Ephrin-A5 modulates the topographic mapping and connectivity of commissural axons in murine hippocampus.

Entorhinal and commissural/associational projections show a non-overlapping distribution in the hippocampus proper and the dentate gyrus. The expression of Ephrins and their Eph receptors in the developing hippocampus indicates that this family of axonal guidance molecules may modulate the formation of these connections. Here we focused on the role of the ephrin-A5 ligand in the development of the main hippocampal afferents. In situ hybridization showed that ephrin-A5 mRNA was detected mainly in the principal cells of the hippocampus proper and in the dentate gyrus throughout postnatal development. Immunocytochemical analyses revealed prominent expression of the EphA3 receptor, a putative receptor for ephrin-A5, in the main cells and the neuropil of the developing hippocampus. Tracing experiments in ephrin-A5(-/-) mice showed that commissural projections were transiently altered in the hippocampus proper at P5, but they were mistargeted throughout the postnatal development in the dentate gyrus. Immunocytochemistry with anti-calbindin antibodies revealed that the dentate mossy fiber projection was not altered in ephrin-A5(-/-) mice. Electron microscopy studies showed alterations in the density of synapses and spines in commissural/associational layers, but not in entorhinal layers, and in the mossy fibers in these animals. Taken together, these findings indicate that ephrin-A5 signaling is involved in the formation and maturation of synapses in the hippocampus.

Pattern of expression of the tetraspanin Tspan-5 during brain development in the mouse.

Here, we report the pattern of expression of the tetraspanin mTspan-5 at various developmental stages. Expression was first seen at E10 and remained until adulthood, with increased levels between P0 and P5. mTspan-5 showed high expression in the cortical areas and Purkinje cells throughout development. Moreover, transcripts were also detected in the developing heart, forelimbs, and hindlimbs.

Mouse Tspan-5, a member of the tetraspanin superfamily, is highly expressed in brain cortical structures.

Using a subtractive hybridization method for the identification of genes related to the development of the murine cerebral cortex, we cloned a mouse homologue of a human tetraspanin family member, Tspan-5. We have isolated a 3.1 Kb cDNA fragment containing the entire coding region. Analysis of the cDNA nucleotide sequence revealed that mouse Tspan-5 shares 98% amino acid sequence identity with its human homologue. The predicted length of the mouse protein is 268 amino acids, with four putative hydrophobic domains with N- and C-intracellular tails, and two extracellular domains. Northern blot analysis of adult mouse tissues showed a single transcript, which is preferentially expressed in the brain. In situ hybridization showed prominent expression of Tspan-5 in the neocortex, the hippocampus, amygdala and in Purkinje cells in the cerebellum. The pattern of expression of Tspan-5 in the mouse brain suggests a role for the tetraspanins in the maintenance of adult brain function.

Autophosphorylation of Tyr397 and its phosphorylation by Src-family kinases are altered in focal-adhesion-kinase neuronal isoforms.

In brain, focal adhesion kinase (FAK) is regulated by neurotransmitters and has a higher molecular mass than in other tissues, due to alternative splicing. Two exons code for additional peptides of six and seven residues ('boxes' 6 and 7), located on either side of Tyr(397), which increase its autophosphorylation. Using in situ hybridization and a monoclonal antibody (Mab77) which does not recognize FAK containing box 7, we show that, although mRNAs coding for boxes 6 and 7 have different patterns of expression in brain, FAK+6,7 is the main isoform in forebrain neurons. The various FAK isoforms fused to green fluorescent protein were all targeted to focal adhesions in non-neuronal cells. Phosphorylation-state-specific antibodies were used to study in detail the phosphorylation of Tyr(397), a critical residue for the activation and function of FAK. The presence of boxes 6 and 7 increased autophosphorylation of Tyr(397) independently and additively, whereas they had a weak effect on FAK kinase activity towards poly(Glu,Tyr). Src-family kinases were also able to phosphorylate Tyr(397) in cells, but this phosphorylation was decreased in the presence of box 6 or 7, and abolished in the presence of both. Thus the additional exons characteristic of neuronal isoforms of FAK do not alter its targeting, but change dramatically the phosphorylation of Tyr(397). They increase its autophosphorylation in vitro and in transfected COS-7 cells, whereas they prevent its phosphorylation when co-transfected with Src-family kinases.

