Propionic and methylmalonic acids inhibit the in vitro phosphorylation of a 85 kDa cytoskeletal protein from cerebral cortex of rats.

In this study we examine the action of methylmalonic (MMA) and propionic (PA) acids, metabolites which accumulate in methylmalonic and propionic acidemias respectively, on the endogenous phosphorylating system associated with the cytoskeletal fraction of cerebral cortex of young rats. Chronic treatment with PA and treatment of tissue slices with MMA or PA are effective in decreasing the in vitro phosphorylation into a 85 kDa cytoskeletal associated protein. We tested the effect of the acids on the endogenous kinase activities by using specific kinase activators and inhibitors. Results demonstrated that the acids interfere with the endogenous cAMP-dependent and Ca2+/calmodulin-dependent kinase activities. Furthermore, in vitro dephosphorylation of the 85 kDa protein was totally inhibited in brain slices treated with the acids. Considering the importance of protein phosphorylation to cellular function, we speculate that alteration in the phosphorylating level of cytoskeletal associated phosphoproteins induced by MMA and PA treatments may somehow be involved in steps leading to brain damage.

Effects of acute and chronic administration of methylmalonic and propionic acids on the in vitro incorporation of 32P into cytoskeletal proteins from cerebral cortex of young rats.

We studied the effects of acute and chronic administration of methylmalonic (MMA) and propionic (PA) acids on the in vitro incorporation of 32P into neurofilament subunits (NF-M and NF-L), alpha and beta tubulins, from cerebral cortex of rats. In the chronic treatment, drugs were administered subcutaneously from day 6-17 post-partum (MMA 0.76-0.89 micromol/g body weight and PA 0.93 micromol/g body weight). In the acute treatment MMA and PA were injected (MMA 3.78 micromol/g body weight and PA 3.90 micromol/g body weight). Control animals received saline in the same volumes. The Triton-insoluble cytoskeletal fraction of control in treated animals was isolated and incubated with 32P-ATP. Our results demonstrate that both drugs were able to inhibit 32P in vitro incorporation into neurofilaments and tubulins. The acute administration of MMA decreased the in vitro 32P incorporation into NF-L and alpha-tubulin subunit, whereas PA administration decreased the 32P in vitro incorporation into NF-M, NF-L, and tubulins. On the other hand, chronic MMA administration induced a decreased 32P in vitro incorporation into NF-M, while chronic treatment with propionate decreased the in vitro phosphorylation of NF-M and alpha-tubulin. This study provides consistent evidence that a decreased phosphorylation of cytoskeletal proteins is induced by MMA and PA metabolites which accumulate in methylmalonic and propionic acidemias respectively. Therefore, it is possible that an altered brain cytoskeletal metabolism could be related with the structural alterations of CNS observed in these disorders.

Alpha-ketoisocaproate increases the in vitro 32P incorporation into intermediate filaments in cerebral cortex of rats.

In this study we investigated the effects of alpha-ketoisocaproic (KIC), alpha-ketoisovaleric (KIV) and alpha-keto-beta-methylvaleric (KMV) acids on the phosphorylation of intermediate filament (IF) proteins of cerebral cortex of rats. Tissue slices were incubated with [32P] orthophosphate in the presence or absence of the acids. The intermediate filament enriched cytoskeletal fraction was isolated and the radioactivity incorporated into neurofilament subunits, vimentin and glial fibrillary acidic protein was measured. Results demonstrated that KIC significantly increased phosphorylation of these proteins whereas the other acids had no effect. Experiments using protein kinase inhibitors indicated that the effect of KIC was mediated by Ca2+/calmodulin- and cAMP-dependent protein kinases. This study provides evidence that KIC, a key metabolite accumulating in maple syrup urine disease, increases phosphorylation of IF proteins.

Methylmalonic and propionic acids increase the in vitro incorporation of 32P into cytoskeletal proteins from cerebral cortex of young rats through NMDA glutamate receptors.

In this study we investigated the effects of methylmalonic acid (MMA) and propionic acid (PA) on the phosphorylation of cytoskeletal proteins of cerebral cortex of rats. Slices of tissue were incubated with 32P-orthophosphate in the presence or absence of glutamate, MMA, PA and ionotropic or metabotropic glutamate receptor agonists. The cytoskeletal fraction was isolated and the radioactivity incorporated into the cytoskeletal proteins was measured. Results demonstrated that the acids, glutamate and NMDA increased the phosphorylation of the proteins studied. However, this effect was not observed for non-NMDA ionotropic agonists or metabotropic agonists. Experiments using glutamate receptor antagonists confirmed that MMA and PA at the same concentrations as found in tissues from propionic or methylmalonic acidemic children increase the phosphorylation of cytoskeletal proteins, possibly via NMDA glutamate receptors. Therefore, it is feasible that these findings may be related to the neurological dysfunction characteristic of these disorders.

Methylmalonic acid reduces the in vitro phosphorylation of cytoskeletal proteins in the cerebral cortex of rats.

