Activators of protein kinase C increase the phosphorylation of the synapsins at sites phosphorylated by cAMP-dependent and Ca2+/calmodulin-dependent protein kinase in the rat hippocampal slice.

Previous studies have shown that activators of protein kinase C (C kinase) produce synaptic potentiation in the hippocampus. For example, the C kinase activator phorbol dibutyrate has been shown to increase transmitter release in the hippocampus. In addition, a role for C kinase in long-term potentiation has been proposed. A common assumption in such studies has been that substrates for C kinase were responsible for producing these forms of synaptic potentiation. However, we have recently shown that phorbol dibutyrate increased the phosphorylated of synapsin II (formerly protein III, Browning et al., 1987) in chromaffin cells (Haycock et al., 1988). Synapsin II is a synaptic vesicle-associated phosphoprotein that is a very poor substrate for C kinase but an excellent substrate for cAMP-dependent and Ca2+/calmodulin-dependent protein kinase. We felt, therefore, that activation of C kinase might lead to activation of a kinase cascade. Thus effects of C kinase activation might be produced via the phosphorylation of proteins that are not substrates for C kinase. In this report we test the hypothesis that activators of C kinase increase the phosphorylation of synapsin II and an homologous protein synapsin I. Our data indicate that PdBu produced dose-dependent increases in the phosphorylation of synapsin I and synapsin II. We also performed phospho-site analysis of synapsin I using limited proteolysis. These studies indicated that PdBu increased the phosphorylation of multiple sites on synapsin I. These sites have previously been shown to be phosphorylated by both cAMP-dependent protein kinase and the multifunctional Ca2+/calmodulin-dependent protein kinase II.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+/calmodulin-dependent protein kinase II immunoreactivity in Lewy bodies

Summary Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) is one of the predominant protein kinases in the brain. We found that CaM kinase II immunoreactivity was concentrated in the peripheral halos of Lewy bodies (LBs) in Parkinson's disease and Lewy body-like hyaline inclusions (LBHIs) in amyotrophic lateral sclerosis. An immunoelectron microscopic examination of LBs revealed that the filaments at the periphery of LBs were decorated with immunopositive deposits. Since CaM kinase II has a broad substrate specificity and can phosphorylate neurofilaments and other cytoskeletal proteins, it may play some role in the formation of LBs and LBHIs through the aberrant phosphorylation of the cytoskeletal elements in these inclusions.Supported by a Grant-in-Aid for Scientific Research on Priority Areas, No. 02240104, from the Ministry of Education, Science and Culture, Japan     

Ca2+/calmodulin-dependent protein kinase II in the spinal cord contributes to neuropathic pain in a rat model of mononeuropathy

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is known to subserve activity-dependent neuronal plasticity in the central nervous system. To examine in vivo the implication of spinal CaMKII activity in the generation and development of neuropathic pain after peripheral nerve injury, we used an animal model of mononeuropathy, the chronic constriction injury (CCI) model, in the rat. We found that, 3 days after CCI, the total CaMKII (tCaMKII) immunoreactivity increased in the superficial laminae of the spinal cord and this increase continued for up to 14 days. The immunoreactivity of phosphorylated CaMKII showed an increase from 1 day after CCI, which preceded the up-regulation of tCaMKII. A non-selective N-methyl-d-aspartate receptor antagonist, MK801, significantly attenuated the increase of tCaMKII and phosphorylated CaMKII. Moreover, intrathecal administration of an inhibitor of CaMKII, KN93, before the CCI surgery attenuated the development of thermal hyperalgesia and mechanical allodynia. In addition, KN93 significantly reduced the nociceptive behavior in phase II of the formalin test. These findings demonstrate that the activity of CaMKII in spinal neurons is elevated after peripheral nerve injury and may be involved in central sensitization. The alteration of CaMKII is considered to be a neuroplastic change that occurs in spinal neurons that contributes to neuropathic pain, suggesting the potential for the development of novel therapeutics for neuropathic pain that target CaMKII.     

Involvement of a Ca/calmodulin-dependent protein kinase II-associated mechanism in the induction of an outward potassium current by quisqualate

The inhibitory action of a glutamate agonist, quisqualate, in association with the intracellular signal transduction, was electrophysiologically examined in identifiedEuhadra neurons. Quisqualate dose-dependently induced a slow outward current (Quis current) which was blocked by tetraethylammonium. This current was suppressed by intracellular injection of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), and was enhanced by a CaMKII inhibitor, KN-62. However, no significant changes in the Quis current were observed when the catalytic subunit of protein kinase A (PKA) or the protein kinase C (PKC) fragment (530–558) was intracellularly applied; or using a PKA inhibitor, H-8 or a PKC inhibitor, staurosporine. These results suggest a novel mechanism linked to CaMKII, by which quisqualate induces an outward potassium current.     

