Developmental changes in Ca2+/calmodulin-dependent protein kinase II in cultures of hippocampal pyramidal neurons and astrocytes

Abstract

We have analyzed Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) localization, activity, and endogenous protein substrates during differentiation and synaptogenesis in cultured hippocampal neurons. Primary cultures from hippocampi from 18 d embryonic rats are composed primarily of pyramidal neurons, with minimal contamination by nonneuronal cells. We have used monoclonal (Mab) and affinity-purified polyclonal antibodies that recognize either or both of the subunits of CaM-kinase II in order to localize the enzyme at progressive stages of neuronal differentiation. Diffuse but specific binding, determined by indirect immunofluorescence analyses, was first detected in cell bodies and growth cones of pyramidal neurons after 4 d in culture. Immunoreactivity increased during the next 3 d of culture, at which time fluorescent, labeling was patchy along neuritic processes. By 10 d, intensely fluorescent, discrete spots were observed along processes and on cell bodies. Astrocyte cultures prepared from newborn rat cortex showed no detectable immunofluorescence with anti-CaM-kinase II antibodies. Cytosolic and particulate fractions from cultured pyramidal neurons and astrocytes were analyzed using immunoblot, in vitro phosphorylation, 2-dimensional gel electrophoresis, and phosphopeptide mapping techniques. Although pure astrocyte cultures contained low levels of Ca2+/CaM-stimulated protein kinase activity, they did not display detectable levels of immunoreactive 50 kDa subunit nor 50 and 60 kDa phosphoproteins analogous to the autophosphorylated subunits of CaM-kinase II. Immunoblot analysis detected the 60 kDa kinase subunit in particulate and cytosolic fractions from 2 d neurons. By contrast, the 50 kDa subunit of CaM-kinase II was not detected in cytosolic or particulate fractions of pyramidal neurons before 4 d in culture. In 2 d pyramidal neuron cultures, only low levels of Ca2+/CaM-stimulated protein phosphorylation were observed. Ca2+/CaM-dependent phosphorylation of 10 d pyramidal cell proteins was 3-5-fold greater than that of 2 d cultures, and included major phosphoproteins of 48, 50, 56, 58/60, 80-86, 90, 120, 138, 175, and 190 kDa. Phosphopeptide maps of 58/60 and 50 kDa phosphoproteins gave patterns very similar to those of the autophosphorylated 60 and 50 kDa subunits, respectively, of purified CaM-kinase II. A phosphoprotein doublet of 83 kDa was identified as synapsin I. Developmental changes in Ca2+/CaM-dependent phosphorylation in pyramidal neuron cultures were very similar to those previously described in subcellular fractions from postnatal rat forebrain.

Spatial and Temporal Regulation of Ca2+/Calmodulin-Dependent Protein Kinase II Activity in Developing Neurons

“…The apparent requirement for ␣CaMKII expression is consistent with the reported low levels of Ca 2ϩ /calmodulin-stimulated protein phosphorylation in 2 DIV cultures of hippocampal neurons (Scholz et al, 1988).…”

Regulation of the Development of Mesencephalic Dopaminergic Systems by the Selective Expression of Glial Cell Line-Derived Neurotrophic Factor in Their Targets

“…No expression was observed in large striatal neurons or non-neural cell types. This neuronal cellular pattern of expression is in keeping with the known expression patterns of CaMKII (Ouimet et al, 1984;Scholz et al, 1988). Thus, transgene expression occurs in a cellular population similar to that identified for endogenous GDNF (Lopez-Martin et al, 1999).…”

Spatial Patterns of Ca2+ Signals Define Intracellular Distribution of a Signaling by Ca2+/Calmodulin-dependent Protein Kinase II

“…Previous studies have demonstrated the existence of CaMKII in astrocytes (26,28), and CaMKII activated by Ca 2ϩ was shown to phosphorylate vimentin in these cells (19,26). Furthermore, they display local and global Ca 2ϩ signaling in response to neurotransmitters (8,29 (Fig.…”