The present work was undertaken to determine the action of methylmalonic acid (MMA), a metabolite, which accumulates in high amounts in methylmalonic acidemia, on the endogenous phosphorylating system associated with the cytoskeletal fraction proteins of cerebral cortex of young rats. We demonstrated that pre-treatment of cerebral cortex slices of young rats with 2.5 mM buffered methylmalonic acid (MMA) is effective in decreasing in vitro incorporation of [32P]ATP into neurofilament subunits (NF-M and NF-L) and alpha- and beta-tubulins. Based on the fact that this system contains cAMP-dependent protein kinase (PKA), Ca2+/calmodulin-dependent protein kinase II (CaMKII) and protein phosphatase 1 (PP1), we first tested the effect of MMA on the kinase activities by using the specific activators cAMP and Ca2+/calmodulin or the inhibitors PKAI or KN-93 for PKA and CaMKII, respectively. We observed that MMA totally inhibited the stimulatory effect of cAMP and interfered with the inhibitory effect of PKAI. In addition, the metabolite partially prevented the stimulatory effect of Ca2+/calmodulin and interfered with the effect of KN-93. Furthermore, in vitro dephosphorylation of neurofilament subunits and tubulins was totally inhibited in brain slices pre-treated with MMA. Taken together, these results suggest that MMA, at the same concentrations found in tissues of methylmalonic acidemic children, inhibits the in vitro activities of PKA, CaMKII and PP1 associated with the cytoskeletal fraction of the cerebral cortex of rats, a fact that may be involved with the pathogenesis of the neurological dysfunction characteristic of methylmalonic acidemia.

The effects of behavioral tasks on the in vitro phosphorylation of intermediate filament subunits of rat hippocampus are mediated by CaMKII and PKA.

Neurofilaments (NF) are the most abundant constituents of the neuronal cytoskeleton, while glial fibrillary acidic protein (GFAP) is a major component of the glial astrocyte cytoskeleton. These proteins can be phosphorylated by different protein kinases and they are regulated in a complex way by phosphorylation. Using a hippocampal cytoskeletal fraction we demonstrated that the behavioral tasks of inhibitory avoidance and habituation can differently alter the in vitro phosphorylation of the 150 kDa (NF-M) and the 68 kDa (NF-L) neurofilament subunits and of the GFAP. In order to verify the effect of habituation and inhibitory avoidance training on the phosphatase activity, we performed the time course-dephosphorylation assay (5-30 min of incubation of the cytoskeletal fraction with 32P-ATP). Subsequently we investigated the effect of these behavioral tasks on the protein kinase activities associated with the cytoskeletal fraction, carring out the 32P incorporation assays in the presence of specific kinase inhibitors. Results suggest that phosphatase activity is not altered in the cytoskeletal fraction by the behavioral tasks and that the increased in vitro phosphorylation of NF-M and NF-L caused by habituation is probably mediated by the Ca2+/calmodulin dependent protein kinase (CaMKII). However, the inhibition of GFAP in vitro phosphorylation caused by inhibitory avoidance training is probably related to the cAMP dependent protein kinase (PKA).

Developmentally regulated in vitro phosphorylation of a 85 kDa triton-insoluble protein of the cerebral cortex of rats.

We studied the ontogeny of concentration and in vitro phosphorylation of an 85 kDa Triton-insoluble protein from cerebral cortex of 7, 15, 21 and 90 day old rats. The Triton-insoluble cytoskeletal fraction contains an 85 kDa basic phosphoprotein different from synapsin 1, as determined by nonequilibrium pH gradient electrophoresis and phosphopeptide mapping with V8 protease. The concentration of the 85 kDa cytoskeletal associated phosphoprotein was analyzed during development. Results indicated that the concentration of this protein oscillated during suckling, presenting a maximal value at day 15 and decreasing again to stabilize at values near those of 7 day old rats, remaining constant in 21 and 90 day old animals. However, in vitro 32P incorporation, expressed as cpm/microgram, presented a developmentally regulated pattern, with maximal values in young rats, declining with age to negligible values in 90 day old animals. The endogenous phosphorylating system responsible for in vitro 32P incorporation into the 85 kDa protein was determined by the addition of specific activators of second-messenger protein kinases (cAMP, Ca2+/ calmodulin and Ca2+/phosphatidylserine/phorbol ester) and a protein phosphatase inhibitor (okadaic acid) to the incubation system. Results suggested that the in vitro phosphorylation system is composed of protein kinase A, Ca2+/calmodulin dependent protein kinase and protein phosphatase 1.

Ca(2+)-mediated phosphorylation and proteolysis activity associated with the cytoskeletal fraction from cerebral cortex of rats.

We describe a Triton-insoluble cytoskeletal fraction extracted from cerebral cortex of young rats retaining an endogenous Ca(2+)-mediated mechanism acting in vitro on Ca2+/calmodulin-dependent protein kinase II (CaM-KII) activity and on phosphorylation and proteolysis of the 150 kDa neurofilament subunit (NF-M), alpha and beta tubulin. Exogenous Ca2+ induced a 70% decrease in the in vitro phosphorylation of the NF-M and tubulins and a 30-50% decrease in the total amount of these proteins. However, when calpastatin was added basal phosphorylation and NF-M and tubulin content were recovered. Furthermore, exogenous Ca2+/calmodulin induced increased in vitro phosphorylation of the cytoskeletal proteins and CaM-KII activity only in the presence of calpastatin, suggesting the presence of Ca(2+)-induced calpain-mediated proteolysis. This fraction could be an interesting model to further studies concerning the in vitro effects of Ca(2+)-mediated protein kinases and proteases associated with the cytoskeletal fraction.

