The binding of divalent metal ions to platelet factor XIII modulates its proteolysis by trypsin and thrombin

We investigated the effect of divalent metal ions on the proteolytic cleavage and activation of platelet Factor XIII by thrombin and trypsin. In the absence of metal ions (5 mM EDTA), trypsin and thrombin rapidly degraded platelet Factor XIII (80 kDa) to low-molecular-mass peptides (50-19 kDa) with simultaneous loss of transglutaminase activity. Divalent metal ions protected Factor XIII from proteolytic inactivation with an order of efficacy of Ca2+ greater than Zn2+ greater than Mg2+ greater than Mn2+. Calcium (2 mM) increased by 10- to 1000-fold the trypsin and thrombin concentrations required to degrade Factor XIII to a 19-kDa peptide. Factor XIIIa formed by thrombin in the presence of 5 mM EDTA had one-half the specific activity of Factor XIIIa formed in the presence of calcium. Factor XIII was cleaved by trypsin in the presence of 5 mM Ca2+ to a 51 +/- 3-kDa fragment that had 60% of the original Factor XIIIa activity. A similar tryptic peptide formed in the presence of 5 mM EDTA did not have transglutaminase activity. In the presence of 5 mM Mg2+, thrombin cleaved Factor XIII to a major 51 +/- 3-kDa fragment that had 60% of the Factor XIIIa activity. Mn2+ (0.1-5 mM) limited trypsin and thrombin proteolysis. The resulting digest containing a population of Factor XIII fragments (50-14 kDa) expressed 50-60% transglutaminase activity of Factor XIIIa. Factor XIII was fully activated by both trypsin and thrombin in the presence of 5 mM Zn2+, resulting in two fragments of 76 and 72 kDa. We conclude that the binding of divalent metal ions to platelet Factor XIII induces conformational changes in the protein that alter its susceptibility to proteolysis and influence the expression of transglutaminase activity.     

Modeling the role of gap junctions between excitatory neurons in the developing visual cortex

Recent experiments in the developing mammalian visual cortex have revealed that gap junctions couple excitatory cells and potentially influence the formation of chemical synapses. In particular, cells that were coupled by a gap junction during development tend to share an orientation preference and are preferentially coupled by a chemical synapse in the adult cortex, a property that is diminished when gap junctions are blocked. In this work, we construct a simplified model of the developing mouse visual cortex including spike-timing-dependent plasticity of both the feedforward synaptic inputs and recurrent cortical synapses. We use this model to show that synchrony among gap-junction-coupled cells underlies their preference to form strong recurrent synapses and develop similar orientation preference; this effect decreases with an increase in coupling density. Additionally, we demonstrate that gap-junction coupling works, together with the relative timing of synaptic development of the feedforward and recurrent synapses, to determine the resulting cortical map of orientation preference.     

Pre-synaptic and post-synaptic localization of EphA4 and EphB2 in adult mouse forebrain

The ephrin receptors EphA4 and EphB2 have been implicated in synaptogenesis and long-term potentiation in the cerebral cortex and hippocampus, where they are generally viewed as post-synaptic receptors. To determine the precise distribution of EphA4 and EphB2 in mature brain synapses, we used subcellular fractionation and electron microscopy to examine the adult mouse forebrain/midbrain. EphA4 and EphB2 were both enriched in microsomes and synaptosomes. In synaptosomes, they were present in the membrane and the synaptic vesicle fractions. While EphA4 was tightly associated with PSD-95-enriched post-synaptic density fractions, EphB2 was easily extracted with detergents. In contrast, both receptors were found in the pre-synaptic active zone fraction. By electron microscopy, EphA4 was mainly detected in axon terminals, whereas EphB2 was more frequently detected in large dendritic shafts, in the hippocampus and cerebral cortex. However, in the ventrobasal thalamus, EphB2 was detected most frequently in axon terminals and thin dendritic shafts. The localization of EphA4 and EphB2 in multiple compartments of neurons and synaptic junctions suggests that they interact with several distinct scaffolding proteins and play diverse roles at synapses.     

