest that FMRP plays a role in the process whereby synaptic activity during development results in structural and functional maturation of the synapse. We hypothesize that synaptic synthesis of FMRP may be essential for activity-based synapse maturation and elimination, a key process in normal brain development.

Metabotropic glutamate receptor-initiated translocation of protein kinase p90rsk to polyribosomes: a possible factor regulating synaptic protein synthesis.

Maintenance of lasting synaptic efficacy changes requires protein synthesis. We report here a mechanism that might influence translation control at the level of the single synapse. Stimulation of metabotropic glutamate receptors in hippocampal slices induces a rapid protein kinase C-dependent translocation of multifunction kinase p90rsk to polyribosomes; concomitantly, there is enhanced phosphorylation of at least six polyribosome binding proteins. Among the polyribosome bound proteins are the p90rsk-activating kinase ERK-2 and a known p90rsk substrate, glycogen synthase kinase 3beta, which regulates translation efficiency via eukaryotic initiation factor 2B. Thus metabotropic glutamate receptor stimulation could induce synaptic activity-dependent translation via translocation of p90rsk to ribosomes.

Fragile X mental retardation protein is translated near synapses in response to neurotransmitter activation.

Local translation of proteins in distal dendrites is thought to support synaptic structural plasticity. We have previously shown that metabotropic glutamate receptor (mGluR1) stimulation initiates a phosphorylation cascade, triggering rapid association of some mRNAs with translation machinery near synapses, and leading to protein synthesis. To determine the identity of these mRNAs, a cDNA library produced from distal nerve processes was used to screen synaptic polyribosome-associated mRNA. We identified mRNA for the fragile X mental retardation protein (FMRP) in these processes by use of synaptic subcellular fractions, termed synaptoneurosomes. We found that this mRNA associates with translational complexes in synaptoneurosomes within 1-2 min after mGluR1 stimulation of this preparation, and we observed increased expression of FMRP after mGluR1 stimulation. In addition, we found that FMRP is associated with polyribosomal complexes in these fractions. In vivo, we observed FMRP immunoreactivity in spines, dendrites, and somata of the developing rat brain, but not in nuclei or axons. We suggest that rapid production of FMRP near synapses in response to activation may be important for normal maturation of synaptic connections.

Abnormal dendritic spines in fragile X knockout mice: maturation and pruning deficits.

Fragile X syndrome arises from blocked expression of the fragile X mental retardation protein (FMRP). Golgi-impregnated mature cerebral cortex from fragile X patients exhibits long, thin, tortuous postsynaptic spines resembling spines observed during normal early neocortical development. Here we describe dendritic spines in Golgi-impregnated cerebral cortex of transgenic fragile X gene (Fmr1) knockout mice that lack expression of the protein. Dendritic spines on apical dendrites of layer V pyramidal cells in occipital cortex of fragile X knockout mice were longer than those in wild-type mice and were often thin and tortuous, paralleling the human syndrome and suggesting that FMRP expression is required for normal spine morphological development. Moreover, spine density along the apical dendrite was greater in the knockout mice, which may reflect impaired developmental organizational processes of synapse stabilization and elimination or pruning.

Calcium ion impedes translation initiation at the synapse.

Stimulation of synaptoneurosome suspensions by the neurotransmitter glutamate gives rise to rapid loading of ribosomes onto mRNA and increased incorporation of amino acids into trichloroacetic acid-precipitable polypeptides. Metabotropic glutamate receptors (mGluRs) are responsible for this effect. Although simultaneous Ca2+ entry and mGluR stimulation do not change the response, entry of Ca2+ 30 s or 3 min before mGluR stimulation markedly depresses the polyribosomal loading. Either NMDA or ionophore (A23187) produces the depression. A calmodulin antagonist, W7, alleviates the effect, suggesting that inactivation of phospholipase A2 by calcium-calmodulin-dependent kinase II is partially responsible for the phenomenon. Thus, interaction between different classes of glutamate receptors affects the control of protein translation at the synapse. This effect may partially explain recent observations of negative interactions between receptor classes in induction of long-term potentiation.

Correspondence between sites of NGFI-A induction and sites of morphological plasticity following exposure to environmental complexity.

To determine if gene regulation may play a role in behaviorally-induced morphological plasticity in the brain, we used in situ hybridization to measure levels of mRNA for the immediate early gene transcription factor NGFI-A (also known as ZENK, zif/268, egr-1 and Krox 24). Brains of periadolescent male rats exposed to 2-4 days of the following behavioral treatments were compared: (1) group housing in a complex environment (EC); (2) individual housing with daily handling (HIC); and (3) individual handling (IC). Quantitative analysis of the autoradiograms revealed that EC rats had significantly higher levels of NGFI-A than IC rats in regions of cortex previously shown to exhibit morphological plasticity (most pronounced in visual cortex), but not in frontal cortex where no dendritic changes have been detected. HIC rats were intermediate between the two groups. These data support an association between structural plasticity and altered patterns of immediate early gene expression.

Morphogenesis in memory formation: synaptic and cellular mechanisms.

We review some of the evidence for structural changes in synapses in response to environmental stimulation. These include changes in synapse number, in distribution of presynaptic vesicles, in synaptic bouton size, and complex changes in the shape and size of synaptic contact zones. Increased numbers of postsynaptic polyribosomal aggregates (PRA) are correlated histologically with developmental plasticity. We discuss the role that dendritically targeted mRNAs and polyribosomes might play in providing rapid, localized synthesis of proteins necessary for structural change. Using synaptoneurosomes, we have demonstrated that depolarization leads to a rapid (1-2 min) increase in PRA and in [35S]methionine incorporation into polypeptides. We have shown that this process is initiated by metabotropic glutamate receptors, which trigger phosphatidyl inositol hydrolysis, leading to release of internal Ca2+ stores and activation of protein kinase C. Entry of external Ca2+, however, seems to downregulate polyribosomal aggregation, via a calmodulin-dependent mechanism, suggesting that translation may be controlled by interaction of ionotropic receptors, voltage-dependent calcium channels, and metabotropic receptors.