Effect of hyperphenylalaninemia chemically induced on in vitro incorporation of 32P into cytoskeletal proteins from cerebral cortex of developing rats.

We studied the effect of hyperphenylalaninemia on in vitro incorporation of 32P into cytoskeletal proteins from cerebral cortex of rats by injecting l-phenylalanine plus alpha-methylphenylalanine subcutaneously from the 6th to the 14th day postpartum. Chronic hyperphenylalaninemia induced an increased in vitro phosphorylation of the 150-kDa neurofilament subunit and tubulins present in the cytoskeletal fraction at the end of the treatment and 3 days after treatment discontinuation. In addition, when in vitro phosphorylation of the cytoskeletal proteins from treated animals was performed in the presence of the drugs we observed a decreased in vitro incorporation of 32P into these proteins. Thus, the effect of l-phenylalanine plus alpha-methylphenylalanine on the endogenous protein kinase and phosphatase activities was examined and the results demonstrated that these drugs have an inhibitory effect on calcium/calmodulin-dependent protein kinase II and protein phosphatase type 1.

Phenylalanine inhibition of the phosphorylation of cytoskeletal proteins from cerebral cortex of young rats is prevented by alanine.

BACKGROUND: Phenylalanine has been considered the main responsible agent for the brain damage that occurs in phenylketonuria. METHODS AND RESULTS: In this work we studied the effect of this amino acid on the in vitro phosphorylation of cytoskeletal proteins of the cerebral cortex of rats. We observed that 2 mM phenylalanine, a concentration usually found in the plasma of phenylketonuric patients, decreased the in vitro 32P incorporation into these proteins. In addition, we investigated the effect of alanine on the inhibition of 32P incorporation into cytoskeletal proteins caused by phenylalanine. We observed that 0.5 mM alanine did not alter 32P incorporation but prevented the inhibition provoked by phenylalanine. CONCLUSION: In case the inhibition of cytoskeletal protein phosphorylation by phenylalanine also occurs in human phenylketonuria, it is possible that alanine supplementation to the phenylalanine-restricted diet may be beneficial to these patients.

In vitro phosphorylation of cytoskeletal proteins in the rat cerebral cortex is decreased by propionic acid.

In the present study we demonstrate that propionic acid (PA), a metabolite that accumulates in large amounts in propionic acidemia, is able to decrease in vitro incorporation of [32P]ATP into neurofilament subunits (NF-M and NF-L) and alpha- and beta-tubulin. Considering that the endogenous phosphorylating system associated with the cytoskeletal fraction contains cAMP-dependent protein kinase (PKA), Ca2+/calmodulin protein kinase II (CaMKII), and protein phosphatase 1 (PP1), we first assayed the effect of the acid on the kinase activities by using the specific activators cAMP and Ca2+/calmodulin or the inhibitors PKAI or KN-93 for PKA and CaMKII, respectively. Results demonstrated that the acid totally inhibited the stimulatory effect of cAMP and interfered with the inhibitory effect of PKAI. In addition, PA partially prevented the stimulatory effect of Ca2+/calmodulin and interfered with the effect of KN-93. In addition, we demonstrated that PA totally inhibited in vitro dephosphorylation of neurofilament subunits and tubulins mediated by PP1 in brain slices pretreated with the acid. Taken together, these results demonstrate that PA inhibits the in vitro activities of PKA, CaMKII, and PP1 associated with the cytoskeletal fraction of the cerebral cortex of rats. This study suggests that PA at the same concentrations found in tissues from propionic acidemic children may alter phosphorylation of cytoskeletal proteins, which may contribute to the neurological dysfunction characteristic of propionic acidemia.

Effects of neonatal cerebral hypoxia-ischemia on the in vitro phosphorylation of synapsin 1 in rat synaptosomes.

Synapsins are phosphoproteins related to the anchorage of synaptic vesicles to the actin skeleton. Hypoxia-ischemia causes an increased calcium influx into neurons through ionic channels gated by activation of glutamate receptors. In this work seven-day-old Wistar rats were submitted to hypoxia-ischemia and sacrificed after 21 hours, 7, 30, or 90 days. Synaptosomal fractions were obtained by Percoll gradients and incubated with 32P (10 microCi/g). Proteins were analysed by SDS-PAGE and radioactivity incorporated into synapsin 1 was counted by liquid scintillation. Twenty-one hours after hypoxia-ischemia we observed a reduction on the in vitro phosphorylation of synapsin 1, mainly due to hypoxia, rather than to ischemia; this effect was reversed at day 7 after the insult. There was another decrease in phosphorylation 30 days after the event interpreted as a late effect of hypoxia-ischemia. No changes were observed at day 90. Our results suggest that decreased phosphorylation of synapsin 1 could be related to neuronal death that follows hypoxia-ischemia.