Biomedical subjects
A Routtenberg
Publications and source records attributed to A Routtenberg.
Glial-derived S100b protein selectively inhibits recombinant beta protein kinase C (PKC) phosphorylation of neuron-specific protein F1/GAP43.
Protein F1/GAP43 is neuron-specific, associated with neurite outgrowth during development and a substrate for PKC. This protein is present in high levels in serotonergic neurons which in culture sprout in response to the glial-derived S100b, the beta-beta homodimer. As an initial step in determining whether S100b acts on F1/GAP43 we studied the regulation by S100b of PKC phosphorylation of F1/GAP43. Either the S100b or a mixture of S100a and S100b, both from a brain glial cell source, inhibited in vitro phosphorylation of purified F1/GAP43 by purified PKC in a dose-dependent manner. Using recombinant PKC subtypes, purified S100b preferentially inhibited the F1/GAP43 phosphorylation by the beta subtype. The IC50 of S100b for beta I and beta II PKC was 8 microM while for alpha and gamma PKC it was 64 microM. S100b inhibition was thus subtype-selective. Histone III-S phosphorylation by the four PKC subtypes was not inhibited by S100b. S100b inhibition was thus substrate-selective. Moreover, the effect of S100b on phosphorylation could not be explained by a direct inhibition of kinase activity. Together with earlier studies implicating a role for S100 in synaptic plasticity and neurite outgrowth, the present results suggest that S100b may regulate such functions through its inhibition of neuron-specific PKC substrate (F1/GAP43) phosphorylation. The regulation of this neuron-specific substrate phosphorylation by glial S100 suggests the potential for a novel neuro-glial interaction. Finally, the location of S100 gene on chromosome 21, trisomic in Down's syndrome, and over-expressed in this disorder, as well as in Alzheimer's disease, suggests a link to cognitive impairments in human.
Protein kinase C and F1/GAP-43 gene expression in hippocampus inversely related to synaptic enhancement lasting 3 days.
The mRNA levels of protein F1 (also known as GAP-43), and protein kinase C (PKC) subtypes were measured 3 days after the induction of long-term enhancement (also known as long-term potentiation) in the hippocampus of chronically prepared conscious rats by quantitative in situ hybridization. Altered mRNA levels correlated significantly with alternations in synaptic efficacy; such correlations have not been reported previously. Rats with greater synaptic enhancement had lower gene expression in the CA3 subfield of F1/GAP-43 and both beta-PKC and gamma-PKC, but not alpha-PKC. For microtubule-associated protein 2 (MAP-2), neurogranin, and the glutamate receptor subtype B-flip, no correlation was observed in any cell field between synaptic enhancement and hybridization to the mRNA. To our surprise, alterations in mRNA levels of F1/GAP-43 and gamma-PKC were highly correlated (r = +0.928, P < 0.001), suggesting coordinate regulation. Since F1/GAP-43 is associated with neurite growth, its lowered expression at 3 days would reduce potential growth, leading to synaptic stabilization. We propose that long-term synaptic change is mediated by gene expression of the very same proteins initially modified posttranslationally.
Brief exposure to a novel environment enhances binding of hippocampal transcription factors to their DNA recognition elements.
The behavioral regulation of transcription factor function in hippocampus, a brain region activated by novelty and important for information storage, has not been previously studied. Sixty min after a 4 min exploration of a novel space by adult albino rats, electrophoretic mobility shift assays revealed a selective increase in specific binding to particular consensus recognition elements. For both AP-1 and CRE, an upper and lower band were observed in the electrophoretic mobility shift assay. The upper band showed increased binding restricted to the 60 min time point after exploration; no increase was observed 30 min or less, or 120 min or more after the novel experience. Increased binding to the lower of either AP-1 or CREB was observed at 30 and 120 min. When the time of exploration was increased from 4 to 15 min, no alterations in either the upper or lower band were observed in the transcription factor binding to either the AP-1 or the CRE element. Since the animals ceased to explore the environment after 10 min, the lack of binding may reflect behavioral habituation, leading to transcription factor deactivation. We conclude that brief, naturalistic stimulation can activate brain transcription factors in a time-delimited fashion, suggesting post-translational control of protein-DNA binding. We propose that promoter elements of target genes critically involved in the storage of information are turned on by environmentally-activated transcription factors.