Enhanced activation of Ca2+/calmodulin-dependent protein kinase II upon downregulation of cyclin-dependent kinase 5-p35

Cyclin-dependent kinase 5 (Cdk5)-p35 is downregulated in cultured neurons by N-methyl-D-aspartate (NMDA) via the proteasomal degradation of p35. However, it is not known where in neurons this downregulation occurs or the physiologic meaning of the reaction. We show the enrichment of Cdk5 and p35 in the postsynaptic density and the NMDA-induced degradation of postsynaptic p35 using brain slices and cultured neurons. To evaluate the role of this downregulation, we examined the relationship between Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activation and Cdk5 downregulation, as events downstream from NMDA stimulation. Glutamate or NMDA stimulation induced CaMKII autophosphorylation over a time course that mirrored the time course of p35 degradation. To simulate the downregulation of postsynaptic Cdk5 in invitro experiments, we used the Cdk5 inhibitor roscovitine. The inhibition of Cdk5 activity by roscovitine enhanced CaMKII autophosphorylation and activation in cultured neurons, and in an isolated postsynaptic-density-enriched fraction. These results suggest that Cdk5 activity suppresses CaMKII activation, and that the downregulation of Cdk5 activity after treatment withNMDA facilitates CaMKII activation, leading to the easier induction of long-term potentiation.     

Immunological evidence that the beta isoform of Ca2+/calmodulin-dependent protein kinase IV is a cerebellar granule cell-specific product of the CaM kinase IV gene.

Ca2+/calmodulin-dependent protein kinase IV (CaM kinase IV) exists as two monomeric isoforms, alpha and beta. In this study, we raised an antibody against the beta isoform and provided immunohistochemical evidence for specific expression of the beta isoform in cerebellar granule cells as a single gene-derived translational product distinct from the alpha isoform. Immunohistochemical examination showed that the beta-immunoreactivity was confined to the nuclei of the cerebellar granule cells, in contrast to the more widespread immunoreactivity for the alpha isoform in both nuclei and cytoplasm of the cerebellar granule cells and many other neurons with dominant nuclear localization. In developing cerebella, the beta-immunoreactivity gradually appeared in the internal granule cells during the postnatal 2nd and 3rd weeks, while the alpha-immunoreactivity had already appeared in the internal granule cells in the 1st postnatal week. Unlike the alpha isoform, beta-immunoreactivity was not detected in the Purkinje cells at any developmental stages. The differential expression of the alpha and beta isoforms suggests that each isoform may be involved in different cerebellar functions.     

Axonal localization of delta Ca/calmodulin-dependent protein kinase II in developing P19 neurons

Ca²⁺/calmodulin-dependent protein kinase, type II (CaMK-II) is an enzyme encoded by four genes (, β, γ and δ) and traditionally associated with synaptic function in the adult central nervous system, but also believed to play a role during neuronal development. P19 mouse embryonic cells are a model system for neurogenesis and primarily express isozymes of δ CaMK-II. It is not yet known whether or where δ CaMK-II is expressed in P19 neurons. Using an antibody specific for the δ CaMK-II C-terminal tail, we detected a 20-fold increase in levels of δ CaMK-II along axons after 8 days of development. This coincides with increased mRNA and protein levels of δ_C CaMK-II, which contains the alternative tail. This follows the initial stages of neurite outgrowth and β₃ tubulin expression, which occur after 4 days. δ CaMK-II co-localizes with the axonal protein GAP-43, but not the dendritic microtubule-associated protein MAP-2, a known substrate of CaMK-II. Like δ CaMK-II, GAP-43 shows increased expression after 8 days. These findings demonstrate developmental regulation of the alternative C-terminal δ CaMK-II exon and implicate endogenous δ CaMK-II in axonal development in embryonic cells.     

