The relationship between neurotrophic factors and CaMKII in the death and survival of retinal ganglion cells.

The scientific discourse relating to the causes and treatments for glaucoma are becoming reflective of the need to protect and preserve retinal neurons from degenerative changes, which result from the injurious environment associated with this disease. Knowledge, in particular, of the signal transduction pathways which affect death and survival of the retinal ganglion cells is critical to this discourse and to the development of a suitable neurotherapeutic strategy for this disease. The goal of this chapter is to review what is known of the chief suspects involved in initiating the cell death/survival pathways in these cells, and what still remains to be uncovered. The least controversial aspect of the subject relates to the potential role of neurotrophic factors in the protection of the retinal ganglion cells. On the other hand, the postulated triggers for signaling cell death in glaucoma remain controversial. Certainly, the restricted flow of neurotrophic factors has been cited as one possible trigger. However, the connections between glaucoma and other factors present in the retina, such as glutamate, long held to be a prospective culprit in retinal ganglion cell death are still being questioned. Whatever the outcome of this particular debate, it is clear that the downstream intersections between the cell death and survival pathways should provide important foci for future studies whose goal is to protect retinal neurons, situated as they are, in the stressful environment of a cell destroying disease. The evidence for CaMKII being one of these intersecting points is discussed.

Molecular cloning and sequence analyses of calcium/calmodulin-dependent protein kinase II from fetal and adult human brain. Sequence analyses of human brain calciuum/calmodulin-dependent protein kinase II.

The aims of this study were to characterize specific mRNAs and the expression pattern for isoforms of calcium/calmodulin-dependent protein kinase II (CaMKII) in the human brain. We cloned and sequenced the CaMKII alpha and beta subunit cDNAs, and used them to study the CaMKII expression in human brain. Four distinct isoforms of CAMKII were isolated. Two of them were characterized as CaMKII alpha and beta subunits. The other two showed similar nucleotide sequences, but one had a 33-bp insertion relative to the alpha subunit, and the other had a 75-bp deletion relative to the beta subunit. These alterations are located within the variable regions. These two isoforms were characterized as CaMKII alphaB and beta(e). Northern blot analysis showed that a 4.4-kb messenger RNA for the alpha isoform and a 3.9-kb messenger RNA for the beta isoform were expressed in both human fetal and adult brain to different degrees. The results indicate that CaMKII expression is developmentally regulated. The CaMKII isoform expression was confirmed in human fetal and adult brain using RT-PCR with specific primers, which flanked the CaMKII variable regions. The CaMKII alpha, alphaB, beta, beta' and beta(e) isoforms were characterized in both human fetal and adult brain.

Visual-mediated regulation of retinal CaMKII and its GluR1 substrate is age-dependent.

Previous studies have shown that multifunctional calcium/calmodulin-dependent protein kinase II (CaMKII) and one of its substrates, the glutamate receptor, are key players in experience-driven synaptic plasticity in several areas of the central nervous system (CNS). To determine if CaMKII and the glutamate receptor are regulated by visual activity in the retina, we compared dark-reared (DR; 1 week) rats with control rats raised in a diurnal light-dark cycle (LD), at the following ages: postnatal day 12 (P12d), 2-month (2m) and 6-month (6m) old. The mRNA levels of CaMKIIalpha and beta were determined by a competitive reverse transcription polymerase chain reaction (competitive RT-PCR) method. The protein levels of these two subunits were evaluated by immunoblots. The data show that the mRNAs for CaMKIIalpha and beta were increased about 8-fold and 10-fold, respectively, in the retinae of DR P12d rats. As for the proteins, 2- and 2.6-fold elevations for CaMKIIalpha and beta, respectively, were evident. The GluR1 subunit of the AMPAR (AMPAR-GluR1) was also evaluated in antibody-treated blots and found to be increased about 2-fold after 1 week of dark rearing in the retinae of P12d rats. This type of experience-driven molecular change was age-dependent, showing less increase in 2m old rats and not present in 6m old rats. Returning DR 2m old rats to the LD environment for 1 week was sufficient to restore the dark-induced changes to the levels of the age-matched LD controls. Based on the data, a theoretical model for activity-dependent modulation of the developing retinal synapses is proposed.

Characterization of apoptosis-genes associated with NMDA mediated cell death in the adult rat retina.