Postsynaptic then presynaptic protein kinase C activity may be necessary for long-term potentiation.
Protein kinase C inhibitor was injected intracellularly by iontophoresis into CA1 somata either before or after long-term potentiation in the hippocampal slice preparation. Two different protein kinase C inhibitors, polymyxin B (PMXB) or 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), injected 10 min before long-term potentiation induction caused potentiated responses to return to baseline 15-35 min after induction without significantly affecting the initial magnitude of potentiation. There was no effect on long-term potentiation persistence when H-7 or PMXB was injected intracellularly 5 min after long-term potentiation induction. In contrast, focal extracellular micro-pressure ejection of protein kinase C inhibitor in the stratum radiatum, 15 or 30 min, but not 60 min after long-term potentiation induction caused decay of long-term potentiation to baseline. This is probably a presynaptic action since intracellular inhibitors injected postsynaptically were ineffective 5 min after long-term potentiation induction. Focal application to stratum pyramidale produced a weaker decay than to stratum radiatum suggesting a Schaffer collateral presynaptic terminal site of action. We propose that activation of postsynaptic protein kinase C activity is necessary for long-term potentiation persistence but this activity persists for less than 5 min after induction. Presynaptic protein kinase C activity is also necessary for persistence and is time-limited to less than 60 min. It is attractive to think that these two events are sequentially activated and employ different protein kinase C subtypes differentially localized to presynaptic or postsynaptic elements.
Phosphorylation of pyruvate dehydrogenase in the hippocampal slice: time course of response to cellular depolarization.
In the rat hippocampal slice, we evaluated the effects of potassium depolarization on phosphorylation of the alpha subunit of the pyruvate dehydrogenase complex (E1 alpha, mol. wt. approximately 41 kDa) using an in vitro back-titration assay. The in vitro back-titration phosphorylation of El alpha was maximal following 30 s of depolarization and remained elevated for up to 3 min following termination of the depolarizing pulse. Possible significance of and mechanisms for the depolarization-induced changes in E1 alpha phosphorylation are discussed.
Contrasting patterns of protein phosphorylation in human normal and Alzheimer brain: focus on protein kinase C and protein F1/GAP-43.
We introduce a new procedure to study kinase substrates in postmortem human brain. By adding purified exogenous protein kinase C (PKC) and the phospholipid phosphatidylserine to brain homogenates in vitro we are able to analyze PKC substrates. A human 53-kDa phosphoprotein is described that appears to be homologous to rat and monkey protein F1 (GAP-43). This identity is based on molecular weight, isoelectric point, phosphorylation by exogenous protein kinase C, enhancement of its phosphorylation by three activators (phospholipids, calcium and phorbol esters), phosphopeptide maps, and cross-reactivity with an antibody raised against rat protein F1. Protein F1 is a PKC substrate associated with synaptic plasticity and nerve growth. Its phosphorylation in rat brain has been correlated with long-term potentiation, an electrophysiological model of memory. In the present study of normal brain, human protein F1 shows an occipitotemporal in vitro phosphorylation gradient. This is consistent with previous observations in nonhuman primates. This gradient is less pronounced in Alzheimer's disease (AD). Changes in the in vitro phosphorylation pattern of three other non-PKC substrates in Alzheimer's disease, including one with characteristics similar to microtubule-associated protein tau, are also reported. These results suggest that protein phosphorylation can be studied in postmortem human brain and that PKC-mediated phosphorylation of protein F1, already linked to synaptic plasticity and memory, may be altered in AD.
Selective expression of protein F1/(GAP-43) mRNA in pyramidal but not granule cells of the hippocampus.