The interaction of plasma fibronectin with fibroblastic cells in suspension

We have examined the interaction of soluble plasma [3H]fibronectin with fibroblastic cells in suspension. Fibronectin labeled by reductive methylation binds to baby hamster kidney cells in serum-free medium in a time-dependent manner at 4, 22, and 37 degrees C, with half-maximal binding occurring in 12-15 min at 22 degrees C. The binding is saturable and reversible. At least 90% of the cell-associated fibronectin is external to the plasma membrane, as judged by trypsin susceptibility of the bound radioactivity. Scatchard analysis of the concentration dependence of binding indicates the presence of a single class of binding sites, even at low input concentrations of fibronectin. There are approximately 5 +/- 1 X 10(5) sites/cell with an apparent dissociation constant of 8.0 +/- 0.5 X 10(-7) M; thus, the binding of soluble fibronectin to these cells is of moderate affinity. This putative fibroblast fibronectin receptor is resistant to trypsin in the presence of physiological concentrations of divalent cations but is susceptible to trypsin in the presence of 5 mM EDTA. Binding of 0.1 mg/ml [3H]fibronectin is 60-80% inhibited by 8 mg/ml unlabeled fibronectin and 95% inhibited by 1 mg/ml purified 75-kDa fibronectin cell-binding domain, but is unaffected by 1 mg/ml 44-kDa collagen-binding domain or 5 mg/ml ovalbumin. The binding parameters determined in this study further define the fibroblast cell-surface fibronectin receptor.     

Epileptogenic actions of GABA and fast oscillations in the developing hippocampus

GABA excites immature neurons and inhibits adult ones, but whether this contributes to seizures in the developing brain is not known. We now report that in the developing, but not the adult, hippocampus, seizures beget seizures only if GABAergic synapses are functional. In the immature hippocampus, seizures generated with functional GABAergic synapses include fast oscillations that are required to transform a naive network to an epileptic one: blocking GABA receptors prevents the long-lasting sequels of seizures. In contrast, in adult neurons, full blockade of GABA(A) receptors generates epileptogenic high-frequency seizures. Therefore, purely glutamatergic seizures are not epileptogenic in the developing hippocampus. We suggest that the density of glutamatergic synapses is not sufficient for epileptogenesis in immature neurons; excitatory GABAergic synapses are required for that purpose. We suggest that the synergistic actions of GABA and NMDA receptors trigger the cascades involved in epileptogenesis in the developing hippocampus.     

Role of androgens and the androgen receptor in remodeling of spine synapses in limbic brain areas

Accumulating evidence indicate that structural synaptic plasticity in limbic areas plays a vital role not only in normal brain functions, such as cognition and mood, but also in the development of neurological and mental disorders. We have learned from studies investigating neuronal remodeling that estrogens have an exceptional synaptogenic potential that seems to be specific to limbic areas of the adult female brain. On the other hand, structural synaptic plasticity in the adult male brain and the synaptogenic effect of androgens received relatively little attention. During the last five years, the Leranth laboratory provided conclusive evidence that the hippocampus and prefrontal cortex of adult male rodents and non-human primates retain considerable structural synaptic plasticity similar to the female, and that androgens are capable of inducing spine synapse growth in both the hippocampus and prefrontal cortex similar to estrogens. Our recent work also demonstrates that androgen-induced remodeling of spine synapses in the prefrontal cortex of adult male rats is dependent, at least to some extent, on functional androgen receptors, while being entirely independent of the androgen receptor in the hippocampus. Based on these findings and on their many beneficial effects, we believe that androgens hold a great and undeservingly neglected therapeutic potential that could be employed to reverse synaptic pathology in various neurocognitive and neuropsychiatric disorders.     

