Differential changes in cell morphology,macromolecular composition and membrane protein profiles of neurons and astrocytes in chronic ethanol treated rats

Cellular morphology, macromolecular composition, (DNA, RNA and Protein content) marker enzyme activities for neurons [neuron specific enolase (NSE)] and astrocytes [glutamine synthetase (GS)] and plasma membrane protein profiles in the bulk isolated neurons and astrocytes from control and ethanol treated rats were studied. One month aged Wistar rats were given ethanol as sole drinking fluid for 10 weeks. Scanning electron microscopy revealed a characteristic cell surface smoothening in astrocytes due to ethanol treatment. DNA levels were unaltered, while RNA and Protein contents were decreased in astrocytes and neurons. Further,³H-leucine incorporation into proteins was decreased in neurons and astrocytes derived from ethanol treated rats indicating reduced protein synthesis in neurons and astrocytes. GS activity was affected severely suggesting impairment in astrocytic functions. Plasma membrane protein composition was analyzed by 2-D electrophoresis. The analysis indicated several protein defects in the plasma membranes of neurons and astrocytes, which might be involved in membrane disorder during ethanol challenge.¹²⁵I-Wheat Germ agglutinin binding studies showed three prominent proteins (160, 116 and 97 kDa) in astrocyte membrane fraction suggesting the possible involvement of N-terminal glycoproteins in altered astrocyte morphology during ethanol ingestion. Impairment in the astrocyte cell functions, protein changes in plasma membrane and cellular morphology studies suggest that astrocytes may be more vulnerable than neurons for ethanol effects.     

Adenylyl Cyclases 1 and 8 Initiate a Presynaptic Homeostatic Response to Ethanol Treatment

Background

Although ethanol exerts widespread action in the brain, only recently has progress been made in understanding the specific events occurring at the synapse during ethanol exposure. Mice deficient in the calcium-stimulated adenylyl cyclases, AC1 and AC8 (DKO), demonstrate increased sedation duration and impaired phosphorylation by protein kinase A (PKA) following acute ethanol treatment. While not direct targets for ethanol, we hypothesize that these cyclases initiate a homeostatic presynaptic response by PKA to reactivate neurons from ethanol-mediated inhibition.

Methodology/Principal Findings

Here, we have used phosphoproteomic techniques and identified several presynaptic proteins that are phosphorylated in the brains of wild type mice (WT) after ethanol exposure, including synapsin, a known PKA target. Phosphorylation of synapsins I and II, as well as phosphorylation of non-PKA targets, such as, eukaryotic elongation factor-2 (eEF-2) and dynamin is significantly impaired in the brains of DKO mice. This deficit is primarily driven by AC1, as AC1-deficient, but not AC8-deficient mice also demonstrate significant reductions in phosphorylation of synapsin and eEF-2 in cortical and hippocampal tissues. DKO mice have a reduced pool of functional recycling vesicles and fewer active terminals as measured by FM1-43 uptake compared to WT controls, which may be a contributing factor to the impaired presynaptic response to ethanol treatment.

Conclusions/Significance

These data demonstrate that calcium-stimulated AC-dependent PKA activation in the presynaptic terminal, primarily driven by AC1, is a critical event in the reactivation of neurons following ethanol-induced activity blockade.     

Alterations in pp60c-src accompany differentiation of neurons from rat embryo striatum.

Cultured neurons from rat embryo striatum were found to contain two structurally distinct forms of pp60c-src. The 60-kilodalton (kDa) form appeared similar to pp60c-src from cultured rat fibroblasts or astrocytes. The 61-kDa form was specific to neurons and differed in the NH2-terminal 18 kDa of the molecule. In undifferentiated neurons the predominant phosphorylated species of pp60c-src was the fibroblast form. Upon differentiation, a second phosphorylated form of pp60c-src was detected. This form had two or more additional sites of serine phosphorylation within the NH2-terminal 18-kDa region of the molecule, one of which was Ser-12. The specific protein-tyrosine kinase activity of the total pp60c-src population increased 14-fold, as measured by autophosphorylation, or 7-fold, as measured by phosphorylation of an exogenous substrate, as striatal neurons differentiated. This elevation in protein kinase activity occurred without a detectable decrease in Tyr-527 phosphorylation or increase in Tyr-416 phosphorylation. Our results support the idea that the expression of the neuron-specific form of pp60c-src and the increase in specific protein kinase activity may be important for neuronal differentiation.     

