Dendritic translocation of RC3/neurogranin mRNA in normal aging, Alzheimer disease and fronto-temporal dementia.

RC3/neurogranin is a postsynaptic protein kinase C (PKC)-/calmodulin-binding substrate implicated in long-term potentiation (LTP) forms of synaptic plasticity. Our previous digoxigenin in situ hybridization (DIG-ISH) studies detected RC3 mRNA in apical dendrites and cell bodies of neurons in the rat cerebral cortex and hippocampus. This observation suggested that RC3 mRNA is selectively translocated to dendrites, where it may be translated locally in response to synaptic activity. To test this hypothesis further, we isolated a full-length cDNA clone of the homologous human RC3 mRNA from a human cortex lambda GT11 library, determined its nucleotide and predicted amino acid sequences, and performed mRNA expression studies in cerebral cortex from normal human patients and from patients with Alzheimer disease (AD) and fronto-temporal dementia (FTD). The human cDNA clone detects a single approximately 1.3 kb mRNA whose nucleotide sequence is 73% similar to the rat nucleotide sequence and 96% similar to its amino acid sequence. DIG-ISH studies detect robust staining of RC3 mRNA in cell bodies of numerous neurons throughout Layers II-VI and in both apical and basal dendrites of pyramidal neurons in human neocortex (temporal/frontal). We conclude that dendritic targeting of RC3 mRNA is conserved in human brain. In AD neocortex tissue, there is little or no evidence for RC3 mRNA translocation to dendrites, while in FTD neocortex, targeting of RC3 mRNA to apical dendrites is preserved. Comparative studies in AD and FTD point to the potential importance of synapse integrity and the dendritic cytoskeleton in RC3 mRNA targeting in the human neocortex.

Functional studies of single-site variants in the calmodulin-binding domain of RC3/neurogranin in Xenopus oocytes.

Single-site variants in the calmodulin-binding domain of RC3/neurogranin were heterologously expressed in Xenopus oocytes to examine their effects on serotonin-evoked currents. RC3 variants serine36 -->alanine (Ser36-->Ala), serine36-->glycine (Ser36-->Gly), and phenylalanine37-->tryptophan (Phe37-->Trp), which bind calmodulin but are deficient in protein kinase C (PKC) phosphorylation, display serotonin-evoked Ca(2+)-dependent Cl- currents in oocytes similar to control oocytes. A serine36-->aspartate (Ser36-->Asp) variant, which does not bind calmodulin and mimics the PKC-phosphorylated state of RC3, significantly enhances serotonin-evoked currents in a manner similar to wild-type. The results suggest that RC3 not only regulates the availability of free calmodulin in a dendritic spine but also, when phosphorylated, independently stimulates G-protein coupled second messenger pathways that generate inositol 1,4,5-trisphosphate (IP3), diacylglycerol (DAG) and intracellular Ca2+.

Localization of RC3 (neurogranin) in rat brain subcellular fractions.

Previous studies have shown that RC3 (neurogranin) is a postsynaptic, protein kinase C (PKC)/calmodulin-binding substrate that accumulates throughout the perikaryal and dendritic cytoplasm and is often closely associated with the postsynaptic density (PSD) in dendritic spines of neostriatal neurons. Here Western immunoblotting studies of rat brain subcellular fractions confirm that RC3 is predominantly a cytosolic protein but is found in lower amounts in membrane-enriched microsomes and synaptosomes. Solubilization of synaptosomes suggests that RC3 may only be loosely associated with the PSD.

G-protein gamma 7 subunit is selectively expressed in medium-sized neurons and dendrites of the rat neostriatum.

We used subtractive hybridization to isolate clones of gamma 7, a 68 residue G-protein gamma subunit. Northern blotting and in situ hybridization reveal that the gamma 7 subunit mRNA is expressed primarily in medium-sized neurons of the neostriatum and nucleus accumbens and neurons of the olfactory tubercle, and at low levels in the dentate gyrus of the hippocampal formation and laminae II-III, and V of the neocortex. The gamma 7 mRNA is translocated into dendrites of neurons in the neostriatum and the dentate gyrus of the hippocampus. gamma 7 is expressed at relatively very low concentrations in peripheral tissues. The selective pattern of gamma 7 expression within the brain is highly reminiscent of those of the striatum-enriched adenylyl cyclase ACST, dopamine receptors, and the alpha subunit of G(olf), suggesting that, in striatum, gamma 7 may be a subunit of a G(olf) alpha-containing G protein that couples dopamine receptors selectively to ACST.

Functional consequences of expression of the neuron-specific, protein kinase C substrate RC3 (neurogranin) in Xenopus oocytes.

RC3 (neurogranin) is a neuron-specific substrate of protein kinase C (PKC) that accumulates predominantly in dendritic spines of forebrain neurons and undergoes long-term potentiation (LTP)-associated increases in PKC-phosphorylation in hippocampal slices. Here the hypothesis that RC3 functions by modulating the IP3/DAG second messenger pathway after its phosphorylation by DAG-activated PKC was tested by heterologous expression in Xenopus oocytes. Acetylcholine-evoked inward chloride (Cl-) currents, dependent on both IP3 release and intracellular calcium (Ca2+), were 2- to 3-fold higher in RC3-injected oocytes than in uninjected control oocytes. RC3-oocytes did not exhibit enhanced currents when preincubated with the protein kinase inhibitor H-7 or when a glycine residue was substituted for serine, the PKC phosphorylation site of RC3. Activation of endogenous oocyte PKC by phorbol esters generated inward Cl- currents in RC3 oocytes but not in control oocytes. RC3-dependent Cl- currents were also elicited by phorbol ester in Ca(2+)-free media. We propose that PKC-phosphorylated RC3 is capable of enhancing the mobilization of intracellular Ca2+ in Xenopus oocytes and, by inference, may play a role in Ca2+ homeostasis in dendrites of forebrain neurons.

Identification of cortexin: a novel, neuron-specific, 82-residue membrane protein enriched in rodent cerebral cortex.

Nucleotide sequence analysis of a cDNA clone of a rat cortex-enriched mRNA identifies a novel integral membrane protein of 82 amino acids. The encoded protein is named cortexin to reflect its enriched expression in cortex. The amino acid sequence of rat cortexin and its mouse homologue are nearly identical (98% similarity), and both contain a conserved single membrane-spanning region in the middle of each sequence. Northern blot analysis shows that cortexin mRNA is brain-specific, cortex-enriched, and present at significant levels in fetal brain, with peak expression in postnatal rodent brain. In situ hybridization studies detect cortexin mRNA primarily in neurons of rodent cerebral cortex, but not in cells of the hindbrain or white matter regions. The function of cortexin may be particularly important to neurons of both the developing and adult cerebral cortex.