Biopsy-derived adult human brain tau is phosphorylated at many of the same sites as Alzheimer's disease paired helical filament tau.

Tau from Alzheimer's disease (AD) paired helical filaments (PHF-tau) is phosphorylated at sites not found in autopsy-derived adult tau from normal human brains, and this suggested that PHF-tau is abnormally phosphorylated. To explore this hypothesis, we examined human adult tau from brain biopsies and demonstrated that biopsy-derived tau is phosphorylated at most sites thought to be abnormally phosphorylated in PHF-tau. These sites also were phosphorylated in autopsy-derived human fetal tau and rapidly processed rat tau. The hypophosphorylation of autopsy-derived adult human tau is due to rapid dephosphorylation postmortem, and protein phosphatases 2A (PP2A) and 2B (PP2B) in human brain biopsies dephosphorylate tau in a site-specific manner. The down-regulation of phosphatases (i.e., PP2A and PP2B) in the AD brain could lead to the generation of maximally phosphorylated PHF-tau that does not bind microtubules and aggregates as PHFs in neurofibrillary tangles and dystrophic neurites.

Thermal denaturation of rat pulmonary and testicular angiotensin-converting enzyme isozymes. Effects of chelators and CoCl2.

In an attempt to assess the biochemical consequences resulting from structural differences between rat pulmonary and testicular angiotensin-converting enzyme, the thermal stability of crude and purified preparations of each enzyme was compared. Structural heterology was verified by molecular weight determinations and by peptide mapping after limited proteolysis with Staphylococcus V8 proteinase. Thermal stability was monitored by changes in catalytic activity following incubations at 55 degrees C in the presence of chelators and CoCl2. Purified pulmonary angiotensin-converting enzyme was more sensitive to inhibition by the chelators EDTA and 1,10-phenanthroline and by the site-directed inhibitor captopril than was the testicular isozyme. Although the pulmonary holoenzyme was unaffected by cobalt, the testicular holoenzyme was inhibited by cobalt in a concentration-dependent manner. Crude pulmonary angiotensin-converting enzyme was significantly more resistant to thermal denaturation than its crude testicular counterpart. The differences in the thermal lability of each isozyme were still present in purified preparations, although the purified enzymes appeared to be more thermally stable than their crude counterparts. Both chelators and cobalt markedly potentiated the thermal denaturation of each isozyme. These data suggest that the structural heterology of the pulmonary and testicular isozymes may affect the interaction of zinc with the respective enzymes and that zinc may contribute to the structural integrity and thermal stability of angiotensin-converting enzyme in each tissue.

Adrenalectomy-induced alterations of calmodulin-dependent hippocampal adenylate cyclase activity: role of guanine nucleotide-binding proteins.

Ca2+/calmodulin-dependent processes are altered by manipulations of the hypothalamic-pituitary-adrenal axis. In particular, adrenalectomy (ADX) attenuates hippocampal, but not cortical, calmodulin-dependent adenylate cyclase activity measured during the active (waking) phase of rats. The involvement of calmodulin- and guanine nucleotide (G)-binding proteins in the effects of ADX on the activity of calmodulin-dependent adenylate cyclase were investigated. In hippocampal membranes, inclusion of the GTP antagonist guanosine 5'-O-(2-thiodiphosphate) (250 microM) caused pronounced inhibition of calmodulin-stimulated adenylate cyclase activity. Guanosine 5(1)-O-(2-thiodiphosphate) had much smaller effects on calmodulin-independent (basal and forskolin-stimulated) enzyme activity. Substitution of Mn2+ for Mg2+ in the assay medium increased basal and forskolin-stimulated adenylate cyclase activity, but abolished calmodulin-dependent activation of this enzyme in both hippocampal and cortical membranes. These treatments blunted ADX-induced attenuation of hippocampal adenylate cyclase. ADX, with or without corticosterone administration (40 mg/kg, sc, once daily), failed to alter either Gi alpha or Gs alpha membrane protein content in either hippocampus or cortex. The levels of major membrane calmodulin-binding proteins in hippocampus and cortex also were not significantly altered by ADX. These results confirm that hormonal and biochemical regulation of calmodulin-dependent adenylate cyclase is distinct from that of other adenylate cyclase family members. Changes in Gs alpha and Gi alpha protein content alone cannot account for the effects of ADX on this enzyme. Overall, our studies suggest that the effects of ADX on calmodulin-dependent adenylate cyclase may occur through a reduction in the absolute amount of the catalytic subunit or an alteration(s) in the efficiency of coupling between adenylate cyclase and its modulators.

