Morphoregulatory functions of the RNA-binding motif protein 3 in cell spreading, polarity and migration.

RNA-binding proteins are emerging as key regulators of transitions in cell morphology. The RNA-binding motif protein 3 (RBM3) is a cold-inducible RNA-binding protein with broadly relevant roles in cellular protection, and putative functions in cancer and development. Several findings suggest that RBM3 has morphoregulatory functions germane to its roles in these contexts. For example, RBM3 helps maintain the morphological integrity of cell protrusions during cell stress and disease. Moreover, it is highly expressed in migrating neurons of the developing brain and in cancer invadopodia, suggesting roles in migration. We here show that RBM3 regulates cell polarity, spreading and migration. RBM3 was present in spreading initiation centers, filopodia and blebs that formed during cell spreading in cell lines and primary myoblasts. Reducing RBM3 triggered exaggerated spreading, increased RhoA expression, and a loss of polarity that was rescued by Rho kinase inhibition and overexpression of CRMP2. High RBM3 expression enhanced the motility of cells migrating by a mesenchymal mode involving extension of long protrusions, whereas RBM3 knockdown slowed migration, greatly reducing the ability of cells to extend protrusions and impairing multiple processes that require directional migration. These data establish novel functions of RBM3 of potential significance to tissue repair, metastasis and development.

Differential translation and fragile X syndrome.

Fragile X syndrome (FXS) is caused by the transcriptional silencing of the Fmr1 gene, which encodes a protein (FMRP) that can act as a translational suppressor in dendrites, and is characterized by a preponderance of abnormally long, thin and tortuous dendritic spines. According to a current theory of FXS, the loss of FMRP expression leads to an exaggeration of translation responses linked to group I metabotropic glutamate receptors. Such responses are involved in the consolidation of a form of long-term depression that is enhanced in Fmr1 knockout mice and in the elongation of dendritic spines, resembling synaptic phenotypes over-represented in fragile X brain. These observations place fragile X research at the heart of a long-standing issue in neuroscience. The consolidation of memory, and several distinct forms of synaptic plasticity considered to be substrates of memory, requires mRNA translation and is associated with changes in spine morphology. A recent convergence of research on FXS and on the involvement of translation in various forms of synaptic plasticity has been very informative on this issue and on mechanisms underlying FXS. Evidence suggests a general relationship in which the receptors that induce distinct forms of efficacy change differentially regulate translation to produce unique spine shapes involved in their consolidation. We discuss several potential mechanisms for differential translation and the notion that FXS represents an exaggeration of one 'channel' in a set of translation-dependent consolidation responses.

Marking synaptic activity in dendritic spines with a calpain substrate exhibiting fluorescence resonance energy transfer.

Excitatory synaptic activity can evoke transient and substantial elevations of postsynaptic calcium. Downstream effects of elevated calcium include the activation of the calcium-dependent protease calpain. We have developed a reagent that identifies dendritic spines in which calpain has been activated. A fusion protein was expressed that contained enhanced yellow and enhanced cyan fluorescent protein (EYFP and ECFP, respectively) linked by a peptide that included the micro-calpain cleavage site from alpha-spectrin. A PDZ-binding site fused to ECFP anchored this protein to postsynaptic densities. The fusion protein exhibited fluorescence resonance energy transfer (FRET), and diminution of FRET by proteolysis was used to localize calpain activity in situ by fluorescence microscopy. Incubation of the fusion protein with calpain in the presence of calcium resulted in the separation of EYFP and ECFP into monomeric fluorophores. In transiently transfected cell lines and dissociated hippocampal neurons, FRET was diminished by raising intracellular calcium levels with an ionophore or with glutamatergic agonists. Calpain inhibitors blocked these changes. Under control conditions, FRET levels in different dendritic spines of cultured neurons and in hippocampal slices were heterogeneous but showed robust decreases upon treatment with glutamatergic agonists. Immunostaining of cultured neurons with antibodies to a spectrin epitope produced by calpain-mediated digestion revealed an inverse correlation between the amount of FRET present at postsynaptic elements and the concentration of spectrin breakdown products. These results suggest that the FRET methodology identifies sites of synaptically induced calpain activity and that it may be useful in analyzing synapses undergoing changes in efficacy.

The pathogenic activation of calpain: a marker and mediator of cellular toxicity and disease states.