Constitutive downregulation protein kinase C epsilon in hSOD1G93A astrocytes influences mGluR5 signaling and the regulation of glutamate uptake

Accumulating evidence indicates that motor neuron degeneration in amyotrophic lateral sclerosis (ALS) is a non‐cell‐autonomous process and that impaired glutamate clearance by astrocytes, leading to excitotoxicity, could participate in progression of the disease. In astrocytes derived from an animal model of ALS (hSOD1^G93A rats), activation of type 5 metabotropic glutamate receptor (mGluR5) fails to increase glutamate uptake, impeding a putative dynamic neuroprotective mechanism involving astrocytes. Using astrocyte cultures from hSOD1^G93A rats, we have demonstrated that the typical Ca²⁺ oscillations associated with mGluR5 activation were reduced, and that the majority of cells responded with a sustained elevation of intracellular Ca²⁺ concentration. Since the expression of protein kinase C epsilon isoform (PKC?) has been found to be considerably reduced in astrocytes from hSOD1^G93A rats, the consequences of manipulating its activity and expression on mGluR5 signaling and on the regulation of glutamate uptake have been examined. Increasing PKC? expression was found to restore Ca²⁺ oscillations induced by mGluR5 activation in hSOD1^G93A‐expressing astrocytes. This was also associated with an increase in glutamate uptake capacity in response to mGluR5 activation. Conversely, reducing PKC? expression in astrocytes from wild‐type animals with specific PKC?‐shRNAs was found to alter the mGluR5 associated oscillatory signaling profile, and consistently reduced the regulation of the glutamate uptake‐mediated by mGluR5 activation. These results suggest that PKC? is required to generate Ca²⁺ oscillations following mGluR5 activation, which support the regulation of astrocytic glutamate uptake. Reduced expression of astrocytic PKC? could impair this neuroprotective process and participate in the progression of ALS.     

Light and electron microscopic distribution of the AMPA receptor subunit,GluR2, in the spinal cord of control and G86R mutant superoxide dismutase transgenic mice

Excitotoxicity has been hypothesized to contribute to amyotrophic lateral sclerosis (ALS) neurodegeneration. The similar pattern of vulnerability in the spinal cord of mutant superoxide dismutase (SOD-1) transgenic mice and mice treated with excitotoxins supports a role for excitotoxicity in the mechanism of degeneration. The distribution of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) class of glutamate receptors (GluRs) with different calcium permeabilities has been proposed as an explanation for this differential vulnerability. GluR2 appears to be the dominant determinant of calcium permeability for AMPA receptors; thus, it is critical for their contribution to excitotoxic mechanisms. In this study, we investigate the distribution of GluR2 immunoreactivity in the spinal cord of control and SOD-1 transgenic mice. GluR2 immunoreactivity is present equally within vulnerable neurons (i.e., motor neurons and calretinin-immunoreactive neurons) as well as nonvulnerable neurons (i.e., calbindin-immunoreactive neurons and dorsal horn neurons). In addition, postembedding immunoelectron microscopy reveals that GluR2 is present in synapses of dorsal and ventral horn neurons and that the percentage of labeled synapses and numbers of immunogold particles per synapse do not vary between these spinal cord regions. Comparing control mice with SOD-1 transgenic mice, at both the light and the electron microscopic levels, the distribution and intensity of GluR2-immunoreactivity do not appear to be altered. These results suggest that the cellular and synaptic distribution of GluR2 is not a determinant of the selective vulnerability observed in SOD-1 transgenic mice or in ALS patients. J. Comp. Neurol. 395:523–534, 1998. © 1998 Wiley-Liss, Inc.     

Ca2+/calmodulin‐dependent protein kinase II in spinal dorsal horn contributes to the pain hypersensitivity induced by γ‐aminobutyric acid type a receptor inhibition

The fast inhibitory synaptic transmission mediated by the γ‐aminobutyric acid type A receptor (GABA_AR) within spinal dorsal horn exerts a gating control over the synaptic conveyance of nociceptive information from the periphery to higher brain regions. Although a large body of evidence has demonstrated that the impairment of GABAergic inhibition alone is sufficient to elicit pain hypersensitivity in intact animals, the underlying mechanisms remain to be characterized. The present study shows that Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) is an important signaling protein downstream of reduced GABAergic inhibition. We found that pharmacological removal of inhibition by intrathecal application of the GABA_AR antagonist bicuculline significantly enhanced the autophosphorylation of CaMKII at Thr286 in spinal dorsal horn of mice. In addition to increased CaMKII activity, bicuculline also promoted CaMKII interaction with N‐methyl‐D‐aspartate (NMDA)‐subtype glutamate receptors and induced the translocation of CaMKII from cytosolic compartments to the synaptosomal membrane fraction. Immunoblotting analysis revealed that the phosphorylation levels of NMDA receptor NR2B subunit at Ser1303 and of AMPA‐subtype glutamate receptor GluR1 subunit at Ser831, two important CaMKII phosphorylation sites, were substantially enhanced after bicuculline application. Behavioral tests illustrated that intrathecal administration of the CaMKII inhibitor KN‐93, NMDA receptor antagonist D‐APV, or AMPA receptor antagonist GYKI 52466 effectively ameliorated the mechanical allodynia evoked by bicuculline. These data thus indicate that CaMKII signaling is critical for the reduced inhibition to evoke spinal sensitization. © 2013 Wiley Periodicals, Inc.     