Identification of calmodulin-binding proteins.

We have outlined and partially characterized a series of biotinylated calmodulin derivatives that may be useful in the study of calmodulin-binding protein expression, physical points of calmodulin-target interaction, and proteolytic mapping of related calmodulin-binding proteins. Biotinylated calmodulins offer several advantages as probes of protein-protein interactions. First, biotinylation can be directed to different amino acid residues. Second, biotinylation can be carried out under mild, near-physiological conditions, reducing the likelihood that conditions of protein modification would destroy biological function. Third, biotinylated proteins are stable, and reagents needed for their preparation and detection are relatively inexpensive. Fourth, the sensitivity of avidin-chromogenic enzyme systems is approaching that of radioactivity, with the added advantage that chromogens can be visualized in a relatively short time with respect to autoradiography. However, as with any protein modification procedure, one must be cautious when interpreting the results obtained with biotinylated proteins. For calmodulin-binding proteins, some interactions are impaired by modification of specific lysyl residues. On the other hand, interaction of biotinylated calmodulin with phosphodiesterase occurs, but this interaction may obscure recognition of the biotin residue by avidin. One approach to circumvent this problem is to have a series of site-directed biotinylated proteins available for use as outlined in this chapter. The choice of which agent to use is determined by the primary sequence of the protein of interest and whether any information is available concerning the effects of chemical modification on structure (i.e., acetylation experiments, modification of free sulfhydryls). In the absence of such information, an empirical approach can be taken. Photobiotin affords an easy means for biotinylation of proteins; however, the sites of modification are not always predictable. NHS-biotin derivatives are readily available and are relatively easy to use. Finally, one may wish to biotinylate the protein while liganded to its normal interacting molecule, in the case of calmodulin, calcium ion is the obvious choice. However, calmodulin could also be biotinylated while bound to a specific binding protein such as calcineurin. The latter method may be of use in determination of changes in reactivities of specific amino acid residues subsequent to binding. Finally, it may prove advantageous to biotinylate genetically engineered calmodulin, yeast calmodulin, or plant calmodulin to further define calmodulin-target protein interactions. Thus, the use of biotinylated calmodulin derivatives may offer insights into a range of structural and functional questions relevant to regulation of specific calmodulin-binding proteins.

Immunohistochemical localization of protein-O-carboxylmethyltransferase in rat brain neurons.

The distribution of the enzyme protein-O-carboxylmethyltransferase (EC 2.1.1.24) has been investigated in the rat brain using both immunohistochemical and biochemical techniques. The enzyme, which carboxylmethylates free aspartic and glutamic acid residues of protein substrates, was localized in neurons, but not other cell types throughout the brain. The highest immunoreactivity was detected throughout the cortex, followed by the hippocampus, the corpus striatum, the thalamus and the amygdala. Immunoreactive cells were detected in other brain regions but were not as prominent as those regions listed above. The distribution of immunoreactivity in the hippocampus was most striking, with considerable labelling of the pyramidal and granule cells in all regions. Numerous pyramidal cells were labelled in the cerebral cortex, with some ascending processes exhibiting immunoreactivity. The corpus striatum was uniformly labelled, suggesting that the enzyme was not localized to any specific neurotransmitter system. The antisera employed in this study was generated against purified bovine brain protein-O-carboxylmethyltransferase and Western immunoblot analysis showed cross reactivity against both rat brain and human erythrocyte forms of the enzyme. Enzyme activity and methyl acceptor protein capacity were examined in 1.5 mm coronal sections of rat brain. The regions with highest enzyme activities were found in cross-sections containing cortex and corpus striatum or cortex and hippocampus. The lowest enzyme activities were noted in slices of brainstem and cerebellum, areas exhibiting low amounts of immunoreactive protein-O-carboxylmethyltransferase. Methyl acceptor protein capacity was highest in slices of cortex and corpus striatum, cortex and hippocampus and was lowest in slices of brainstem and cerebellum. These results demonstrate that protein-O-carboxylmethyltransferase has an unique neuronal pattern of distribution in the rodent central nervous system, and suggest that the carboxylmethylation of proteins may be of functional significance in these neurons.