Over-activation of calpain, a ubiquitous calcium-sensitive protease, has been linked to a variety of degenerative conditions in the brain and several other tissues. Dozens of substrates for calpain have been identified and several of these have been used to measure activation of the protease in the context of experimentally induced and naturally occurring pathologies. Calpain-mediated cleavage of the cytoskeletal protein spectrin, in particular, results in a set of large breakdown products (BDPs) that are unique in that they are unusually stable. Over the last 15 years, measurements of BDPs in experimental models of stroke-type excitotoxicity, hypoxia/ischemia, vasospasm, epilepsy, toxin exposure, brain injury, kidney malfunction, and genetic defects, have established that calpain activation is an early and causal event in the degeneration that ensues from acute, definable insults. The BDPs also have been found to increase with normal ageing and in patients with Alzheimer's disease, and the calpain activity may be involved in related apoptotic processes in conjunction with the caspase family of proteases. Thus, it has become increasingly clear that regardless of the mode of disturbance in calcium homeostasis or the cell type involved, calpain is critical to the development of pathology and therefore a distinct and powerful therapeutic target. The recent development of antibodies that recognize the site at which spectrin is cleaved has greatly facilitated the temporal and spatial resolution of calpain activation in situ. Accordingly, sensitive spectrin breakdown assays now are utilized to identify potential toxic side-effects of compounds and to develop calpain inhibitors for a wide range of indications including stroke, cerebral vasospasm, and kidney failure.

Allosteric modulation of AMPA-type glutamate receptors increases activity of the promoter for the neural cell adhesion molecule, N-CAM.

To study regulation in vivo of the promoter for the neural cell adhesion molecule, N-CAM, we have used homologous recombination to insert the bacterial lacZ gene between the transcription and translation initiation sites of the N-CAM gene. This insertion disrupts the gene and places the expression of beta-galactosidase under the control of the N-CAM promoter. Animals homozygous for the disrupted allele did not express N-CAM mRNA or protein, but the pattern of beta-galactosidase expression in heterozygous and homozygous embryos was similar to that of N-CAM mRNA in wild-type animals. The homozygotes exhibited many of the morphological abnormalities observed in previously reported N-CAM knockout mice, with the exception that hippocampal long-term potentiation in the Schaffer collaterals was identical in homozygous, heterozygous, and wild-type animals. Heterozygous mice were used to examine the regulation of the N-CAM promoter in response to enhanced synaptic transmission. Treatment of the mice with an ampakine, an allosteric modulator of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that enhances normal glutamate-mediated synaptic transmission, increased the expression of beta-galactosidase in vivo as well as in tissue slices in vitro. Similar treatments also increased the expression of N-CAM mRNA in the heterozygotes. The effects of ampakine in slices were strongly reduced in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an AMPA receptor antagonist. Taken together, these results indicate that facilitation of AMPA receptor-mediated transmission leads to activation of the N-CAM promoter and provide support for the hypothesis that N-CAM synthesis is regulated in part by synaptic activity.

Distinct distributions of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits and a related 53,000 M(R) antigen (GR53) in brain tissue.

Polyclonal antibodies against specific carboxy-terminal sequences of known alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits (GluR-4) were used to screen regional homogenates and subcellular fractions from rat brain. Affinity purified anti-GluR1 (against amino acids 877-899), anti-GluR2/3 (850-862), and anti-GluR4a and anti-GluR4b (868-881) labeled distinct subunits with the expected molecular weight of approximately 105,000. These antigens were shown to have distinct distributions in the brain. While GluR2/3 epitopes had a distribution profile similar to that of the presynaptic marker synaptophysin, GluR1 was notable for its abundance in the hippocampus and its relatively low density in neocortical areas, and GluR4 was highly enriched in cerebellar tissue. An additional antigen (glutamate receptor-related, GR53) of lower molecular weight (50,000-59,000) was recognized in rat, human, frog, chick and goldfish brain samples by anti-GluR4a as well as by anti-GluR1 at, an antibody that specifically recognizes the extracellular aminoterminal domain of GluR1 (amino acids 163-188). Both antibodies also labeled antigens of approximately 105,000 mol. wt in brain tissue from all species tested. The approximately 53,000 mol. wt antigen was concentrated 10-20-fold in synaptic membranes vs homogenates across rat brain regions. Both the 105,000 and the 53,000 mol. wt proteins were also concentrated in postsynaptic densities, and neither of the two antigens were evident in seven non-brain tissue samples. These data indicate that AMPA receptors have regionally different subunit combinations and that some AMPA receptor composites include proteins other than the conventional 105,000 mol. wt GluR subunits.

Stable maintenance of glutamate receptors and other synaptic components in long-term hippocampal slices.