FAK+ and PYK2/CAKbeta, two related tyrosine kinases highly expressed in the central nervous system: similarities and differences in the expression pattern.

Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2/cell adhesion kinase beta (PYK2/CAKbeta) are related, non-receptor, cytoplasmic tyrosine kinases, highly expressed in the central nervous system (CNS). In addition, FAK+ is a splice isoform of FAK containing a 3-amino acid insertion in the carboxy-terminal region. In rat hippocampal slices, FAK+ and PYK2/CAKbeta are differentially regulated by neurotransmitters and depolarization. We have studied the regional and cellular distribution of these kinases in adult rat brain and during development. Whereas PYK2/CAKbeta expression increased with postnatal age and was maximal in the adult, FAK+ levels were stable. PYK2/CAKbeta mRNAs, detected by in situ hybridization, were expressed at low levels in the embryonic brain, and became very abundant in the adult forebrain. Immunocytochemistry of the adult brain showed a widespread neuronal distribution of FAK+ and PYK2/CAKbeta immunoreactivities (ir). PYK2/CAKbeta appeared to be particularly abundant in the hippocampus. In hippocampal neurons in culture at early stages of development, FAK+ and PYK2/CAKbeta were enriched in the perikarya and growth cones. FAK+ extended to the periphery of the growth cones tips, whereas PYK2/CAKbeta appeared to be excluded from the lamellipodia. During the establishment of polarity, a proximal-distal gradient of increasing PYK2/CAKbeta-ir could be observed in the growing axon. In most older neurons, FAK+-ir was confined to the cell bodies, whereas PYK2/CAKbeta-ir was also present in the processes. In vitro and in vivo, a subpopulation of neurons displayed neurites with intense FAK+-ir. Thus, FAK+ and PYK2/CAKbeta are differentially regulated during development yet they are both abundantly expressed in the adult brain, with distinctive but overlapping distributions.

Thyroid hormone regulates reelin and dab1 expression during brain development.

The reelin and dab1 genes are necessary for appropriate neuronal migration and lamination during brain development. Since these processes are controlled by thyroid hormone, we studied the effect of thyroid hormone deprivation and administration on the expression of reelin and dab1. As shown by Northern analysis, in situ hybridization, and immunohistochemistry studies, hypothyroid rats expressed decreased levels of reelin RNA and protein during the perinatal period [embryonic day 18 (E18) and postnatal day 0 (P0)]. The effect was evident in Cajal-Retzius cells of cortex layer I, as well as in layers V/VI, hippocampus, and granular neurons of the cerebellum. At later ages, however, Reelin was more abundant in the cortex, hippocampus, cerebellum, and olfactory bulb of hypothyroid rats (P5), and no differences were detected at P15. Conversely, Dab1 levels were higher at P0, and lower at P5 in hypothyroid animals. In line with these results, reelin RNA and protein levels were higher in cultured hippocampal slices from P0 control rats compared to those from hypothyroid animals. Significantly, thyroid-dependent regulation of reelin and dab1 was confirmed in vivo and in vitro by hormone treatment of hypothyroid rats and organotypic cultures, respectively. In both cases, thyroid hormone led to an increase in reelin expression. Our data suggest that the effects of thyroid hormone on neuronal migration may be in part mediated through the control of reelin and dab1 expression during brain ontogenesis.

Alternatively spliced focal adhesion kinase in rat brain with increased autophosphorylation activity.

pp125 focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase transducing signals initiated by integrin engagement and G protein-coupled receptors, is highly expressed in brain. FAK from brain had a higher molecular weight and an increased autophosphorylation activity, than from other tissues. In addition to a 9-base insertion in the 3'-coding region, which defines FAK+, rat striatal FAK mRNAs contained several additional short exons, coding for peptides of 28, 6, and 7 residues, respectively (termed boxes 28, 6, and 7), surrounding the autophosphorylated Tyr-397. In transfected COS 7 cells, the presence of boxes 6 and 7 conferred an increased overall tyrosine phosphorylation, a higher phosphorylation of Tyr-397 assessed with a phosphorylation state-specific antibody, and a more active autophosphorylation in immune precipitates. The presence of box 28 did not alter further these parameters. Two-dimensional phosphopeptide maps of hippocampal FAK were identical to those of FAK+6,7. The presence of the various exons did not alter the interaction of FAK with c-Src, n-Src, or Fyn. Thus, several splice isoforms of FAK are preferentially expressed in rat brain, some of which have an increased autophosphorylation activity, suggesting that FAK may have specific properties in neurons.