Calcium-dependent interaction of calcineurin with Bcl-2 in neuronal tissue.

Calcineurin, a calmodulin-dependent protein phosphatase, regulates transcription and possibly apoptosis. Previous studies demonstrated that in baby hamster kidney-21 cells after co-transfection calcineurin interacts with Bcl-2, thereby altering transcription and apoptosis. Using co-immunoprecipitation and subcellular fractionation techniques, we observed that calcineurin occurred as a complex with Bcl-2 in various regions of rat and mouse brain. The calcineurin-Bcl-2 complex was identified in mitochondrial, nuclear, microsomal and cytosol fractions. In vitro induction of hypoxia and aglycia or N-methyl-D-aspartate treatment markedly altered both extent of complex formation and its subcellular localization. These observations suggest that Bcl-2 either sequesters calcineurin, that calcineurin dephosphorylates Bcl-2, or that Bcl-2 shuttles calcineurin to specific substrates. Calcineurin also co-immunoprecipitated with the inositol-tris-phosphate receptor. This interaction increased after in vitro hypoxia/aglycia. In Bcl-2 (-/-) mice, interactions between calcineurin- and inositol-tris-phosphate receptor occurred less frequently than in wild-type mice under both control and hypoxic conditions. Experiments involving cell-free systems, as well as brain slices treated with thapsigargin or with N-methyl-D-aspartate suggested that calcium and calmodulin activation of calcineurin leads to interactions between calcineurin and Bcl-2. These data indicate that during times of cellular stress and damage, Bcl-2 targets activated calcineurin to specific compartments and substrates.

Trimethyltin-induced neuronal damage in the rat brain: comparative studies using silver degeneration stains, immunocytochemistry and immunoassay for neuronotypic and gliotypic proteins.

Trimethyltin is a neurotoxicant which produces a distinct pattern of neuronal cell death following peripheral administration of a single dose (8 mg/kg, i.p.) in rats. The cupric-silver degeneration stain was used to produce an atlas documenting the distribution and time course of trimethyltin-induced neuronal damage in adult, male Long-Evans rats. Animals were examined at survival times of 1, 2, 3, 4, 5, 7, 10 and 18 days after intoxication. The earliest degeneration was observed at day 1 in the intermediate and ventral divisions of the lateral septal nucleus, followed by development of degeneration on days 2-4 in neuron populations including the septohippocampal nucleus, septohypothalamic nucleus, anterior olfactory nucleus, bed nucleus of the stria terminalis, endopiriform nucleus, parafascicular nucleus, superior colliculus, interstitial nucleus of the posterior commissure, inferior colliculus, pontine nuclei, raphe nuclei, pars caudalis of the spinal trigeminal nucleus, the caudal aspect of nucleus tractus solitarius, dorsal vagal motor nucleus, granule cells in the dentate gyrus, pyramidal cells in CA fields of the hippocampus, and of neurons in the subiculum, pyriform cortex, entorhinal cortex and neocortex (mainly layer Vb and VI). This was followed by degenerative changes on days 5-7 in other structures, including the amygdaloid nuclei, the ventral posterolateral and ventral posteromedial thalamic nuclei and the periaqueductal gray. The distribution of terminal degeneration from these neurons indicate that specific pools of cells are affected in each structure, and the time course suggests somatofugal degeneration. The trimethyltin damage was also assessed with immunocytochemical visualization of a neuronotypic protein, protein-O-carboxyl methyltransferase and a radioimmunoassay for glial fibrillary acidic protein. Protein-O-carboxyl methyltransferase immunoreactivity was altered in neuronal populations damaged by trimethyltin, but did not appear to be either as sensitive or selective an assay of neuronal damage as the silver stain, especially at short survival times. Glial fibrillary acidic proteins were dramatically elevated 21 days after trimethyltin intoxication, particularly in areas of extensive damage. These studies revealed advantages and problems encountered in the use of each technique in assessing neurotoxic effects, forming a basis for discussion of the relative merits of using a battery of specific molecular probes for neurotoxicity evaluations.

Alterations in hippocampal expression of SNAP-25, GAP-43, stannin and glial fibrillary acidic protein following mechanical and trimethyltin-induced injury in the rat.