Calcium/calmodulin-dependent protein kinase II containing a nuclear localizing signal (CaMKII-alphaB) is altered in retinal neurons exposed to N-methyl-D-aspartate (NMDA). AIP (myristoylated autocamtide-2-related inhibitory peptide), a specific inhibitor of CaMKII provides neuroprotection against NMDA-mediated neurotoxicity. In this study, gene-arrays were used to investigate which apoptosis-associated genes are altered after exposure to NMDA. The data indicate an increased expression (2-7-fold) of five such genes encoding proteins that could be involved in NMDA induced cell death. The up-regulated genes are: FasL; GADD45; GADD153; Nur77 and TNF-R1. Treatment with AIP blocked their altered expression. The results suggest that multiples genes are involved in NMDA-induced excitotoxicity and that AIP, a specific inhibitor for CaMKII, regulates the expression of these apoptosis-associated genes in the retina.

Expression of ionotropic glutamate receptors in the retina of the rdta transgenic mouse.

BACKGROUND: The expression of retinal CaMKII is up-regulated in the retina of the rdta mouse in which rod photoreceptors are genetically ablated. As ionotropic glutamate receptors are known substrates of CAMKII, this study set out to determine if the protein levels of ionotropic glutamate receptors in the rdta mouse retina are also affected. RESULTS: The NMDA receptor subunits (NR1, NR2A/B) and the GluR1; AMPA receptor subunit (GluR1) were examined in immunolabeled western blots. The results demonstrate that the amounts of NR1 and NR2A/B receptor subunits are significantly increased in crude synaptic membrane fractions isolated from retinae of the rdta mice when compared to their normal, littermate controls. The GluR1 receptor subunit and its phosphorylation are simultaneously increased in retinae of the rdta mice. CONCLUSIONS: These data indicate that the NMDA receptors and AMPA (GluR1) receptors are altered in the retinae of rdta mice that lack rod photoreceptors. Because the rods are lost at an early stage in development, it is likely that these results are indicative of synaptic reorganization in the retina.

Calcium/calmodulin-dependent protein kinase II containing a nuclear localizing signal is altered in retinal neurons exposed to N-methyl-D-aspartate.

This study investigated N-methyl-D-aspartate (NMDA) mediated cell death and its possible regulation by calcium/calmodulin-dependent protein kinase II (CaMKII) in the adult rat retina. To investigate cell death, the terminal deoxyribonucleotidyltransferase (TdT)-mediated biotin-16-dUTP nick-end labelling (TUNEL) method was used to detect fragmented DNA in fixed tissue sections of rat retina. The TUNEL assay confirmed that apoptosis occurs in the inner nuclear layer (INL) and ganglion cell layer (GCL) following NMDA injection. The level of antibody binding to CaMKII-alpha, the activity of CaMKII, and the mRNA level for the alpha(B) subunit of CaMKII were found to be elevated for short time periods (30 min, 2 h) after a single intravitreal injection of NMDA. In contrast to this, there was a decrease in CaMKII activity and in the CaMKII-alpha(B) mRNA levels at longer time periods (24 h) following injection of NMDA. These effects were specific for the mRNA for the alpha(B) subunit, an alternatively spliced product of the CaMKII-alpha gene, that contains a nuclear localizing signal (NLS) known to target this protein to the nucleus. It is suggested that regulated expression of CaMKII-alpha(B) could be involved in the NMDA-mediated cell death in retinal neurons.

Neuroprotective effect of AIP on N-methyl-D-aspartate-induced cell death in retinal neurons.

Excessive activation of glutamate receptors mediates neuronal death, but the intracellular signaling pathways that mediate this type of neuronal death are only partly understood. Previously, we have demonstrated that calcium/calmodulin-dependent protein kinase II-alpha(B) (CaMKII-alpha(B)) containing a nuclear localizing signal but not CaMKII-alpha is altered in retinal neurons exposed to N-methyl-D-aspartate (NMDA). The present study describes a prospective function of CaMKII-alpha(B) in signal transduction leading to apoptosis. The terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-16-dUTP nick-end labelling (TUNEL) method was used to detect fragmented DNA in fixed tissue sections of rat retina. The TUNEL assay confirmed that cell death occurs in the inner nuclear and ganglion cell layers following injection of 4 mM NMDA. A specific AIP (myristoylated autocamtide-2-related inhibitory peptide) with proven cell permeability inhibits CaMKII activity in vivo. Neuroprotection achieved by 500 microM AIP was complete when administered 2 h before and coincident with the NMDA application. Additionally, 100 microM of AIP protects only partially against the NMDA-induced excitotoxicity. The conformationally active fragment of caspase-3 (17 kDa), known to be involved in neuronal apoptosis was apparent within 30 min and at 2 h postinjection with NMDA. This activation was inhibited by 500 microM AIP when administered 2 h before and coincident with the NMDA application. The results suggest that CaMKII-alpha(B) isoform plays a role in excitotoxicity-induced neuronal apoptosis.