Inhibition of phosphotyrosine phosphatases reveals candidate substrates of the PDGF receptor kinase

In normal fibroblasts stimulated by platelet derived growth factor (PDGF), PDGF receptors are transiently phosphorylated on tyrosine and represent the major phosphotyrosine containing protein. The phosphate of the phosphotyrosine groups turns over rapidly, and extensive evidence indicates a dynamic balance between phosphorylation and dephosphorylation reactions. Thus, the effect of an inhibitor of phosphatases, orthovanadate, on the pattern of the tyrosine phosphorylations induced by PDGF in Swiss 3T3 fibroblasts was investigated. Western blot analysis with antibodies against phosphotyrosine indicated that whereas in unstimulated cells no phosphotyrosine containing proteins were detected, treatment of cells with orthovanadate alone elicited the slow phosphorylation of several proteins including a 170 kDa component that was recognized to be the phosphorylated PDGF receptor. Addition of PDGF to cells shortly pretreated with vanadate highly increased the intensity of the 170 kDa band corresponding to the phosphorylated receptor and caused its stabilization during time. In addition, the phosphorylation on tyrosine of other proteins (molecular mass 116, 80, 73, 60, 50 and 39 kDa) was also induced. Both the receptor and the other tyrosine phosphorylated proteins appeared to be associated with the detergent insoluble matrix.     

Mutualism based on stress: selective synthesis and phosphorylation of a stress protein by an intracellular symbiont.

The effects of a temperature shift-up and various metabolic inhibitors on the protein synthesis of an endosymbiont isolated from the pea aphid were studied. The syntheses of at least three major polypeptides were stimulated transiently immediately after a temperature shift-up, and treatment with ethanol and heavy metals (Cd2+ and As2+). One of these proteins, the 63 kDa heat-shock protein (63-kDa HSP), was immunoprecipitated with antiserum raised against symbionin, which is selectively synthesized by the endosymbiont harbored by the aphid bacteriocytes. The 63 kDa heat-shock protein has a molecular mass of 800 kDa and is more acidic than symbionin. It was also shown that symbionin is subject to phosphorylation in vivo and in vitro after a temperature shift-up. It was thought likely that forms of environmental stress such as heat shock and metabolic inhibitors stimulate the synthesis of a phosphorylated form of symbionin. It was also suggested that the in vitro phosphorylation of symbionin is due to its own catalytic activity. Since symbionin is a homolog of the Escherichia coli groEL protein, a stress protein, it is likely that the endosymbiont suffers stress when harbored by the bacteriocytes and responds in a similar manner to environmental stress when outside these cells.     

Regulation of DNA methylation by ethanol induces tissue plasminogen activator expression in astrocytes

Alcohol exposure affects neuronal plasticity in the adult and developing brain. Astrocytes play a major role in modulating neuronal plasticity and are a target of ethanol. Tissue plasminogen activator (tPA) is involved in modulating neuronal plasticity by degrading the extracellular matrix proteins including fibronectin and laminin and is up‐regulated by ethanol in vivo. In this study we explored the hypothesis that ethanol affects DNA methylation in astrocytes thereby increasing expression and release of tPA. It was found that ethanol increased tPA mRNA levels, an effect mimicked by an inhibitor of DNA methyltransferase (DNMT) activity. Ethanol also increased tPA protein expression and release, and inhibited DNMT activity with a corresponding decrease in DNA methylation levels of the tPA promoter. Furthermore, it was observed that protein levels of DNMT3A, but not DNMT1, were reduced in astrocytes after ethanol exposure. These novel studies show that ethanol inhibits DNA methylation in astrocytes leading to increased tPA expression and release; this effect may be involved in astrocyte‐mediated inhibition of neuronal plasticity by alcohol.

     

Effect of tumour promoting agents on protein phosphorylation in human placenta

• 1.1. The effects of okadaic acid (OA) and phorbol-12-myristate-13-acetate (PMA) on protein phosphorylation were studied in human term placentas.
• 2.2. When samples treated with tumour promoters were compared with untreated samples, the phosphorylation of a 135 kDa protein was significantly decreased; OA also produced a decrease in phosphorylation of a 24 kDa protein.
• 3.3. Both substances produced an alteration in the proportions of bands of masses 170, 65 and 24 kDa, relative to total phosphorylation: PMA treatment also affected the band of mass 135 kDa.
• 4.4. Placental cell extracts were also subjected to Western blotting with a protein kinase C (PKC) antibody, reportedly specific for the α- and β-isoforms.
• 5.5. Two immunoreactive proteins were detected; an 80 kDa band, presumably corresponding to the α- or β-PK.C, and a 64 kDa protein, which could be a degradation production of the 80 kDa protein or it could correspond to another form of the enzyme. The expression of PKC did not change on treatment with PMA.