A set of well-defined antisera against neuronal and glial proteins were used to characterize patterns of protein expression in rat hippocampus following transection of the fimbira-fornix and perforant pathways or after administration of the selective neurotoxicant trimethyltin (8 mg/kg, i.p.). SNAP-25 (synaptosomal protein, mol. wt 25,000) is a neuron-specific, developmentally regulated presynaptic protein, stannin is a protein enriched in cells sensitive to trimethyltin, and GAP-43 (growth-associated protein, mol. wt 43,000) is associated with axonal growth and regeneration. Glial fibrillary acidic protein is an astrocyte-specific intermediate filament protein and a marker for reactive gliosis. SNAP-25 immunoreactivity was altered following both neurotoxicant and mechanical injury. Three days after fimbria-fornix/perforant path lesions, there was a loss of SNAP-25 immunoreactivity in hippocampal efferent pathways and in the lesioned entorhinal cortex. By day 12, there was evidence of reinnervation of hippocampal subfields by SNAP-25-immunopositive commissural afferent fibers. On day 3, immunoblots showed the appearance of SNAP-25a, a developmental isoform produced by alternative splicing of nine amino acids in exon 5, in lesioned tissues. This isoform declined by day 12 and was not found in contralateral control hippocampus or non-lesioned brain regions. Stannin immunoreactivity was unchanged, while GAP-43 was prominent on day 12 post-lesion. Glial fibrillary acidic protein immunoreactivity indicated gliosis near the site of pathway transection. In contrast, trimethyltin induced a marked loss of stannin immunoreactivity in hippocampal neurons seven days after injection. Trimethyltin increased glial fibrillary acidic protein staining in the hippocampus and other damaged regions. SNAP-25 immunoreactivity was markedly increased in mossy fibers and other hippocampal fields seven days following trimethyltin. Immunoblot analysis showed that only the adult SNAP-25b isoform was expressed after trimethyltin intoxication. These data suggest that SNAP-25 is a useful marker for presynaptic damage. Furthermore, reexpression of developmental isoforms of SNAP-25a may precede functional reinnervation when the postsynaptic target remains intact.

In vitro hypoxia and excitotoxicity in human brain induce calcineurin-Bcl-2 interactions.

Although pathogenesis of neuronal ischemia is incompletely understood, evidence indicates apoptotic neuronal death after ischemia. Bcl-2, an anti-apoptotic and neuroprotective protein, interacts with calcineurin in non-neuronal tissues. Activation of calcineurin, which is abundant in the brain, may play a role in apoptosis. Using co-immunoprecipitation experiments in biopsy-derived, fresh human cortical and hippocampal slices, we examined possible interactions between calcineurin and Bcl-2. Calcineuin-Bcl-2 interactions increased after exposure in vitro to excitotoxic agents and conditions of hypoxia/aglycia. This interaction may shuttle calcineurin to substrates such as the inositol-1,4,5-tris-phosphate receptor because under these experimental conditions interactions between calcineurin and inositol-1,4,5-tris-phosphate receptor also increased. A specific calcineurin inhibitor, FK-520, attenuated insult-induced increases in calcineurin-Bcl-2 interactions and augmented caspase-3 like activity. These data suggest that Bcl-2 modulates neuroprotective effects of calcineurin and that calcineurin inhibitors increase ischemic neuronal damage.

Changes in expression of tyrosine hydroxylase immunoreactivity in human SMS-KCNR neuroblastoma following retinoic acid or phorbol ester-induced differentiation.

Human SMS-KCNR cells differentiated in response to either retinoic acid or phorbol esters; differentiated cells extended numerous, complex neurites and showed reduced proliferation. Tyrosine hydroxylase (TH) immunoreactivity was measured in this cell line following treatment with retinoic acid (1-10 microM), 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 16-160 nM), or combinations of these agents. After 21 days of treatment with either TPA or retinoic acid (RA), TH immunoreactivity was measured in this using densitometric scans of Western blots, was doubled relative to untreated or serum-deprived SMS-KCNR cultures. Increases in TH immunoreactivity could be detected after 6 days of treatment. Treatment with RA for 3 days followed by phorbol esters for an additional 3 days resulted in a 3-fold increase in TH immunoreactivity at day 6; reversing the order of drug treatment did not have this effect. Treatment of cultures with the divalent cationophore A23187 caused treated cells to retract neurites; expression of TH immunoreactivity was decreased relative to drug-treated and control cultures. These results suggest that retinoic acid treatment may 'prime' SMS-KCNR cells for the subsequent effects of phorbol esters, and indicate that the patterns of biochemical differentiation induced by TPA or RA are different.