Paranodin, a glycoprotein of neuronal paranodal membranes.

Ranvier nodes are flanked by paranodal regions, at the level of which oligodendrocytes or Schwann cells interact closely with axons. Paranodes play a critical role in the physiological properties of myelinated nerve fibers. Paranodin, a prominent 180 kDa transmembrane neuronal glycoprotein, was purified and cloned from adult rat brain, and found to be highly concentrated in axonal membranes at their junction with myelinating glial cells, in paranodes of central and peripheral nerve fibers. The large extracellular domain of paranodin is related to neurexins, and its short intracellular tail binds protein 4.1, a cytoskeleton-anchoring protein. Paranodin may be a critical component of the macromolecular complex involved in the tight interactions between axons and myelinating glial cells characteristic of the paranodal region.

Regulation of a neuronal form of focal adhesion kinase by anandamide.

Anandamide is an endogenous ligand for central cannabinoid receptors and is released after neuronal depolarization. Anandamide increased protein tyrosine phosphorylation in rat hippocampal slices and neurons in culture. The action of anandamide resulted from the inhibition of adenylyl cyclase and cyclic adenosine 3', 5'-monophosphate-dependent protein kinase. One of the proteins phosphorylated in response to anandamide was an isoform of pp125-focal adhesion kinase (FAK+) expressed preferentially in neurons. Focal adhesion kinase is a tyrosine kinase involved in the interactions between the integrins and actin-based cytoskeleton. Thus, anandamide may exert neurotrophic effects and play a role in synaptic plasticity.

Cloning of focal adhesion kinase, pp125FAK, from rat brain reveals multiple transcripts with different patterns of expression.

Focal adhesion kinase (pp125FAK, or FAK) is a cytoplasmic tyrosine kinase enriched in focal adhesions. We have screened a rat striatum cDNA library with a PCR-amplified cDNA probe specific for FAK messenger. Sequencing of multiple clones revealed the existence of three different 5'-leader sequences resulting from the combination of 5 conserved boxes. One of them contains a potential alternative initiation site, 78 base pairs upstream of that previously described. Another is 89% identical to a human genomic sequence located on chromosome 3. Most positive clones contained an insertion coding for three amino acids (Pro-Trp-Arg) in the region responsible for focal adhesion targeting. We propose to name this variant of the protein FAK +. The pattern of expression of the multiple forms of FAK was studied by RT-PCR and Southern hybridization with specific primers and probes. The different 5'-leader sequences were always found in the same proportions. In contrast, FAK + mRNA was found at very low levels in non-nervous tissues, whereas it was highly expressed in all brain regions. In cells in culture, it was present in astrocytes and enriched in neurons. These results demonstrate the existence of multiple forms of FAK transcript and protein, one of which, FAK +, may play a specific role in the nervous system.

Focal adhesion kinase in rat central nervous system.

Focal adhesion kinase (pp125FAK, FAK) is a 125 kDa non-receptor tyrosine kinase enriched in focal adhesions of various cell types, where it is thought to transduce signals triggered by contact with the extracellular matrix. We have studied the expression and localization of FAK in rat CNS. Immunoblotting, immunohistochemistry and in situ hybridization revealed the presence of FAK in all regions of the adult brain and demonstrated its enrichment in specific neuronal populations of the cerebral and cerebellar cortex, as well as in the hippocampus. During development, FAK protein levels were highest around birth in cerebral cortex and caudate putamen and decreased in the adult. In situ hybridization revealed enrichment of FAK mRNA in the ventricular germinative and external layers during the last period of embryonic growth. In primary cultures FAK immunoreactivity was localized in focal adhesions in astrocytes, whereas in developing neurons the highest levels were found in growth cones and perikarya. In the growth cone, FAK immunoreactivity colocalized with actin filaments. In mature neurons FAK appeared to be distributed in the whole cytoplasm, with no enrichment in any cellular compartment. Our results demonstrate the presence of high levels of FAK in rat CNS, maximal during development but persistent in the adult. Its enrichment in growth cones suggests that it may play a role in neurite outgrowth, as well as in plasticity in the adult.