     

Ethanol alters astrocyte development: A study of critical periods using primary cultures

Using astrocytes obtained from 21-day-old rat fetuses, in primary culture, we have analyzed the effect of prenatal alcohol consumption on DNA and protein synthesis of astrocytes during their development. The variation in sensitivity of astrocytes to ethanol in vitro during the proliferation and maturation periods was also assessed. Control astrocytes showed peaks of DNA and protein synthesis at 8 and 15 days, respectively. A significant decrease in both DNA and protein synthesis was found in astrocytes from fetuses prenatally exposed to ethanol. This effect on DNA synthesis was also observed when control astrocytes were exposed to ethanol (100mM) in vitro during the entire culture period. The effects on astrocytes of short term (48h) exposure to ethanol during the proliferation or differentiation periods on the above mentioned parameters and on the cell cycle as well as the possible recovery from these effects were also evaluated. Decreases in DNA and protein synthesis were found in both periods. However, DNA synthesis and content were more affected in astrocytes exposed to ethanol during the proliferation period. This effect correlates with an accumulation of cells in the G_o/G₁ phase of the cell cycle. On the other hand, when cells exposed to ethanol were cultured in alcohol-free medium to assess recovery, only cells exposed to ethanol during days 4 to 6 still showed DNA ethanol-induced effects at 21 days. In conclusion, our results show that ethanol consumption during gestation induces serious damage to cortical astrocyte progenitor cells. Our results further demonstrate that although astrocytes are more sensitive to the toxic effect of alcohol during the proliferation period, exposure to ethanol during glial maturation also alters their normal development.     

Kinase activity and protein phosphorylation in control and malignant hyperthermic skeletal muscle

1. Native 6% Laemmli gels were used to resolve 7 protein kinase activity bands in control and malignant hyperthermia (MH)-susceptible porcine and human skeletal muscle extracts. 2. MH-susceptible samples were consistently more active than the controls. 3. Following halothane treatment, a 43 kDa component displayed increased phosphorylation by a calcium-calmodulin dependent kinase in MH-susceptible vs control human samples. 4. Increased phosphorylation of additional endogenous protein components of molecular mass 116 and 60 kDa was observed.     

Growth stimulation by serum in Entamoeba histolytica is associated with protein tyrosine dephosphorylation

Very little protein tyrosine phosphorylation was observed in growing (exponential-phase) Entamoeba histolytica cells by immunoblotting and quantitative immunofluorescence. After 1 h of serum deprivation, two proteins (42 and 38 kDa in SDS-PAGE) were tyrosine phosphorylated and two more proteins (96 and 63 kDa) also showed tyrosine phosphorylation when examined after 4 h of serum deprivation. Intense enhancements of anti-phosphotyrosine immunofluorescence levels were observed during this period of serum withdrawal. Membrane-associated tyrosine kinase activity reached a peak (3.5-fold increase) 1 h after serum deprivation and decreased thereafter reaching a basal level by 2 h of serum deprivation. Interestingly, tyrosine kinase activities remained unaffected by serum stimulation (2-60 min) of serum-deprived cells. Also, during this period of serum stimulation tyrosine phosphorylated proteins of serum-deprived cells were dephosphorylated. Tyrosine phosphatase activities were suppressed during serum deprivation and on serum addition to serum-deprived cells tyrosine phosphatase activities increased significantly. Our data attest that protein tyrosine phosphorylation was associated with growth inhibition of E. histolytica and serum stimulation of E. histolytica produced tyrosine phosphatase activation and protein tyrosine dephosphorylation.     

Regulation of ERK Phosphorylation by Ethanol in Fetal Cortical Neurons

Using fetal cortical neurons and phospho-specific MAP kinase antibody, we investigated the modulation of MAP kinase pathway by ethanol. Our results show that acute ethanol inhibited, while chronic treatment increased, the phosphorylation of MAP kinase per se; likewise AP-5 (a competitive antagonist for NMDA receptors) also increased the basal phosphorylation of MAP kinase following chronic treatment for 5 days. However, chronic ethanol or AP-5 induced MAP kinase phosphorylation was inhibited by KN-62 (calcium calmodulin dependent kinase inhibitor), suggesting the possible involvement of CaM (Calcium calmodulin) kinase. Immunoblot analysis revealed an upregulation of CaM kinase content in chronic ethanol and AP-5 treated cells. These results indicate that acute ethanol may inhibit, while chronic ethanol treatment increases, the basal phosphorylation of MAP kinase per se.     