Use of avidin-biotin subtractive hybridization to characterize mRNA common to neurons destroyed by the selective neurotoxicant trimethyltin.

Trimethyltin (TMT), a selective neurotoxicant, destroys a distinct subpopulation of neurons which possess no known biochemical or anatomic linkage. However, TMT-sensitive neurons may share common gene products related to susceptibility. In an effort to isolate mRNAs common to TMT-sensitive neurons, avidin/biotin based-subtractive hybridization was used to generate a cDNA library specifically related to TMT-toxicity. Out of 50 cDNAs, two clones hybridized only to poly(A+) mRNA isolated from the brains of saline-treated rats. Two of these cDNAs, p9T10 and p9T19, were used for in situ hybridization; both hybridized to hippocampus, limbic cortex, amygdala and other regions destroyed by TMT, suggesting that these probes identified mRNA enriched in TMT-sensitive neurons. The patterns of in situ hybridization coupled with the loss of p9T10 and p9T19 hybridization to mRNA isolated from the brains of TMT-treated rats suggests that one or both of these two clones may represent mRNA found in neurons damaged by TMT. The combination of selective neurotoxic lesions followed by cDNA subtractive hybridization should prove to be a useful strategy for the isolation of gene products from specific neuronal populations.

Protein carboxylmethylation and nervous system function.

Enzymatic protein-O-carboxylmethylation transfers methyl groups from S-adenosylmethionine to aspartyl and/or glutamyl residues of various methyl acceptor proteins. The function of this post-translational modification of protein, originally detected as "methanol-forming" activity in pituitary gland, has remained enigmatic in nervous tissue. Theories concerning the function of protein methylation have focused on possible roles in neurotransmitter release, neurophysin carboxylmethylation, regulation of calmodulin and calmodulin-binding proteins, chemotaxis, processing of precursor peptides, and repair/recognition of racemized D-amino acids. However, difficulties in establishing quantitative and temporal relationships between methylation and the biochemical event described have led to controversies. Similarly, the alkaline lability of the carboxylmethyl ester bond has led to difficulties in using the high resolution gel electrophoresis systems so successfully used in characterization of other post-translational events. Recent studies localizing protein-O-carboxylmethyltransferase to neurons in the rat brain suggest that this enzyme may be involved in signal transduction in the CNS. Alternative theories concerning protein methylation will be discussed and future directions for research in this area will be outlined.

Tyrosine hydroxylase: purification from PC-12 cells, characterization and production of antibodies.

Tyrosine hydroxylase has been purified to homogeneity from cultured PC-12 cells. The protein migrates as a single band with a molecular weight of 60,000 on sodium dodecyl sulfate polyacrylamide electrophoresis. Two-dimensional electrophoresis of the pure enzyme resolves three spots (each with molecular weights of 60,000) with isoelectric points of 5.4, 5.8 and 5.9. This charge heterogeneity cannot be explained by the presence of sugar or lipid moieties on the enzyme. Amino acid analysis indicates a relatively high content of hydrophobic amino acids and a lower serine content than other preparations of tyrosine hydroxylase. The enzyme hydroxylates tryptophan at approximately 1% of its rate of tyrosine hydroxylation but will not catalyze the hydroxylation of phenylalanine. Polyclonal antibodies were produced in rabbits against pure tyrosine hydroxylase and were judged to be monospecific by Western blot analysis. The IgG fraction was isolated from serum, and when coupled to cyanogen bromide activated Sepharose, could be used to purify tyrosine hydroxylase from crude extracts in a single step. The antiserum proved to be very useful in immunoprecipitation and immunocytochemical experiments with tyrosine hydroxylase.

Comparative studies on the distribution of protein-o-carboxylmethyltransferase and tyrosine hydroxylase in rat brain by immunocytochemistry.