Activation of mitogen-activated protein kinase by muscarinic receptors in astroglial cells: role in DNA synthesis and effect of ethanol.

Mitogen-activated protein kinase (MAPK) can be phosphorylated by mitogens binding to G-protein-coupled receptors and is considered a major pathway involved in cell proliferation. In this study, we report on the activation of MAPK by muscarinic acetylcholine receptors in astroglial cells, namely the 1321N1 human astrocytoma cell line, primary rat cortical astrocytes, and fetal human astrocytes. Carbachol caused a rapid and transient phorphorylation of MAPK (ERK1/2) in all cell types, with an increase in MAPK activity, without changing the levels of MAPK proteins. Human astrocytoma cells were used to characterize the effect of carbachol on MAPK. Experiments with M2- and M3-receptor subtype-selective antagonists, and with pertussis toxin, indicated that the M3 subtype is responsible for activating MAPK in glial cells. Pretreatment of cells with the protein kinase C (PKC) inhibitor bisindolylmaleimide I, or downregulation of PKC by 24-h treatment with the phorbol ester TPA inhibited carbachol-induced MAPK activation. Additional experiments with PKC alpha- or PKC epsilon-specific compounds indicated that the epsilon isozyme of PKC is primarily involved in MAPK activation by carbachol. Chelation of calcium also inhibited MAPK activation by carbachol. Two MEK (MAPK kinase) inhibitors inhibited carbachol-induced DNA synthesis but only at concentrations that exceeded those sufficient to block carbachol-induced MAPK activation. Ethanol (< or =200 mM) had no effect on MAPK when present alone and did not affect carbachol-induced MAPK activation under various experimental conditions, although it inhibits carbachol-induced DNA synthesis at low concentrations (10-100 mM). These results suggest that activation of MAPK by carbachol may be necessary but not sufficient for its mitogenic effect in astroglial cells, and that does not represent a target for ethanol-induced inhibition of DNA synthesis elicited by muscarinic receptors.     

Current application of neurofilaments in amyotrophic lateral sclerosis and future perspectives

Motor neuron disease includes a heterogeneous group of relentless progressive neurological disorders defined and characterized by the degeneration of motor neurons.Amyotrophic lateral sclerosis is the most common and aggressive form of motor neuron disease with no effective treatment so far.Unfortunately,diagnostic and prognostic biomarkers are lacking in clinical practice.Neurofilaments are fundamental structural components of the axons and neurofilament light chain and phosphorylated neurofilament heavy chain can be measured in both cerebrospinal fluid and serum.Neurofilament light chain and phosphorylated neurofilament heavy chain levels are elevated in amyotrophic lateral sclerosis,reflecting the extensive damage of motor neurons and axons.Hence,neurofilaments are now increasingly recognized as the most promising candidate biomarker in amyotrophic lateral sclerosis.The potential usefulness of neurofilaments regards various aspects,including diagnosis,prognosis,patient stratification in clinical trials and evaluation of treatment response.In this review paper,we review the body of literature about neurofilaments measurement in amyotrophic lateral sclerosis.We also discuss the open issues concerning the use of neurofilaments clinical practice,as no overall guideline exists to date;finally,we address the most recent evidence and future perspectives.     

Ca/calmodulin-dependent protein kinase II in postsynaptic densities after reversible cerebral ischemia in rats

Compartmentalization of protein kinases and association of the enzyme with strategic cellular substrates may be important for regulating signal transduction in neurons. Cerebral ischemia produced by transient 20 min occlusion of common carotid and vertebral arteries in rats caused a dramatic (3-fold) increase in Ca²⁺/Calmodulin-dependent protein kinase II (CaM-KII) in the fraction enriched in postsynaptic density (PSDf), the compartment of the neuron that is involved in signal transduction. This change in compartmentalization was not reversible for up to 24 h after termination of the occlusion and was followed by reduction of CaM-KII to 50% of control content one week after the insult. The observed changes in CaM-KII content did not represent general protein redistribution in PSDf after ischemia since there were no parallel changes in PSDf actin concentration. The redistribution of CaM-KII coincided with gradual (up to 80%) reduction of its activity in PSDf as tested using specific peptide substrate and endogenous CaM-KII substrates. This work provides evidence that ischemia disturbs CaM-KII distribution and activity in PSDf and this may lead to long lasting disruption of signal transduction at the synaptic level.     