alphaCaMKII Is essential for cerebellar LTD and motor learning

Activation of postsynaptic alpha-calcium/calmodulin-dependent protein kinase II (alphaCaMKII) by calcium influx is a prerequisite for the induction of long-term potentiation (LTP) at most excitatory synapses in the hippocampus and cortex. Here we show that postsynaptic LTP is unaffected at parallel fiber-Purkinje cell synapses in the cerebellum of alphaCaMKII(-/-) mice. In contrast, a long-term depression (LTD) protocol resulted in only transient depression in juvenile alphaCaMKII(-/-) mutants and in robust potentiation in adult mutants. This suggests that the function of alphaCaMKII in parallel fiber-Purkinje cell plasticity is opposite to its function at excitatory hippocampal and cortical synapses. Furthermore, alphaCaMKII(-/-) mice showed impaired gain-increase adaptation of both the vestibular ocular reflex and optokinetic reflex. Since Purkinje cells are the only cells in the cerebellum that express alphaCaMKII, our data suggest that an impairment of parallel fiber LTD, while leaving LTP intact, is sufficient to disrupt this form of cerebellar learning.     

Involvement of afadin in the formation and remodeling of synapses in the hippocampus

In the hippocampus, synapses are formed between mossy fiber terminals and CA3 pyramidal cell dendrites and comprise highly developed synaptic junctions (SJs) and puncta adherentia junctions (PAJs). Dynamic remodeling of synapses in the hippocampus is implicated in learning and memory. Components of both the nectin-afadin and cadherin-catenin cell adhesion systems exclusively accumulate at PAJs. We investigated the role of afadin at synapses in mice in which the afadin gene was conditionally inactivated in hippocampal neurons. In these mutant mice, the signals for not only nectins, but also N-cadherin and β-catenin, were hardly detected in the CA3 area, in addition to loss of the signal for afadin, resulting in disruption of PAJs. Ultrastructural analysis revealed an increase in the number of perforated synapses, suggesting the instability of SJs. These results indicate that afadin is involved not only in the assembly of nectins and cadherins at synapses, but also in synaptic remodeling.     

Regional differences in glutaminase activation by phosphate and calcium in rat brain: Impairment in aged rats and implications for regional glutaminase isozymes

Regional regulation of glutaminase by phosphate and calcium was examined in the temporal cortex (TCX), striatum (STR) and hippocampus (HIPP) from adult and aged male F344 rats. Phosphate-dependent glutaminase activity in adult rats was significantly lower (35–43%) in the HIPP (100 and 150 mM) and STR (150 mM) compared to PAG activity in the TCX. Phosphate activation in aged rats was 50–60% lower in the HIPP at concentrations greater than 25 mM compared to the aged TCX or STR. PAG activity in the TCX and STR was unaffected by age, but was significantly reduced (30–50%) in the HIPP from aged rats at phosphate concentrations of 25 mM and greater when compared to adult rats. In adult rats at concentrations of CaCl₂ above 1 mM, PAG activity was significantly lower (60–75%) in the STR and HIPP when compared to the TCX. In aged rats, PAG activity (1 mM CaCl₂) in the HIPP was significantly less (50%) than STR PAG activity in aged rats. Diminished PAG activity was seen only in the TCX (2.5 mM; 32%), and the HIPP (0.5 mM; 25% and 1 mM; 38%) at higher calcium concentrations compared to adult. Phosphate-independent calcium activation of PAG occurred in the HIPP but not in either the TCX or the STR. Addition of phosphate resulted in a synergistic activation of PAG in the STR and TCX, but not in the HIPP. These findings suggest that PAG is regionally regulated by phosphate and calcium, and this regulation is impaired in aged rats. These data also support the hypothesis that isozymes of PAG exist with different regulatory properties.Abbreviation PAG Phosphate-activated glutaminase - L-glutamine amidohydrolase EC 3.5.1.2 - TCX temporal cortex - STR striatum - HIPP hippocampus - F344 Fischer-344 rat     