The distribution of protein-O-carboxylmethyltransferase and tyrosine hydroxylase immunoreactivity in brain was compared with the use of highly specific polyclonal antibodies prepared against the native form of each enzyme. Protein-O-carboxylmethyltransferase was found in brain areas rich in catecholamine neurons as identified by tyrosine hydroxylase immunoreactivity. Rabbit anti-protein-O-carboxylmethyltransferase labeled cell bodies in the locus coeruleus, substantia nigra, and paraventricular nucleus whereas rabbit anti-tyrosine hydroxylase prepared against highly purified, native tyrosine hydroxylase from cultured PC12 cells labelled cell bodies in the same brain regions. In addition, the antibody to tyrosine hydroxylase made possible the visualization of very fine cortical processes containing tyrosine hydroxylase and very dense neuronal networks throughout the nigrostriatal pathway. The coincidence of protein-O-carboxylmethyltransferase and tyrosine hydroxylase in catecholamine rich brain areas provide an anatomical basis for the possibility that protein-O-carboxylmethyltransferase could modulate catecholaminergic neurotransmission.

Localization and characterization of stannin: relationship to cellular sensitivity to organotin compounds.

The cDNA encoding the protein stannin was isolated previously via subtractive hybridization, using differential expression after trimethyltin (TMT) intoxication, as a basis for isolating mRNA which may be expressed in TMT-sensitive cells. Initial characterization revealed a novel gene product which was differentially expressed in several tissues sensitive to TMT. In the current study, biochemical and molecular techniques were used to quantitate stannin expression at the cellular and subcellular levels. Northern blot analysis showed that the stannin 3.0 kb mRNA transcript was present, in decreasing amounts, in: spleen, hippocampus, neocortex, cerebellum, striatum, midbrain, kidney and lung. Liver, heart, skeletal muscle and testis showed no detectable expression of stannin mRNA. Immunoblot analysis using antipeptide antisera raised against stannin indicated a high level of expression in spleen, followed by brain and kidney. Stannin mRNA was present during early brain development and consolidated by post-natal day (PND) 20 to patterns and levels seen in adults. In situ hybridization studies showed widespread neuronal expression of stannin mRNA at PND 1, which shifted to a restricted pattern of expression in specific regions by PND 20. Stannin was partially purified from rodent brain and spleen using cation exchange, sizing and hydrophobic interaction chromatography. It behaved as a monomer throughout purification. Stannin was also expressed in a baculovirus system, using a series of constructs containing the entire cDNA, 1.0 kb of DNA flanking the open reading frame, and a 400 bp open reading frame minimal construct. While all constructs expressed stannin, the best expression was seen with the entire cDNA. Based on current findings, we suggest that stannin expression is necessary but not sufficient for TMT toxicity.

Protein-O-carboxylmethyltransferase in the rat brain: high regional levels in the substantia nigra, locus coeruleus and paraventricular nucleus.

Immunocytochemical techniques were used to localize protein-O-carboxylmethyltransferase in the rat brain. Particularly high levels of immunoreactive protein-O-carboxylmethyltransferase were found in the paraventricular and supraoptic nucleus, the substantia nigra and the locus coeruleus. The enhanced expression of the methyltransferase in these brain regions suggests that protein carboxylmethylation is of particular importance in these areas. These findings are consistent with previous biochemical studies which suggest that protein methylation plays a role in presynaptic monoaminergic neurons and in the release and/or processing of neurohypophyseal peptides.

Glial DNA synthesis and cell proliferation in the lesioned frontal cortex of the rat.

Proliferation of rat neurological cells was quantified following a lesion of the frontal cortex, with the rate of incorporation of intraventricularly administered [3H]thymidine ([3H]TdR) into cortical DNA serving as an index of glial proliferation. Incorporation of [3H]uridine into the corresponding RNA fractions did not serve this purpose. The intraventricular route of administration of thymidine greatly reduced the amount of [3H]TdR needed to label DNA relative to systemic injection. The rate of incorporation of [3H]TdR into DNA was linear for 75 min post-injection. Significantly more [3H]TdR was incorporated into DNA of the lesioned frontal cortex than that of the contralateral control cortex, during the first 4 days post-trauma. The majority of the acid-insoluble radioactivity (from [3H]TdR) was localized in the nuclear subcellular fraction of the cortex. Experiments indicated that the enhanced incorporation of [3H]TdR was not the result of altered metabolism or pool sizes of TdR in the lesioned cortex. Histological analysis indicated that there was a significant increase in the number of glial cells in the lesioned cortex by day 4 post-lesion, which corresponded to the increase in DNA synthetic activity. It was concluded that mechanical trauma to the frontal cortex of the rat results in an increase in the number of glial cells at and near the lesion which is accompanied by an increase in incorporation of [3H]TdR into cortical DNA. This method of measuring posttraumatic DNA synthesis has several advantages over autoradiography.

