Narayan R. Bhat

Transient ischemia stimulates glial fibrillary acid protein and vimentin gene expression in the gerbil neocortex, striatum and hippocampus

Astrocytic activation plays a major role in homeostatic maintenance of the central nervous system in response to neuronal damage. To assess the reactivity of astrocytes in transient cerebral ischemia of the gerbil, we studied the levels of glial fibrillary acidic protein (GFAP) and its mRNA. GFAP mRNA increased by 4 h after carotid artery occlusion, reached peak levels by 72 h with a 12-fold increase over control and then started declining as early as 96 h postischemia. An examination of the specific regions of the brain revealed an increase in GFAP mRNA associated with the forebrain, midbrain, hippocampus and striatum. GFAP mRNA in the non-ischemic cerebellum however, remained expressed at constitutively low levels. Immunoblot analysis with anti-GFAP antibodies demonstrated a 2- to 3-fold increase in the protein after 24 and 48 h of reperfusion. Pretreatment with pentobarbital and 1-(5-oxohexyl)-3-methyl-7-propyl xanthine (HWA 285), the drugs that have been shown to protect against ischemic damage, prevented the increase in GFAP mRNA in the cortex following ischemic injury. Forebrain ischemia also induced vimentin mRNA and protein quantities by 12 h of reperfusion in the cortex. The levels of c-fos and preproenkephalin mRNA increased rapidly within 1 h after ischemic injury, demonstrating a temporal difference in mRNA changes following ischemia. These results indicate that an increase in GFAP and vimentin, the two glial intermediate filament proteins in the area of the ischemic lesion may be associated with a glial response to injury.

Developmental changes in enzymes involved in dolichyl phosphate metabolism in cultured embryonic rat brain cells.

Abstract: The rates of synthesis of dolichol‐linked oligosaccharide intermediates and protein N‐glycosylation increased substantially during a developmental period corresponding to glial differentiation in primary cultures of embryonic rat brain. In this study developmental changes in three enzymes involved in dolichyl phosphate (Dol‐P) metabolism have been examined by in vitro assays and correlated with the induction pattern for lipid intermediate synthesis and protein N‐glycosylation. Dolichyl pyrophosphate (Dol‐P‐P) phosphatase activity was relatively low during the first 9 days in culture, but it increased significantly between days 9 and 25. Dol‐P‐P phosphatase did not change appreciably between days 22 and 30 in culture. A kinetic analysis of the developmental change in Dol‐P‐P phosphatase activity revealed that the Vmax increased 10‐fold between days 4 and 22, and there was also a significant change in the apparent Km for Dol‐P‐P. Dolichol kinase activity increased during the period (9–15 days) when there was a significant induction in oligosaccharide‐lipid synthesis and protein N‐glycosylation, and then declined in parallel with lipid intermediate synthesis and protein N‐glycosylation. Dol‐P phosphatase activity was present at relatively low levels for the first 9 days in culture, but it increased steadily between days 9 and 30. A kinetic comparison of the activity in membrane fractions from brain cells cultured for 9 and 25 days indicated that there was a 10‐fold increase in enzyme protein with unaltered affinity for Dol‐P. The results suggest that elevated dolichol kinase activity enhances the rate of lipid intermediate synthesis, and subsequent reciprocal changes in dolichol phosphorylation‐dephosphorylation are a regulatory factor in the deactivation of oligosaccharide‐lipid synthesis, and consequently of protein N‐glycosylation, during the period following glial differentiation in primary cultures of embryonic rat brain cells.

Induction of N-Glycosylation Activity in Cultured Embryonic Rat Brain Cells

Developmental changes in protein N‐glycosylation activity have been studied using cultures of dissociated fetal rat brain cells as an in vitro model system. These cultures undergo an initial phase of neurite outgrowth and cell proliferation (4–6 days in culture), followed by a period of cellular differentiation. N‐Glycosylation activity has been measured by assaying the incorporation of [2‐3H]‐mannose into dolichol‐linked oligosaccharides and glyco‐protein over a period of 1–25 days in culture. This study revealed a marked induction of N‐glycosylation activity beginning at approximately 1 week of culture. [2‐3H]‐Mannose incorporation into the oligosaccharide‐lipid intermediate fraction and glycoprotein reached maximal values between 12 and 16 days of culture and declined thereafter. The major dolichol‐linked oligosaccharide labeled by the brain cell cultures was shown to be Glc3Man9GlcNAc2 by HPLC analysis. Parallel incorporation studies with [3H]leucine showed that the increase in protein N‐glycosylation was relatively higher than a concurrent increase in cellular protein synthesis observed during the induction period. Maximal labeling of glycoprotein corresponded to the period of glial differentiation, as indicated by a sharp rise in the marker enzymes, 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (an oligodendroglial marker) and glutamine synthetase (an astroglial marker). The results describe a developmental activation of the N‐glycosylation pathway and suggest a possible relationship between N‐linked glycoprotein assembly and the growth and differentiation of glial cells.

Direct assay of membrane-associated protein kinase C activity in B lymphocytes in the presence of Brij 58.

This paper describes a simple and direct procedure for assaying Ca(2+)-dependent protein kinase C (PKC) activity in membrane fractions isolated from purified murine B lymphocytes (B cells) treated with phorbol 12-myristate 13-acetate (PMA). The results indicate that membrane-bound PKC in B cells, treated with PMA, can be measured directly in the presence of 0.5% Brij 58 by assaying the transfer of 32P from [gamma-32P]ATP to histone type III-S. This method obviates the need for partial purification of the protein kinase by ion-exchange chromatography prior to assaying PKC activity. The properties of membrane-associated PKC activity in B cells have been characterized, and the kinetics of PMA-induced translocation of PKC in cultured murine B cells, the rat glial tumor clone C6, and primary neonatal osteoblastic cells have been defined by this direct assay. The results obtained with B cells and the other cell lines indicate that this direct assay procedure could be useful for studies on the factors controlling PKC translocation in a variety of cultured mammalian cells.

LINKING CARDIOMETABOLIC DISORDERS TO SPORADIC AD: A PERSPECTIVE ON POTENTIAL MECHANISMS AND MEDIATORS

J. Neurochem. (2010) 115, 551–562.

Linking cardiometabolic disorders to sporadic Alzheimer’s disease: a perspective on potential mechanisms and mediators: Metabolic disorders and Alzheimer’s

J. Neurochem. (2010) 115, 551–562.

Embryonic rodent brain cells in culture.

Because of its cellular complexity and regional heterogeneity, the mammalian central nervous system is not easily amenable for experimental analysis. The study of the developing brain becomes even more complicated because of the differential growth rates of different parts of the brain. Primary culture techniques involving dissociation of discrete regions of the developing brain into component cells offer an excellent opportunity to study the regulation of growth and differentiation of neural cells and to investigate their biochemical, morphological, and physiological behavior under well-defined conditions. Methods are available now to isolate and grow individual classes of neural cells (i.e., neurons, astrocytes, oligodendrocytes), thereby enabling one to study the cellular behavior at the individual level and to uncover the nature of cell-cell interactions that presumably govern cell differentiation.