Metabotropic glutamate receptors trigger postsynaptic protein synthesis.

K+ depolarization or addition of glutamate to a synaptoneurosome preparation triggers a rapid increase in size of polyribosomal aggregates isolated by centrifugation of lysate through 1 M sucrose. The profile of response to the glutamate analogues quisqualate, ibotenate, and 1-aminocyclopentane-1,3-dicarboxylate corresponds to that of metabotropic receptors. Glutamate stimulation is mimicked by the diacylglycerol analogue 1-oleoyl-2-acetylglycerol and by the protein kinase C activator phorbol dibutyrate. The phospholipase blockers 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate and quinacrine reduce the late phase of the response. The protein kinase C inhibitor calphostin C suppresses the response to 1-aminocyclopentane-1,3-dicarboxylate. These data indicate that glutamatergic synapses upregulate postsynaptic protein synthesis via metabotropic glutamate receptors coupled to the phosphatidylinositol second-messenger system. This mechanism could underlie the reported involvement of metabotropic glutamate receptors in long-term potentiation and other forms of neural plasticity.

Potassium ion stimulation triggers protein translation in synaptoneurosomal polyribosomes.

Synaptoneurosomes, a subcellular fraction of resealed presynaptic plus postsynaptic entities, contain polyribosomal aggregates and are capable of protein synthesis. Upon treatment with K(+), which has been shown to cause depolarization, we observed a rapid increase followed by a decrease in the size of polyribosomal complexes. These were shown by sucrose density gradient analysis to represent a shift of part of the total RNA into polyribosomes; accelerated incorporation of amino acids into polypeptides was also demonstrated. Calcium chelators in the medium reduce the steady-state size of polyribosomal complexes, but do not eliminate the response to depolarization, while intracellular calcium chelators have more profound effects, suggesting that there is mobilization of intracellular calcium stores. Thus depolarization-related changes may affect binding and release of translation initiation complexes with messenger RNA found near synaptic junctions.

The Xenopus laevis estrogen receptor: sequence homology with human and avian receptors and identification of multiple estrogen receptor messenger ribonucleic acids.

We have isolated and sequenced a cDNA clone encompassing the entire protein coding region of the Xenopus laevis estrogen receptor (xER). The Xenopus ER, the first steroid hormone receptor to be sequenced from a cold-blooded organism, exhibits two regions of striking amino acid homology with the human and avian ERs. In the putative DNA binding region, the amino acid sequence of the xER differs from those of the human and avian ERs at only one of 83 amino acids. The putative hormone binding region contains 44 and 46 amino acid blocks in which the sequence is identical in the Xenopus and human ERs. Blot hybridizations of Xenopus liver RNA suggest that the xER is encoded by four mRNAs with lengths of approximately 9, 6.5, 2.8, and 2.5 kilobases. In contrast, hybridization of human RNA to a human ER cDNA clone reveals only a single major ER RNA, approximately 6.7 kilobases in length.

Rearrangement of antigen receptor genes is defective in mice with severe combined immune deficiency.

A process unique to lymphocyte differentiation is the rearrangement of genes encoding antigen-specific receptors on B and T cells. A mouse mutant (C.B-17scid) with severe combined immune deficiency, i.e., that lacks functional B and T cells, shows no evidence of such gene rearrangements. However, rearrangements were detected in Abelson murine leukemia virus-transformed bone marrow cells and in spontaneous thymic lymphomas from C.B-17scid mice. Most of these rearrangements were abnormal: approximately 80% of Igh rearrangements deleted the entire Jh region, and approximately 60% of TCR beta rearrangements deleted the entire J beta 2 region. The deletions appeared to result from faulty D-to-J recombination. No such abnormal rearrangements were detected in transformed tissues from control mice. The scid mutation may adversely affect the recombinase system catalyzing the assembly of antigen receptor genes in developing B and T lymphocytes.

Nude mice are nonpermissive towards the anti-dextran response of congenic cells.

Nude mice bearing allotype Ighb on a BALB/c genetic background (= CB nu/nu) are nonresponders to alpha (1----3)dextran (Dex), in contrast to BALB/c or BALB/c nu/nu. Although CB nu/nu mice accept transplants of congenic BALB/c, or BALB nu/nu lymphocytes, as shown by the expression of donor allotype Igha, they are not permissive for a primary anti-Dex response by the grafted cells. BALB/c or BALB nu/nu cells, however, give a strong anti-Dex response when grafted onto irradiated CB nu/nu or CB 23 (Ighb) euthymic mice. A thymus-independent, radiation-sensitive suppressor cell population is postulated, which specifically hinders the anti-Dex response, and which is exhibited by strains bearing that portion of chromosome 12 which codes for CH allotype Ighb, not containing the germ-line anti-Dex V/D genes. The suppressive action of Ighb lymphocytes could be demonstrated directly in staggered co-transfer experiments.

Demonstration that milk cells invade the suckling neonatal mouse.

Mouse milk cells were stained with rhodamine or fluorescein isothiocyanate and fed to young suckling mice. By visual examination of serial sections and by flow cytofluorometry, we were able to demonstrate directly the presence of these cells in peripheral tissues. It was estimated that at least 0.1% of the fed cells might infiltrate the young mouse, which is initially immunologically defenseless. This is in accordance with evidence from many sources for activity of maternally-derived lymphoid cells in young rodents.