Nectin: an adhesion molecule involved in formation of synapses

The nectin-afadin system is a novel cell-cell adhesion system that organizes adherens junctions cooperatively with the cadherin-catenin system in epithelial cells. Nectin is an immunoglobulin-like adhesion molecule, and afadin is an actin filament-binding protein that connects nectin to the actin cytoskeleton. Nectin has four isoforms (-1, -2, -3, and -4). Each nectin forms a homo-cis-dimer followed by formation of a homo-trans-dimer, but nectin-3 furthermore forms a hetero-trans-dimer with nectin-1 or -2, and the formation of each hetero-trans-dimer is stronger than that of each homo-trans-dimer. We show here that at the synapses between the mossy fiber terminals and dendrites of pyramidal cells in the CA3 area of adult mouse hippocampus, the nectin-afadin system colocalizes with the cadherin-catenin system, and nectin-1 and -3 asymmetrically localize at the pre- and postsynaptic sides of puncta adherentia junctions, respectively. During development, nectin-1 and -3 asymmetrically localize not only at puncta adherentia junctions but also at synaptic junctions. Inhibition of the nectin-based adhesion by an inhibitor of nectin-1 in cultured rat hippocampal neurons results in a decrease in synapse size and a concomitant increase in synapse number. These results indicate an important role of the nectin-afadin system in the formation of synapses.     

Ischemia Induces a Reduction in the Content of Ulinastatin-Like Substance in the Murine Hippocampus

Effects of ischemia on the content of a ulinastatin (UT)-like substance in the murine cerebral cortex and hippocampus were studied. At 24 h post-ischemia, a significant (p < 0.05) decrease in the content of UT-like substance in the hippocampus but not the cerebral cortex and a concurrent increase in the activity of -calpain were observed. In in vitro experiments, a decrease was registered in the content of UT-like substance in the hippocampus in the presence of calcium. This decrease was inhibited by both EDTA and calpastatin treatments. These results implicate the destruction of UT-like substance by -calpain in the ischemic hippocampus.     

Proteolytic activation of the canine cardiac sarcoplasmic reticulum calcium pump

Mild trypsin treatment of canine cardiac microsomes consisting largely of sarcoplasmic reticulum vesicles produced a severalfold activation of oxalate-facilitated calcium uptake. The increase in calcium uptake was associated with an increase in ATP hydrolysis. Proteases other than trypsin were also effective although to a lesser degree. Trypsin produced a shift of the Ca2+ concentration dependency curve for calcium uptake toward lower Ca2+ concentrations, which was almost identical with that produced by phosphorylation of microsomes by cyclic AMP dependent protein kinase when the trypsin and the protein kinase were present at maximally activating concentrations. The Hill numbers (+/- SD) of the Ca2+ dependency after treatment of microsomes with trypsin (1.5 +/- 0.1) or protein kinase (1.7 +/- 0.1) were similar and were not significantly different from those for untreated control microsomes (1.6 +/- 0.1 and 1.8 +/- 0.1, respectively). Autoradiograms of sodium dodecyl sulfate-polyacrylamide electrophoretic gels indicate that 32P incorporation into phospholamban (Mr 27.3K) or its presumed monomeric subunit (Mr 5.5K) was markedly reduced when trypsin-treated microsomes were incubated in the presence of cyclic AMP dependent protein kinase and [gamma-32P]ATP compared to control microsomes incubated similarly but pretreated with trypsin inhibitor inactivated trypsin. The activation of calcium uptake by increasing concentrations of trypsin was paralleled by the reduction of phosphorylation of phospholamban. Trypsin treatment of microsomes previously thiophosphorylated in the presence of cyclic AMP dependent protein kinase and [gamma-35S]thio-ATP did not result in a loss of 35S label from phospholamban, which suggests that phosphorylation of phospholamban protects against trypsin attack.(ABSTRACT TRUNCATED AT 250 WORDS)     

Separation of the epithelial and mesenchymal components of the newt limb regenerate with salt.

After incubation of isolated forelimb regenerates of Notophthalmus (Triturus) viridescens at all developmental stages for 60 minutes at 37 degrees C in a salt medium containing 111 mM sodium chloride, 5.6 mM potassium chloride and 100 mM sodium phosphate buffer at pH 7.5, the wound epithelium of each regenerate was removed intact from its underlying mesenchymal component. The suggestion is made that the salt medium is an effective epithelial-mesenchymal separating agent due to a combination of its hypertonicity, high ionic strength and the fact that the medium precipitates calcium as calcium phosphate. Attempts to dissect away the epithelium from the mesenchyme after incubation of isolated regenerates in sodium phosphate containing 1% or 3% Difco 1:250 trypsin, 10 mM EDTA or 150 units collagenase/ml medium were unsuccessful. Epidermis of adult newt forelimb skin was removed only after extended incubation of the forelimbs in the salt medium for three hours at 37 degrees C or after freezing isolated forelimbs in buffer and subsequent thawing.     