Protein F1/GAP-43 is a protein kinase C substrate associated with axonal growth and synaptic plasticity. We used in situ hybridization in rat brain to determine the cellular distribution of its gene expression. Throughout the septotemporal axis of the adult hippocampus, pyramidal cells express F1/GAP-43 mRNA, but granule cells do not. To determine if F1/GAP-43 expression in granule cells ever occurs, we studied its expression in development during mossy fiber outgrowth, when expression should be maximal. Quantitation of relative hybridization levels in the hippocampus revealed a modest increase in granule cell F1/GAP-43 mRNA coincident with mossy fiber outgrowth. But even the peak hybridization in granule cells on day 16 was 75% less than in pyramidal cells. The distribution of grains was over the entire granule cell layer at day 9, but was restricted by day 20 to the inner aspect of the layer, the site of the youngest cells which are still sending out axonal processes. Cell-selective expression of F1/GAP-43 within a particular brain structure was not restricted to the hippocampus. In cerebellum, F1/GAP-43 hybridization was detected in granule cells but not Purkinje cells; in olfactory bulb, mitral cells but not internal granule cells; in habenula, cells in the lateral but not medial nucleus; in substantia nigra, pars compacta cells but not cells in pars reticulata. Neurons containing biogenic amines exhibited intense F1/GAP-43 hybridization: substantia nigra pars compacta (dopamine), the locus coeruleus (norepinephrine), and dorsal raphe (serotonin). In contrast, cholinergic neurons exhibited little (basal forebrain) or no (medial habenula) hybridization. F1/GAP-43 expression is not restricted to a specific cell type and is not correlated with axon length. High F1/GAP-43 expression is apparent in many neurons having either neuromodulatory or memory storage functions. We propose that F1/GAP-43 is important for accelerating process outgrowth and synaptic remodeling, rather than directing growth itself.
A tale of two contingent protein kinase C activators: both neutral and acidic lipids regulate synaptic plasticity and information storage.
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Neuron-specific protein F1/GAP-43 shows substrate specificity for the beta subtype of protein kinase C.
We determined whether the beta or gamma protein kinase C (PKC) subtypes implicated in long-term potentiation (LTP) selectively regulates protein F1 phosphorylation. Purified bovine PKC subtypes and recombinant PKC subtypes activated by phosphatidylserine (PS) and calcium were tested for their relative ability to phosphorylate purified rat protein F1 (a.k.a. GAP-43). After equalizing enzyme activity against histone, the recombinant beta II PKC phosphorylated protein F1 to a 6 fold greater extent than the recombinant gamma PKC. Bovine beta I PKC phosphorylated protein F1 to a 3 fold greater extent than bovine gamma PKC. Even when PS was replaced by lipoxin B4, which can selectively increase gamma PKC activity, beta I PKC was still superior to gamma PKC in phosphorylating protein F1. Taken together with previous cellular studies of brain showing parallel levels of expression of beta PKC mRNA and protein F1 mRNA, the present results make it attractive to propose that beta PKC regulates protein F1 phosphorylation during the development of synaptic plasticity.
Protein kinase C activity and substrate (F1/GAP-43) phosphorylation in developing cat visual cortex.
Protein kinase C (PKC) and substrate proteins such as F1/GAP-43 have been previously implicated in the synaptic plasticity of long-term potentiation (LTP). As a first step in determining whether they participate in the plasticity observed during the critical period of visual cortex development, we have studied cytosol and membrane PKC activity as well as the endogenous phosphorylation of visual cortical proteins in cat cortical areas 17, 18 at postnatal days 1 and 3, weeks 1, 3, 5, 7, 9, 13, 28 and 51, and adult year 5. There was an 8.4 to 10.9 fold increase in cytosolic PKC activity relative to day 1 level during the critical period of synaptic plasticity (weeks 3-13) which then dramatically decreased back to 2.5-fold of day 1 level by week 51. This was near the adult level of cytosolic PKC. Since there was an increase of 1.8- to 2.1-fold in membrane PKC activity during the critical period, this argues against a PKC translocation event and for an increase in enzyme synthesis. Endogenous phosphorylation in the same visual cortex tissue revealed an increase in protein F1 phosphorylation during the critical period. This level of PKC substrate activity was maintained in the adult providing a mechanism for plasticity in adult cat visual cortex.
Long-term potentiation in intact infant rat hippocampus.
In rats 14-28 days of age, high-frequency stimulation of the perforant path granule cell synapse in the dentate gyrus produced long-term potentiation of the population spike that was comparable in magnitude (150-250% of baseline) and duration (120 min) to that produced in adult animals with the same stimulation paradigm. In contrast, potentiation of the excitatory postsynaptic potential occurred inconsistently.
Inhibition of protein kinase C blocks two components of LTP persistence, leaving initial potentiation intact.