Structure and expression of Spk-1, an src-related gene product found in the planarian Dugesia (G) tigrina.

A cDNA clone encoding a 497 amino acid protein 75% similar to most Src proteins has been isolated from the planarian Dugesia (Girardia) tigrina (Platyhelminthes; Turbellaria) by PCR followed by screening procedures. This gene product has been designated Spk-1 as it is the first Src-related kinase isolated in a planarian. The predicted amino acid sequence of Spk-1 suggest that it is anchored to the plasma membrane and that it interacts with other phosphotyrosine proteins. Spk-1 is expressed in both intact and regenerating organisms as an mRNA transcript of about 1.9 kb. Planarians, which conserve most features of the common ancestor to protostomian and deuterostomian phyla, are the most primitive triploblastic organisms from which a protein tyrosine kinase gene product has been isolated. The presence of this gene product in such a primitive organism, and its presumed role, are discussed.

Hepatic endothelial lipase activity in neonatal rat liver.

Hepatic endothelial lipase (HEL) activity is as high in the neonatal (1-day old) rat liver as in adults. Most of the HEL activity is located at the capillaries since 75% of the total activity is released by heparin or collagenase perfusion. The residual activity (non-releasable) is located in hepatocytes and not in hemopoietic cells, which are the major cell type in neonatal liver. Per mg of protein, the HEL activity is 50% higher in neonatal than in adult hepatocytes. We suggest that neonatal hepatocytes have an increased capacity to synthesize and secrete HEL activity, so maintaining a high activity in the whole organ. It might contribute to the hepatic uptake of cholesterol from circulating lipoproteins, in a period in which endogenous cholesterol synthesis is known to be inhibited in the liver.

Lipoprotein lipase activity in neonatal-rat liver cell types.

The lipoprotein lipase activity in the liver of neonatal (1 day old) rats was about 3 times that in the liver of adult rats. Perfusion of the neonatal liver with collagenase decreased the tissue-associated activity by 77%. When neonatal-rat liver cells were dispersed, hepatocyte-enriched (fraction I) and haemopoietic-cell-enriched (fraction II) populations were obtained. The lipoprotein lipase activity in fraction I was 7 times that in fraction II. On the basis of those activities and the proportion of both cell types in either fraction, it was estimated that hepatocytes contained most, if not all, the lipoprotein lipase activity detected in collagenase-perfused neonatal-rat livers. From those calculations it was also concluded that haemopoietic cells did not contain lipoprotein lipase activity. When the hepatocyte-enriched cell population was incubated at 25 degrees C for up to 3 h, a slow but progressive release of enzyme activity to the incubation medium was found. However, the total activity (cells + medium) did not significantly change through the incubation period. Cycloheximide produced a time-dependent decrease in the cell-associated activity. Heparin increased the amount of lipoprotein lipase activity released to the medium. Because the cell-associated activity was unchanged, heparin also produced a time-dependent increase in the total activity. In those cells incubated with heparin, cycloheximide did not affect the initial release of lipoprotein lipase activity to the medium, but blocked further release. The cell-associated activity was also decreased by the presence of cycloheximide in those cells. It is concluded that neonatal-rat hepatocytes synthesize active lipoprotein lipase.

Acid lipase activity in neonatal rat liver cell types. Effect of starvation.

The acid lipase activity in the liver of neonatal (1-day-old) rats was studied. It was found that (i) in whole liver, the activity was 50% lower than in adult rats; (ii) in neonatal livers, the activity was 7.7-fold higher in hepatocytes than in hemopoietic cells; (iii) neonatal hepatocytes contained about 25% of the activity detected in adult hepatocytes; (iv) all the differences disappeared when expressed per mg of protein; and (v) starvation did not affect the activity either in adult or in neonatal rat liver.