Developmental expression of neuronal calmodulin-binding proteins in rat brain.

The developmental patterns of calmodulin-binding proteins (CaM-BPS) in rat brain were examined using biotinylated calmodulin overlays of one- and two-dimensional gels. Hippocampus showed the earliest onset of CaM-BP expression (postnatal day 5; PND5), followed by cerebral cortex and striatum, both of which had detectable levels of CaM-BPs by PND7. Cerebellum had the latest onset of CaM-BP expression; CaM-BPs were not detectable until PND9. Very few CaM-BPs were present in brain before PND5 and all regions reached near adult levels by PND20. However, several unique CaM-BPs were seen in embryonic brain and these proteins may have an important role in developing neurons. These data suggest an orderly, complex expression of CaM-BPs which increases during times of synaptogenesis and synaptic maturation.

Expression of the calmodulin-dependent protein phosphatase, calcineurin, in rat brain: developmental patterns and the role of nigrostriatal innervation.

The distribution of neurons expressing the calmodulin-dependent protein phosphatase, calcineurin (CN) was characterized in developing and adult rat brain using a combination of immunocytochemical, immunoblot and in situ hybridization approaches. Immunoblot analysis revealed a strong increase postnatally in CN protein expression. Four differently-charged isoforms of CN were observed in adult brain with apparent regional differences in isoform expression. Immunocytochemistry showed highest levels of CN in hippocampus, striatum, substantia nigra, amygdala and septal nuclei with immunoreactivity first appearing in striatum and septal nuclei, followed by hippocampus, neocortex and limbic structures. In situ hybridization demonstrated that mRNA for the catalytic subunit of CN was seen as early as postnatal day (PND) 1 in striatum, cortex and hippocampus. Since immunoreactivity was not detectable until day 4, this suggests that mRNA expression may precede that of protein by several days in these regions. Lesioning of developing and adult nigrostriatal dopamine neurons either with 6-hydroxydopamine or by surgical hemitransection had little effect on expression of CN, suggesting that CN expression is not influenced transsynaptically by dopamine. Collectively, these findings demonstrate that CN protein and mRNA expression are subject to regional and temporal control during brain development suggesting that specific synaptic connections may influence CN gene expression. However, in striatum, dopaminergic innervation does not appear to affect CN levels.

Developmental expression of calmodulin-dependent cyclic nucleotide phosphodiesterase in rat brain.

The patterns of expression of calmodulin-dependent cyclic nucleotide phosphodiesterase (CaM-PDE) have been studied in developing and adult rat brain using affinity-purified polyclonal antibodies against CaM-PDE. An immunocytochemical map of adult brain regions expressing CaM-PDE, constructed from serial coronal brain sections, illustrated that CaM-PDE was expressed in specific neuronal subpopulations throughout the adult rat brain. Immunoblot analysis coupled with subcellular fractionation indicated that CaM-PDE was primarily localized to cytoplasmic fractions, with a small amount associated with synaptosomal membranes. Immunoblots from developing brain indicated that CaM-PDE expression increased dramatically during postnatal days 7-20 (PND 7-20); parallel increases in CaM-PDE enzyme activity occurred during this same time. Immunocytochemical studies indicated that several distinct patterns of CaM-PDE expression occurred during development. Neocortex showed low levels of CaM-PDE immunoreactivity in neuronal somata of layers III, V and VI on PND 4 that increased by PND 11; the adult somatodendritic pattern of immunoreactivity was observed by PND 60. Similar patterns were observed in cerebellar Purkinje cells, with somatodendritic staining observed by PND 12. By contrast, caudate-putamen, the inferior olive and the hypoglossal nuclei expressed high levels of CaM-PDE on PND 4, with levels considerably lower in the adult animal. The different patterns of expression suggest that in neocortex and cerebellum, CaM-PDE increases during the period of neuronal differentiation and active synaptogenesis, while in the caudate-putamen, inferior olive and hypoglossal nucleus, high levels may be required early in development.