Protein serine and threonine phosphorylation,hyperactivation and acrosome reaction in in vitro capacitated hamster spermatozoa

Monoclonal antibodies against phosphoserine and phosphothreonine were used in the present study to investigate the changes in serine and threonine phosphorylation respectectively during capacitation of hamster spermatozoa. Immunoblot analysis of hamster spermatozoa capacitated in TALP, a medium that supports capacitation, showed that a set of four proteins of molecular weight 56, 63, 66, and 100 kDa was phosphorylated both at the serine and threonine residues. In addition, five other proteins of molecular weight 32, 39, 45, 53, and 61 kDa were phosphorylated specifically at the threonine residues. Of these nine proteins, the 100 kDa protein showed a time dependent or capacitation-dependent decrease in intensity which coincided with the percentage acrosome-reacted spermatozoa. In contrast, the 49 and 63 kDa threonine phosphorylated proteins showed increased phosphorylation coinciding with capacitation. H8 (a serine and threonine kinase inhibitor) had a transient effect on the phosphorylation of these two phosphothreonine proteins but inhibited acrosome reaction substantially all through the treatment period. Okadaic acid (OA) (a serine and threonine protein phosphatase inhibitor) inhibited hyperactivation but had no effect on acrosome reaction. In fact, OA stimulated acrosome reaction. Finally the immunofluorescence studies indicated localization of the serine phosphorylated proteins in tail as well as in head of the capacitated hamster spermatozoa whereas the threonine phosphorylated proteins were localized mostly in the tail of the spermatozoa. The findings of the present study suggest that serine/threonine phosphorylation and the enzymes responsible for regulating the level of phosphorylation play an important role in capacitation and capacitation-associated events namely hyperactivation and acrosome reaction. However, further studies are needed in order to establish the exact role of these proteins in capacitation of spermatozoa.     

Fertilization promoting peptide and adenosine, acting as first messengers, regulate cAMP production and consequent protein tyrosine phosphorylation in a capacitation-dependent manner

Fertilization promoting peptide (FPP) and adenosine have been shown to act as first messengers, regulating availability of the second messenger cAMP by initially stimulating cAMP production in uncapacitated spermatozoa and then inhibiting it in capacitated cells. This study investigated possible capacitation-related changes in protein tyrosine phosphorylation in response to FPP and adenosine. Time-dependent changes in phosphorylation of proteins of approximately 30-140 kDa were observed in both uncapacitated and capacitated suspensions, the general level of phosphorylation being markedly greater in capacitated cells. In the presence of FPP, phosphorylation was stimulated in uncapacitated but inhibited in capacitated spermatozoa, compared with untreated control samples. Adenosine, cholera toxin, and CGS-21680, a stimulatory A(2a) adenosine receptor agonist, also stimulated phosphorylation in uncapacitated spermatozoa, while Gln-FPP, a competitive inhibitor of FPP, blocked responses to FPP. In capacitated cells, FPP's inhibition of phosphorylation was abolished when cells were treated with FPP in the presence of pertussis toxin. Consistent with the capacitation-dependent effects of FPP and adenosine on cAMP production, these results support the hypothesis that FPP and adenosine modulate sperm function by regulating the AC/cAMP signaling pathway and, consequently, protein tyrosine phosphorylation. Of particular significance is the identification of several phosphoproteins showing FPP-induced alterations in phosphorylation. In uncapacitated spermatozoa, proteins of approximately 116, 95, 82, 75, 66, 56, and 42 kDa showed increased phosphorylation, while in capacitated cells, phosphoproteins of approximately 116, 95, 82, 75, 70, 66, 56, and 50 kDa showed decreased phosphorylation. This suggests that these particular proteins may be involved in stimulation and arrest of capacitation, respectively.     

