Pick's disease is a subset of fronto-temporal dementia characterised by severe atrophy of the temporal and frontal lobes due to marked neuronal loss accompanied by astrocytic gliosis enriched in glial acidic protein. The remaining neurones have intracytoplasmic inclusions composed of hyperphosphorylated tau, called Pick bodies, in addition to hyperphosphorylated tau in astrocytes and oligodendrocytes. Gel electrophoresis and western blotting using markers of glycoxidation (advanced glycation end products, N-carboxyethyl-lysine and N-carboxymethyl-lysine: AGE, CEL, CML, respectively) and lipoxidation (4-hydroxy-2-nonenal: HNE, and malondialdehyde-lysine: MDAL) were used in the frontal and occipital cortex in three Pick's disease cases and three agematched controls. In Pick's disease, increased AGE, CML, CEL, HNE and MDAL bands of about 50 kDa were observed in the frontal cortex (but not in the occipital cortex) in association with increased density of glial acidic protein bands. Bi-dimensional gel electrophoresis and western blotting also disclosed increased amounts and numbers of glial acidic protein isoforms in the frontal cortex in Pick's disease. Moreover, redox proteomics showed glycoxidation, as revealed with anti-CEL antibodies and lipoxidation using anti-HNE antibodies, of at least three glial acidic protein isoforms. The present results demonstrate that glial acidic protein is a target of oxidative damage in the frontal cortex in Pick's disease.
A panel of four anti-MAP1B antibodies have been used to study the presence and post-translational modification of MAP1B in primary cultures of glial cells. Two antibodies (150 and 125) recognize phosphorylated epitopes whereas the other two (531 and 842) recognize non-phosphorylated phosphorylatable epitopes on the MAP1B molecule. Immunofluorescence and Western blot analysis with antibodies 531 and 842 revealed the presence of small amounts of MAP1B-like immunoreactivity in type 1 astrocytes and a greater content in more differentiated glial cells found in long-term cultures. By immunofluorescence, these latter cells gave positive immunostaining with antibody 125, which recognizes a phosphorylated epitope phosphorylated by casein kinase II. Antibody 150, which reacts to a phosphorylated epitope on the MAP1B molecule, did not show any detectable immunoreactivity in glial cells cultures, either by immunofluorescence or Western blot. All four antibodies recognized hippocampal neurones in culture, with especially intense immunostaining in cell bodies and axons, and reacted strongly with protein present in hippocampal neurones extracts showing an electrophoretic mobility similar to that of brain MAP1B. In mixed optic nerve glial cell cultures, anti-galactocerebroside (GalC) positive cells gave also positive staining with antibodies 531 and 125. We propose that MAP1B is present in cultures of glial cells in moderate amounts and with a phosphorylation state different than in neurones. Thus, less differentiated glial cells, such as type 1 astrocytes, have a small amount of MAP1B, mainly in a non-phosphorylated form, which is spread diffusely in the cytoplasm and probably does not interact with microtubules. More differentiated glial cells, such as oligodendrocytes, show a greater content in MAP1B which, at least in part, is phosphorylated by a casein kinase II-like activity.
SummaryThe development of highly asymmetrical neurones from undifferentiated neuroblasts involves the ex tension of processes (axon and dendrites), that depends on the assembly of an inner microtubule scaffolding. Clonal cell lines of neuronal origin, N2A and NIE-115 neuroblastoma cells, have been chosen as model systems to study the modifications of microtubule protein which accompany the out growth of axon-like processes (neurites). Neuroblas toma cells grow as proliferating and undifferentiated cells in standard culture medium but can be con sidered as committed neuronal precursors. Thus, they are characterized by a high content of tubulin, including the minor neuronal-specific fiS isoform, and of MAPs including MAP1B and tau-like proteins. Serum withdrawal from the culture medium results in the extension of axon-like processes which is paralleled by a net increase in the amount of assembled tubulin. However, there is not any in crease in the total amount of either tubulin or major MAPs which suggests an involvement of other regulatory factors in the promotion of microtubule assembly. Of relevance in this respect is the fact that /53-tubulin, MAP1B, and tau-like proteins become phosphorylated during neurite extension.A casein kinase II-like enzyme may be involved in some of these phosphorylation events. This enzyme is primarily localized to the nuclei in undifferentiated neuroblastoma cells, whereas a wider distribution of the enzyme between the nucleus and the cytoplasm is found in differentiating neuroblastoma cells. It thus appears plausible that a modified sorting of casein kinase II into the nucleus and the cytoplasm may be involved in the triggering of the phosphorylation of microtubule proteins during neuroblastoma cell differentiation.
Fructose-1,6-bisphosphatase has been studied in adult mouse brain of different ages using an antibody directed against the liver isoform. The presence of liver fructose-1,6-bisphosphatase in cerebellum, cerebral cortex, and hippocampus was assayed using Western blot and different immunocytochemical techniques. Immunocytochemistry with peroxidase reaction product was used to locate fructose-1,6-bisphosphatase in both neurons and astrocytes in the same areas, as well as in the rest of the brain, at light and electron microscope levels. Double immunofluorescence with neuronal or astrocytic markers confirmed the neuronal and astrocytic location of fructose-1,6-bisphosphatase in confocal microscope images. At the subcellular level, fructose-1,6-bisphosphatase was located in the nuclear and cytoplasmic compartments of both neurons and astrocytes, at all ages studied. Ultrastructurally, immunostaining appeared as small patches in the nucleus and the cytosol. In addition, immunostaining was present over the outer mitochondrial membrane, the plasma membrane, and the membranes of the rough endoplasmic reticulum and nuclear envelope, but not over Golgi membranes. In the neuropil fructose-1,6-bisphosphatase was located in dendritic spines, as well as in abundant astrocytic processes that, in some cases, surrounded immunopositive synapses. The possible role of fructose-1,6-bisphosphatase in neurons and astrocytes is discussed.
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