Calcium Calmodulin Dependent Phosphorylation of Proteins: Fetal Cortical Neurons and Adult Cortex

In the present investigation, changes in the calcium calmodulin-dependent phosphorylation of proteins have been examined in murine fetal cortical neurons and adult cortex. An approximately 80-kD protein in the fetal neurons was not phosphorylated/dephosphorylated in a calmodulin-dependent manner. However, this protein was phosphorylated by PMA both in the presence and absence of calcium. These data suggest that calmodulin inhibits the phosphorylation of a approximately 80-kD protein by inhibiting PKC in murine fetal cortical neurons but not in the adult cortex. More importantly, we demonstrate that the calmodulin-mediated inhibition of phosphorylation was restored by preincubating the cortical neurons with KN-62, a CaM kinase inhibitor.     

Developmental and Regional Studies of the Metabolism of Inositol 1,4,5-Trisphosphate in Rat Brain

Coupling of CNS receptors to phosphoinositide turnover has previously been found to vary with both age and brain region. To determine whether the metabolism of the second messenger inositol 1,4,5-trisphosphate also displays such variations, activities of inositol 1,4,5-trisphosphate 5'-phosphatase and 3'-kinase were measured in developing rat cerebral cortex and adult rat brain regions. The 5'-phosphatase activity was relatively high at birth (approximately 50% of adult values) and increased to adult levels by 2 weeks postnatal. In contrast, the 3'-kinase activity was low at birth and reached approximately 50% of adult levels by 2 weeks postnatal. In the adult rat, activities of the 3'-kinase were comparable in the cerebral cortex, hippocampus, and cerebellum, whereas much lower activities were found in hypothalamus and pons/medulla. The 5'-phosphatase activities were similar in cerebral cortex, hippocampus, hypothalamus, and pons/medulla, whereas 5- to 10-fold higher activity was present in the cerebellum. The cerebellum is estimated to contain 50-60% of the total inositol 1,4,5-trisphosphate 5'-phosphatase activity present in whole adult rat brain. The localization of the enriched 5'-phosphatase activity within the cerebellum was examined. Application of a histochemical lead-trapping technique for phosphatase indicated a concentration of inositol 1,4,5-trisphosphate 5'-phosphatase activity in the cerebellar molecular layer. Further support for this conclusion was obtained from studies of Purkinje cell-deficient mutant mice, in which a marked decrement of cerebellar 5'-phosphatase was observed. These results suggest that the metabolic fate of inositol 1,4,5-trisphosphate depends on both brain region and stage of development.     

Involvement of Nerve Growth Factor and Its Receptor in the Regulation of the Cholinergic Function in Aged Rats

The role of nerve growth factor (NGF) and its receptor (NGFR) in the regulation of cholinergic activity has been studied during the aging process. NGFRs were quantified in cortical membranes using a radioactive binding assay. NGF levels and choline acetyltransferase (ChAT) activity were determined in cortex, hippocampus, neostriatum, and septum. These assays were performed in both adult (6-month-old) and aged (36-month-old) rats. High- and low-affinity 125I-NGF binding sites were present in cortex of adult and aged rats. Furthermore, we observed a decrease in number and affinity of both NGFRs in aged rats. ChAT activity in these rats was lower (approximately 30%) than in adult rats in all the brain regions examined. NGF levels were not modified in cortex and hippocampus and were decreased in neostriatum (55%) and septum (35%). In conclusion, our results suggest that, during the aging process, the cholinergic impairment is related to a decrease in NGF levels in neostriatum but not in cortex and hippocampus. The reduction in level of NGF protein in septum could be due to a decrease in number of high-affinity 125I-NGF binding sites.     