The S100B Protein Inhibits Phosphorylation of GFAP and Vimentin in a Cytoskeletal Fraction from Immature Rat Hippocampus

The S100B protein belongs to a family of small Ca²⁺-binding proteins involved in several functions including cytoskeletal reorganization. The effect of S100B on protein phosphorylation was investigated in a cytoskeletal fraction prepared from immature rat hippocampus. An inhibitory effect of 5 M S100B on total protein phosphorylation, ranging from 25% to 40%, was observed in the presence of Ca²⁺ alone, Ca²⁺ plus calmodulin or Ca²⁺ plus cAMP. Analysis by two dimensional electrophoresis revealed a Ca²⁺/calmodulin-dependent and a Ca²⁺/cAMP-dependent inhibitory effect of S100B, ranging from 62% to 67% of control, on the phosphorylation of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. The fact that S100B binds to the N-terminal domain of GFAP and that the two proteins are co-localized in astrocytes suggests a potential in vivo role for S100B in modulating the phosphorylation of intermediate filament proteins in glia.     

Second messenger and protein phosphorylation mechanisms underlying opiate addiction: Studies in the rat locus coeruleus

We have studied the role of second messenger and protein phosphorylation pathways in mediating changes in neuronal function associated with opiate addiction in the rat locus coeruleus. We have found that chronic opiates increase levels of the G-protein subunits Gi and Go, adenylate cyclase, cyclic AMP-dependent protein kinase, and a number of phosphoproteins (including tyrosine hydroxylase) in this brain region. Electrophysiological data have provided direct support for the view that this up-regulation of the cyclic AMP system contributes to opiate tolerance, dependence, and withdrawal exhibited by these neurons. As the adaptations in G-proteins and the cyclic AMP system appear to occur at least in part at the level of gene expression, current efforts are aimed at identifying the mechanisms, at the molecular level, by which opiates regulate the expression of these intracellular messenger proteins in the locus coeruleus. These studies will lead to an improved understanding of the biochemical basis of opiate addiction.Special issue dedicated to Dr. Paul Greengard     

Release of ciliary neurotrophic factor from cultured astrocytes and its modulation by cytokines

CNTF rescues various types of lesioned neurons in vivo, and it needs to be released from astrocytes into the extracellular space to have the effect. However, direct evidence for CNTF release has not been unequivocally demonstrated. We hypothesized that the rapid sequestration by CNTF receptor present on cultured astrocytes might be the cause of the inability to detect CNTF released into astrocyte-conditioned medium (ACM). Therefore, we measured CNTF immunoreactivity in medium conditioned by astrocytes treated with phosphatidylinositol-specific phospholipase C (PI-PLC) which was used to prevent released CNTF from binding to the CNTF receptor, since PI-PLC cleaves glycosyl-phosphatidylinositol anchor of CNTFR, the unique component involved in CNTF binding. CNTF was not detectable in untreated ACM, but was detectable in PI-PLC-treated ACM. These results together with the evidence that PI-PLC treatment did not have a toxic effect on astrocytes prove the fact that CNTF can be released from astrocytes without cell lysis. Subsequently, the effect of cytokines such as IL-1, TNF-, and EGF on CNTF release was examined. These cytokines increased CNTF protein levels in ACMs without increasing CNTF protein levels in astrocyte-extracts, indicating that they enhanced CNTF release from astrocytes.     

Evolution of several cytoskeletal proteins of astrocytes in primary culture: Effect of prenatal alcohol exposure

In the present work we have analyzed, using immunoblotting and immunofluorescence techniques, the evolution of several cytoskeletal proteins during the development of astrocytes in primary culture. The effect of prenatal exposure to alcohol on these proteins was also evaluated. Microtubular protein -tubulin decreased approximately 47% from 4 to 7 days after which its content remained practically constant. Immunofluorescence studies showed also that the content of -tubulin was greater at day 4 of culture. This increase in fluorescence was coincident with the presence of globular particles which were found in interphase astrocytes and stained with both anti - and anti--tubulin. These structures appeared only in proliferating cells. Glial fibrillary acidic protein (GFAP) and vimentin were analyzed as intermediate filament (IF) proteins. GFAP, in cytoskeletal preparations, increased regularly for 14 days followed by a decrease to day 21. In contrast, vimentin showed a progressive increase throughout the entire culture period. Fluorescence studies revealed some differences between the IF distribution patterns of GFAP and vimentin.In astrocytes obtained from rats prenatally exposed to ethanol, decreases in the amounts of all the cytoskeletal proteins studied were found during the entire culture period. In these cells a striking disorganization of cytoskeleton was also observed. The alcohol-induced decrease of GFAP in cultured astrocytes was also found when this protein was studied in preparations from whole brain developed in vivo.Special issue dedicated to Dr. Santiago Grisolia