Regulation of calcium/calmodulin-dependent protein kinase II in the adult rat retina is mediated by ionotropic glutamate receptors.

This study is concerned with the transmitter-mediated regulation of the alpha(50 kDa) and beta(60 kDa) subunits of calcium calmodulin dependent protein kinase II (CamKII) in the adult rat retina. The level of antibody binding to the CamKII and the activity of CamKII were found to be increased after intravitreal injection of glutamate. Changes in the levels of the antibody-binding to the subunits of CamKII were observed in different subcellular fractions of the retina with a maximum response observed in crude synaptic membrane fractions. The glutamate mediated increases in CamKII were specific and blocked by 3,5-Dimethyl-1 adamantanamine; 3,5-Dimethylamantadine (Memantine), (+/-) 2-Amino-5-Phosphopentonic (AP-5) and 6-Cyano-7-Nitroquinoxaline-2,3-Dione (CNQX) but not with dl -2-Amino-3-Phosphono-Propionic (AP-3). The results indicate that the retinal neurotransmitter, glutamate, can regulate retinal CamKII activity through ionotropic but not metabotropic glutamate receptors. NMDA-receptors were found to be necessary but insufficient to stimulate CamKII. A model in which cooperative interaction between NMDA and non-NMDA glutamate receptors/ion channels is presented to explain the glutamate stimulated increases in CamKII activity in the retina.

Ependymal and choroidal cells in culture: characterization and functional differentiation.

During the past 10 years, our teams developed long-term primary cultures of ependymal cells derived from ventricular walls of telencephalon and hypothalamus or choroidal cells (modified ependymal cells) derived from plexuses dissected out of fetal or newborn mouse or rat brains. Cultures were established in serum-supplemented or chemically defined media after seeding on serum-, fibronectin-, or collagen-laminin-coated plastic dishes or semipermeable inserts. To identify and characterize cell types growing in our cultures, we used morphological features provided by phase contrast, scanning, and transmission electron microscopy. We used antibodies against intermediate filament proteins (vimentin, glial fibrillary acidic protein, cytokeratin, desmin, neurofilament proteins), actin, myosin, ciliary rootlets, laminin, and fibronectin in single or double immunostaining, and monoclonal antibodies against epitopes of ependymal or endothelial cells, to recognize ventricular wall cell types with immunological criteria. Ciliated or nonciliated ependymal cells in telencephalic cultures, tanycytes and ciliated and nonciliated ependymal cells in hypothalamic cultures always exceeded 75% of the cultured cells under the conditions used. These cells were characterized by their cell shape and epithelial organization, by their apical differentiations observed by scanning and transmission electron microscopy, and by specific markers (e.g., glial fibrillary acidic protein, ciliary rootlet proteins, DARPP 32) detected by immunofluorescence. All these cultured ependymal cell types remarkably resembled in vivo ependymocytes in terms of molecular markers and ultrastructural features. Choroidal cells were also maintained for several weeks in culture, and abundantly expressed markers were detected in both choroidal tissue and culture (Na+-K+-dependent ATPase, DARPP 32, G proteins, ANP receptors). In this review, the culture models we developed (defined in terms of biological material, media, substrates, duration, and subculturing) are also compared with those developed by other investigators during the last 10 years. Focusing on morphological and functional approaches, we have shown that these culture models were suitable to investigate and provide new insights on (1) the gap junctional communication of ependymal, choroidal, and astroglial cells in long-term primary cultures by freeze-fracture or dye transfer of Lucifer Yellow CH after intracellular microinjection; (2) some ionic channels; (3) the hormone receptors to tri-iodothyronine or atrial natriuretic peptides; (4) the regulatory effect of tri-iodothyronine on glutamine synthetase expression; (5) the endocytosis and transcytosis of proteins; and (6) the morphogenetic effects of galactosyl-ceramide. We also discuss new insights provided by recent results reported on in vitro ependymal and choroidal expressions of neuropeptide-processing enzymes and neurosecretory proteins or choroidal expression of transferrin regulated through serotoninergic activation.