Developmental changes of protein substrates of Ca / calmodulin-dependent protein kinase II in teh rat forebrain

We previously reported that the level of Ca²⁺ / calmodulin-dependent protein kinase II (CaM kinase II) α and β proteins increases with postnatal age. In the present study, we investigated the development changes in whole protein substrates of CaM kinase II as compared with those of cAMP-dependent protein kinase (A-kinase) in the rat forebrain. Protein substrates were phosphorylated with [γ-³³P]ATP, and analysed by two-dimensional gel electrophoresis. More than 50 substrates for CaM kinase II were found in the soluble and particulate fractions The phosphorylation level of more than 15 substrates increased in the particulate fraction during development. Similarly, that of more than 3 substrates increased in the soluble fraction. Some substrates for A-kinase also increased during development, although some decresed. These findings suggest that the expression of some substrates is regulated during development and that the phosphorylation reaction involves the regulation of neuronal development.     

A Nitric Oxide Synthase Activity Selectively Stimulated by NMDA Receptors Depends on Protein Kinase C Activation in Mouse Striatal Neurons

In mouse striatal neurons in primary culture, the maximal increase in intracellular cyclic guanosine monophosphate level evoked by N -methyl- d -aspartic acid (NMDA) receptor activation was twice that induced by kainate, KCI and ionomycin. Quisqualate was almost inactive. All responses were mediated by nitric oxide (NO) production since they were blocked by haemoglobin (a NO scavenger) and by _L- N^G -monomethylarginine and _L- N^G nitroarginine, the effects of both arginine analogues being reversed by an excess of L-arginine. Several results indicate that NMDA receptors stimulate a specific NO synthase activity. This specifically NMDA-activated NO synthase was blocked by nanomolar concentrations of _L- N^G nitroarginine, whereas the responses evoked by other agents, including kainate, KCI and ionomycin, were only blocked by micromolar concentrations of this NO synthase inhibitor. The NMDA response could not be totally reproduced by an increase in cytosolic calcium (Ca²⁺) alone. In contrast, in the presence of staurosporine, an inhibitor of protein kinases C (PKC), as well as after desensitization of PKC induced by long-term treatment with the phorbol ester, phorbol-12, 13-dibutyrate, NMDA-stimulated NO production was selectively reduced, reaching the level evoked by kainate or Ca²⁺ increase. In conclusion, our results suggest that in striatal neurons, NMDA selectively stimulates a NO synthase activity which is inhibited by low concentrations of _L- N^G nitroarginine, through a mechanism involving PKC.     

(-)-Epigallocatechin gallate, the most active polyphenolic catechin in green tea, presynaptically facilitates Ca2+-dependent glutamate release via activation of protein kinase C in rat cerebral cortex

(-)-Epigallocatechin gallate (EGCG), the main polyphenolic constituent of green tea, has been reported to improve cognitive decline. Considering the central glutamatergic activity is crucial to cognitive function, the objective of this study was to investigate the effect of EGCG on the release of endogenous glutamate using nerve terminals purified from rat cerebral cortex. Results showed that the release of glutamate evoked by 4-aminopyridine (4AP) was facilitated by EGCG in a concentration-dependent manner, and this effect resulted from an enhancement of vesicular exocytosis and not from an increase in Ca2+-independent efflux via glutamate transporter. Examination of the effect of EGCG on cytoplasmic free Ca2+ concentration ([Ca2+]c) revealed that the facilitation of glutamate release could be attributed to an increase in Ca2+ influx through N- and P/Q-type voltage-dependent Ca2+ channels. Consistent with this, the EGCG-mediated facilitation of 4AP-evoked glutamate release was significantly prevented in synaptosomes pretreated with a combination of the N- and P/Q-type Ca2+ channel blockers. Additionally, inhibition of protein kinase C (PKC) by treatment with Ro318220 significantly reduced the facilitatory effect of EGCG on 4AP-evoked glutamate release and phosphorylation of PKC or its presynaptic target myristoylated alanine-rich C kinase substrate (MARCKS). These results suggest that EGCG effects a facilitation of glutamate release from glutamatergic terminals by positively modulating N- and P/Q-type Ca2+ channel activation through a signaling cascade involving PKC. In this EGCG/PKC signaling cascade facilitating glutamate release, the regulation of cytoskeleton dynamics was also indicated to be involved by disruption of cytoskeleton organization with cytochalasin D occluded the EGCG-mediated facilitation of 4AP-evoked glutamate release.