Cultured hippocampal slices retain many in vivo features with regard to circuitry, synaptic plasticity, and pathological responsiveness, while remaining accessible to a variety of experimental manipulations. The present study used ligand binding, immunostaining, and in situ hybridization assays to determine the stability of AMPA- and NMDA-type glutamate receptors and other synaptic proteins in slice cultures obtained from 11 day postnatal rats and maintained in culture for at least 4 weeks. Binding of the glutamate receptor ligands [3H]AMPA and [3H]MK-801 exhibited a small and transient decrease immediately after slice preparation, but the binding levels recovered by culture day (CD) 5-10 and remained stable for at least 30 days in culture. Autoradiographic analyses with both ligands revealed labeling of dendritic fields similar to adult tissue. In addition, slices at CD 10-20 expressed a low to high affinity [3H]AMPA binding ratio that was comparable with that in the adult hippocampus (10:1). AMPA receptor subunits GluR1 and GluR2/3 and an NMDA receptor subunit (NMDAR1) exhibited similar postcutting decreases as that exhibited by the ligand binding levels, followed by stable recovery. The GluR4 AMPA receptor subunit was not evident during the first 10 CDs but slowly reached detectable levels thereafter in some slices. Immunocytochemistry and in situ hybridization techniques revealed adult-like labeling of subunit proteins in dendritic processes and their mRNAs in neuronal cell body layers. Long-term maintenance was evident for other synapse-related proteins, including synaptophysin, neural cell adhesion molecule isoforms (NCAMs), and an AMPA receptor related antigen (GR53), as well as for certain structural and cytoskeletal components (e.g., myelin basic protein, spectrin, microtubule-associated proteins). In summary, following an initial and brief depression, many synaptic components were expressed at steady-state levels in long-term hippocampal slices, thus allowing the use of such a culture system for investigations into mechanisms of brain synapses.

Induction of beta-amyloid-containing polypeptides in hippocampus: evidence for a concomitant loss of synaptic proteins and interactions with an excitotoxin.

Long-term cultures of brain slices were used to test if the lysosomotropic agent chloroquine induces beta-amyloid-related peptides in hippocampus and if such effects are accompanied by other manifestations of brain aging. Chloroquine administration resulted in the appearance of a carboxyl-terminal fragment of the beta-amyloid precursor protein (APP); the 27-kDa antigen was detectable after 24 h, increased rapidly for 6-10 days, and was eliminated upon drug washout. Immunocytochemical analyses showed that beta-amyloid immunoreactivity accumulated in the perikarya of pyramidal neurons, primarily in the form of punctate bodies. These effects were accompanied by a correlated loss (and recovery) of the presynaptic marker synaptophysin and by a delayed reduction of postsynaptic glutamate receptors, while cytoskeletal proteins were unchanged. Acute administration of chloroquine had no evident effects on synaptic responses but prolonged applications caused a decrease in the maximum amplitude of field potentials. Finally, a brief pretreatment with the excitotoxin kainic acid had little effect with regard to APP fragments or synaptophysin, but altered the events following from a subsequent infusion of chloroquine. Buildup of the 27-kDa APP fragment and loss of synaptophysin were more rapid and, more importantly, did not reverse upon washout of chloroquine. These findings indicate that lysosomal dysfunction in hippocampus results in the accumulation of a particular APP fragment and suggest that this event, or a variable correlated with it, is linked to the loss of synaptic proteins. They also raise the possibility that certain aspects of brain aging reflect a synergism between lysosomal disturbances and excitotoxicity.

Spectrin breakdown products increase with age in telencephalon of mouse brain.

Calcium activated proteolysis of brain spectrin produces characteristic breakdown products (BDPs), the concentrations of which increase markedly in many instances of brain pathology. Results reported here indicate that levels of the BDPs rise with age (3-30 months) in the telencephalon but not in the hindbrain of Balb/c mice. These observations suggest that spectrin breakdown is a pathologic biochemical marker which increases with age in some but not all brain regions.

Evidence that matrix recognition contributes to stabilization but not induction of LTP.

Slices of hippocampus were incubated with Arg-Gly-Asp (RGD) peptides known to block members of the integrin class of matrix receptors. Though the peptides caused no detectable difference in the amount of long-term potentiation (LTP) expressed in the CA1 field 1-2 min after induction with high frequency stimulation, they did produce a reversible, dose dependent decay of LTP over a period of 40 min. This effect was not obtained with various non-RGD control peptides. These results suggest that stabilization of LTP requires adhesive interactions via specific matrix recognition sites, whereas induction and expression do not.

Antibodies to the alpha v beta 3 integrin label a protein concentrated in brain synaptosomal membranes.

Immunochemical methods were used to test whether vitronectin receptors exist in synaptosomal membranes (SPMs) and hence are positioned to play a role in synaptic adhesion. Antibodies against the alpha v beta 3 integrin detected proteins in brain homogenates that correspond to conventional integrin subunits. Conversely, these antigens were not found in SPMs prepared from the same brain tissue. The antibodies did, however, express strong immunoreactivity towards a 27 kDa polypeptide that was greatly concentrated in SPMs from major brain regions and that was not found in tissues other than brain. This is an example of an integrin epitope contained in a synaptic polypeptide that is too small to be a conventional matrix receptor, thus, suggesting the possibility that synaptic adhesion involves unusual proteins.