Synthesis and regulation of lipoprotein lipase in the hippocampus

Lipoprotein lipase (LPL) expression was determined in adult rat hippocampus and compared to enzyme expression in other brain regions. Hippocampus LPL mRNA levels were at least 2.5-fold higher than those detected in the cerebral cortex, cerebellum, and remaining brain regions. Enzyme mass and activity levels in the hippocampus were also increased to a similar degree. De novo synthesis of LPL in the hippocampus was confirmed by [35S]methionine-labeling of the tissue and identification of a 57 kDa protein obtained by immunoprecipitation. Addition of an excess amount of bovine LPL completely prevented the immunoprecipitation of this protein. The effect of nutritional modulations on brain LPL activity was determined after a 12-h fast. While no significant changes were observed in other regions of the brain, hippocampus LPL activity in fasted rats increased by 60% compared to the fed control group. Simultaneously, fasting reduced adipose LPL activity by 60%. Intraperitoneal injection of ACTH over a 5-day period had no effect on hippocampus LPL activity, while adipose LPL levels increased 2.3-fold and heart LPL levels decreased 1.4-fold. We conclude that LPL is synthesized, active and regulated in a tissue-specific manner in the adult rat hippocampus.     

Membrane junctions in Xenopus eggs: their distribution suggests a role in calcium regulation

We have observed the presence of membrane junctions formed between the plasma membrane and cortical endoplasmic reticulum of mature, unactivated eggs of xenopus laevis. The parallel, paired membranes of the junction are separated by a 10-mn gap within which electron-dense material is present. This material occurs in patches with an average center-to-center distance of approximately 30 nm. These junctions are rare in immature (but fully grown) oocytes (approximately 2 percent of the plasma membrane is associated with junctions) and increase dramatically during progesterone-induced maturation. Junctions in the mature, unactivated egg are two to three times more abundant in the animal hemisphere (25-30 percent of the plasma membrane associated with junction) as compared with the vegetal hemisphere (10-15 percent). Junction density decreases rapidly to values characteristic of immature oocytes in response to egg activation. The plasma membrane-ER junctions of xenopus eggs are strikingly similar in structure to membrane junctions in muscle cells thought to be essential in the triggering of intracellular calcium release from the sarcoplasmic reticulum. In addition, the junctions’ distinctive, animal-vegetal polarity of distribution, their dramatic appearance during maturation, and their disapperance during activation are correlated with previously documented patterns of calcium-mediated events in anuran eggs. We discuss several lines of evidence supporting the hypothesis that these junctions in xenopus eggs are sites that transduce extracellular events into intracellular calcium release during fertilization and activation of development.     

Alterations of fibroblast interactions with fibronectin and laminin during chick embryo development

Eight day (8-d CEF) and 16 day old chick embryo fibroblasts (16-d CEF) obtained after a mild trypsin treatment (50 micrograms/ml in Ca2+ and Mg2+-free PBS, plus 10 mM EDTA) for 10 min at 37 degrees C present the same number of fibronectin (FN) binding sites at their surface (approximately 550,000 sites per cell) with a Kd approximately equal to 1.40 microM in both cases. Furthermore, FN interacted with high molecular weight plasma membrane proteins (150,000 and 125,000) insensitive to trypsin treatment. Both 8-d and 16-d CEF adhered and spread to the same extent on a fibronectin coated substratum (80% of the CEF adhered in 60 min). In contrast, 8-d and 16-d CEF behaved differently towards laminin (LM). 8-d CEF exhibited approximately 5500 binding sites per cell with a Kd of 1.5 nM (Codogno P., Doyennette, M.-A. and Aubery M., 1987, Experimental Cell Research, 169, 478-489.) and were highly sensitive to trypsin treatment, whereas 16-d CEF do not express cell surface binding sites for laminin. Differences were also observed in the adhesive capacities of 8-d and 16-d CEF on LM substrata: 8-d CEF adhered and spread on LM in a very specific manner (60% of the cells adhere in 60 min) and 16-d CEF did not adhere to LM even after long periods of incubation exceeding 360 min.