Effects of six weeks of chronic ethanol administration on lactic acid accumulation and high energy phosphate levels after experimental brain injury in rats.

The effects of 6 weeks of chronic ethanol administration on the lateral fluid percussion (FP) brain injury-induced regional accumulation of lactate and on the levels of total high-energy phosphates were examined in rats. In both the chronic ethanol diet (ethanol diet) and pair-fed isocaloric sucrose control diet (control diet) groups, tissue concentrations of lactate were elevated in the cortices and hippocampi of both the ipsilateral and contralateral hemispheres at 5 min after brain injury. In both diet groups, concentrations of lactate were elevated only in the injured left cortex and the ipsilateral hippocampus at 20 min after FP brain injury. No significant differences were found in the levels of lactate in the cortices and hippocampi of sham animals and brain-injured animals between the ethanol and control diet groups at 5 min and 20 min after injury. In the ethanol and control diet groups, tissue concentrations of total high-energy phosphates (ATP + PCr) were not affected in the cortices and hippocampi at 5 min and 20 min after lateral FP brain injury. No significant differences were found in the levels of total high-energy phosphates in the cortices and hippocampi of the sham and brain-injured animals between the ethanol and control diet groups at 5 min and 20 min after injury. Histologic studies revealed a similar extent of damage in the cortex and in the CA3 region of the ipsilateral hippocampus in both diet groups at 14 days after lateral FP brain injury. These findings suggest that 6 weeks of chronic ethanol administration does not alter brain injury-induced accumulation of lactate, levels of total high energy phosphates, and extent of morphological damage.

Modifications of ependymal cells membranes by galactocerebrosides in cell culture.

In this paper we have demonstrated that treatment of ependymal cells in culture by galactocerebrosides induced a decrease in plasma membrane fluidity and an increase of EGF binding sites. We have shown in a previous work that galactocerebroside in vitro and in vivo caused an important morphological change in ependymal cells that grew into an astrocytic shape after a five day treatment. We discuss the hypothesis that the first event in morphological effect could be a modification of plasma membrane followed by important changes in molecules distribution.

A study of in vitro and in vivo morphological changes of ependymal cells induced by galactocerebrosides.

Ependymal cells in culture and in vivo were treated with mixture of galactocerebrosides. Galactocerebroside is the major glycolipid of myelin and in demyelinating diseases is found in cerebrospinal fluid. Morphological changes induced by this treatment were examined by microscopy at both optical and ultrastructural levels. In vitro, cilia, microvilli, and junctions between the cells disappeared, processes containing intermediate filaments developed, and the cells lost characteristics typical of ependymal cells and became more astrocyte-like. As shown by vital staining with a fluorescent compound and by nuclear incorporation of bromodeoxyuridine, cells did not proliferate during the period of galactocerebroside treatment and the morphological transformation was restricted to the ependymal cells. In contrast, asialoganglioside-GM1 and sulfatides had no effect on ependymal cell morphology. Some of the in vitro observations could be reproduced in vivo. Junctions between ependymal cells disappeared and intercellular spaces appeared between these cells and the cerebral parenchyma at the basolateral side of the ependymal layer. At the apical side, morphological modifications of junctions and cilia were less evident. As these experimental conditions resemble those existing during demyelination the morphological changes described may account for perturbations of the physiological functions of the ependymal cell.

Monolayer cultures of ependymal cells on porous bottom dishes. A tool for transport studies across the brain cerebrospinal barrier.

We have studied the conditions to obtain ependymal cell cultures on porous bottom dishes and we succeeded to culture in a complete defined medium a continuous layer of primary ependymal cells from newborn rat cerebral hemispheres. This monolayer is composed of non-ciliated (35%) and ciliated ependymal cells (55%), with only a small contamination by astrocytes, oligodendrocytes and fibroblasts (10%). These cells grown on the microporous membrane are oriented and form a layer with an apical side and a basolateral side. We have demonstrated by using Trypan blue that between 14 and 24 days in culture the cells have formed a continuous monolayer. The presence of tight junctions between the cells has been shown by electron microscopy. Using immunocytochemical methods, we have studied the expression of glial fibrillary acidic protein (GFAP) and vimentin in these cultures.