Carla Simone Fogaca Lisboa

In VitroPhosphorylation 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.

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 of32P 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 that proteolysis could be facilitated by diminished phosphorylation levels of NF-M and NF-L.

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.