Protein kinase C (PKC) activity is increased following hippocampal long-term potentiation (LTP; Akers et al., 1986). A similar increase in PKC activity is measured following the induction of a long-lasting potentiation with abbreviated high-frequency stimulation (HFS) in combination with PKC-activating phorbol esters (Colley et al., 1989). Because phorbol esters have no effect on the initial potentiation produced with HFS, and because PKC activity appears to be related to the persistence of LTP and not to the initial change, we concluded that PKC regulates a post-initiation component of LTP. To define the time domain in which PKC activation is necessary for LTP, we studied the effect of the PKC inhibitors polymyxin B (PMXB) and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) micropressure ejected at different time points before and after the induction of LTP. LTP was produced in intact rats with HFS of the perforant path, and inhibitor ejections were made in the molecular layer of the dentate gyrus. PMXB, which at lower doses is a selective inhibitor of PKC, had no effect on initial potentiation, yet caused decay of the potentiated response to baseline within 2 hr. Decay occurred when PMXB was ejected 15 min before and 15 and 30 min after HFS. PMXB, at either low or high doses, was ineffective in blocking LTP persistence at time points greater than 30 min after HFS. Low doses of H-7 produced similar effects to those of PMXB. However, in contrast to a high dose of PMXB, a high dose of H-7 inhibited the persistence of LTP when delivered 240 min after HFS.(ABSTRACT TRUNCATED AT 250 WORDS)
Dietary cis-fatty acids that increase protein F1 phosphorylation enhance spatial memory.
Activation of protein kinase C (PKC) facilitates long-term potentiation (LTP), a model of memory, and increases its substrate protein F1 (aka GAP43) phosphorylation in direct relation to synaptic enhancement. Unsaturated fatty acids (c-FAs) which activate purified PKC, when injected into hippocampus, enhance LTP. To determine if dietary c-FAs could alter memory itself as well as brain PKC substrate (F1) metabolism, rats were maintained for 10 weeks on fatty acid diets enriched in mono-unsaturated oleic acid (OA; 20% olive oil, w/w), or a mono- and di-unsaturated mixture of oleate/linoleate (O/L; 20% corn oil), or a saturated fatty acid diet of laurate/myristate (L/M; 20% hydrogenated coconut oil). The O/L diet group was superior to the OA and L/M groups in spatial memory performance after the first two weeks of acquisition and in later achievement of criterion performance. The O/L diet had a significantly higher hippocampal protein F1 in vitro phosphorylation than in both the OA and L/M in trained and non-trained animals. Significantly, animals that made fewer errors showed higher F1 phosphorylation (r = -0.70). Diet both increases brain PKC substrate phosphorylation and enhances maze learning, confirming the feasibility of enhancing learning and memory by dietary regimens derived from basic neurochemical studies of synaptic plasticity.
Phosphoproteins localized to presynaptic terminal linked to persistence of long-term potentiation (LTP): quantitative analysis of two-dimensional gels.
Previous findings suggest: (1) that altering protein kinase C (PKC) activity alters the persistence of long-term potentiation (LTP) in the intact hippocampal formation; and (2) that PKC activity is directly correlated with persistence of LTP in vivo as measured by the in vitro phosphorylation of two major PKC substrates in adult hippocampus, protein F1 and 80k. Using quantitative analysis of two-dimensional gels, we report here two additional phosphoproteins of 72 and 55 kDa which were directly correlated to persistence of LTP induced in the intact dorsal hippocampal formation. The phosphorylation of both proteins in response to addition of different kinase stimulators was distinct from that of protein F1 and 80k. Moreover, neither protein was a substrate for exogenous PKC. The physicochemical properties of these phosphoproteins suggest they are identical to the previously described synaptic vesicle proteins IIIa and IIIb, and as such are immunologically indistinguishable. Because proteins IIIa and IIIb are known to be phosphorylated by a Ca2+/calmodulin (CaM)-stimulated kinase, and protein F1 is known to be a plasma membrane-associated protein (P-57) which releases bound CaM in response to phosphorylation by PKC, the present findings suggest a potential mechanism in which PKC-mediated changes in plasma membrane proteins produce CaM kinase-mediated changes in synaptic vesicle proteins through a phosphorylation cascade. These membrane/vesicle alterations are postulated to underlie the increased synaptic efficacy which marks persistent LTP.
