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).

Effects of brain ischemia on intermediate filaments of rat hippocampus.

Neurofilaments subunits (NF-H, NF-M, NF-L) and glial fibrillary acidic protein (GFAP) were investigated in the hippocampus of rats after distinct periods of reperfusion (1 to 15 days) following 20 min of transient global forebrain ischemia in the rat. In vitro [14Ca]leucine incorporation was not altered until 48 h after the ischemic insult, however concentration of intermediate filament subunits significantly decreased in this period. Three days after the insult, leucine incorporation significantly increased while the concentration NF-H, NF-M, and NF-L were still diminished after 15 days of reperfusion. In vitro incorporation of 32P into NF-M and NF-L suffered immediately after ischemia, but returned to control values after two days of reperfusion. GFAP levels decreased immediately after ischemia but quickly recovered and significantly peaked from 7 to 10 days after the insult. These results suggest that transient ischemia followed by reperfusion causes proteolysis of intermediate filaments in the hippocampus, and the proteolysis could be facilitated by diminished phosphorylation levels of NF-M and NF-L.