Dose-dependent phorbol ester facilitation or blockade of hippocampal long-term potentiation: relation to membrane/cytosol distribution of protein kinase C activity.
We have proposed that the translocation/activation of protein kinase C (PKC) in synergism with a Ca2+-mediated event plays an essential role in hippocampal long-term potentiation (LTP). In a previous study, we saw no effect of PKC-activating phorbol esters alone on baseline responses, although it has been reported by others to enhance synaptic transmission. To resolve this discrepancy, we investigated the dose-response to phorbol esters of both baseline and potentiated granule cell responses elicited with perforant path stimulation. It was confirmed that iontophoretic ejection of phorbol ester to the dentate hilus, which alone had no effect on baseline responses, prolonged the persistence of potentiation produced by 2 trains of 400 Hz stimulation. These data support the proposed synergistic model in which the effects of phorbol ester and high frequency stimulation together produce a long-lasting potentiation of synaptic activation. A similar synergism was observed with ejection of a lower dose of phorbol ester into the perforant path synaptic zone in the molecular layer. Higher doses delivered to the synaptic zone without 400 Hz stimulation were sufficient to enhance baseline synaptic responses, but these doses inhibited the initial potentiation induced with 2 trains of 400 Hz stimulation delivered immediately after ejection. There was at times a slowly developing enhancement observed after the initial blockade. Thus, induction of a persistent synaptic enhancement was observed without initial potentiation. Measurement of PKC activity in membrane and cytosol indicated that PKC activation is only associated with the persistence phase of LTP. In contrast, there was no change in PKC subcellular distribution associated with the blockade of initial potentiation by higher doses of PDBu.
cis-Fatty acids, which activate protein kinase C, attenuate Na+ and Ca2+ currents in mouse neuroblastoma cells.
1. Activation of protein kinase C (PKC) by phorbol esters or diacylglycerols has been shown to modulate a number of ionic currents carried by Ca2+, K+ and Cl-. Recently, it has been demonstrated that PKC may be activated by cis-fatty acids in the absence of either phospholipid or Ca2+. We wished to determine if this new class of PKC-activating compound would also modulate ionic currents. To this end we applied the whole-cell voltage-clamp technique to N1E-115 neuroblastoma cells. 2. Analysis of families of currents evoked under voltage clamp by depolarizing steps from a holding potential of -85 mV during external application of 5 microM-oleate (a cis-fatty acid) showed a 36% reduction of the peak inward current with no shift in either the peak or the reversal potential of the current-voltage relation and no alteration of outward current. 3. External application of the cis-fatty acids oleate, linoleate and linolenate reversibly attenuated voltage-dependent Na+ current with approximate half-maximal dose values of 2, 3, and 10 microM respectively. Oleate was approximately 2 times more potent when applied internally (ED50 = 1 microM). Externally applied elaidate (a trans-isomer of oleate) and stearate (a saturated fatty acid) which do not activate PKC, had no effect. Since cis-fatty acids are known to fluidize membranes, as well as to activate PKC, we sought to dissociate these functions by applying compounds that fluidize membranes but do not activate PKC: methyloleate and lysophosphatidylcholine. Neither compound affected Na+ current when applied externally at concentrations of 1-50 microM. 4. In contrast to cis-fatty acids, three classical PKC activators, phorbol-12.13-dibutyrate (PDB), phorbol-12.13-diacetate (PDA), and 1.2-oleoylacetylglycerol (OAG) were found to have no effect on the voltage-dependent Na+ current when applied externally at 10 nM-1 microM (phorbol esters) or 1-150 microM (OAG) for incubation periods up to 1 h. 5. External application of the PKC inhibitors polymyxin B, H-7, sphingosine and staurosporine blocked the attenuation of the Na+ current by cis-fatty acid in a dose-dependent manner, with maximal inhibition occurring at doses of 50, 10, 200 and 0.1 microM, respectively. The cyclic nucleotide-dependent protein kinase inhibitor H-8 was much less effective in blocking the cis-fatty acid effect. Polymyxin B and staurosporine were more potent when applied internally. 6. Chronic (24 h) exposure to 1 microM phorbol-12-myristate-13-acetate (TPA) was employed to down-regulate PKC.(ABSTRACT TRUNCATED AT 400 WORDS)