Layer-specific activity of tissue non-specific alkaline phosphatase in the human neocortex.

The ectoenzyme tissue non-specific alkaline phosphatase (TNAP) is mostly known for its role in bone mineralization. However, in the severe form of hypophosphatasia, TNAP deficiency also results in epileptic seizures, suggesting a role of this enzyme in brain functions. Accordingly, TNAP activity was shown in the neuropil of the cerebral cortex in diverse mammalian species. However in spite of its clinical significance, the neuronal localization of TNAP has not been investigated in the human brain. By using enzyme histochemistry, we found an unprecedented pattern of TNAP activity appearing as an uninterrupted layer across diverse occipital-, frontal- and temporal lobe areas of the human cerebral cortex. This marked TNAP-active band was localized infragranulary in layer 5 as defined by quantitative comparisons on parallel sections stained by various techniques to reveal the laminar pattern. On the contrary, TNAP activity was localized in layer 4 of the primary visual and somatosensory cortices, which is consistent with earlier observations on other species. This result suggests that the expression of TNAP in the thalamo-recipient granular layer is an evolutionary conserved feature of the sensory cortex. The observations of the present study also suggest that diverse neurocognitive functions share a common cerebral cortical mechanism depending on TNAP activity in layer 5. In summary, the present data point on the distinctive role of layer 5 in cortical computation and neurological disorders caused by TNAP dysfunctions in the human brain.

Alteration of the pro-oxidant xanthine oxidase (XO) in the thalamus and occipital cortex of patients with schizophrenia.

OBJECTIVES: Mounting evidence shows that oxidative stress (OS) and the purine/adenosine system play a key role in the pathophysiology of schizophrenia. Lately, our group pointed out that not only antioxidants, but also the prooxidant system plays an important role in neuro-psychiatric disorders. Xanthine oxidase (XO) is an enzyme of special interest in this context, since it acts as a prooxidant, but its main product is a vastly important antioxidant, uric acid (UA). Furthermore, XO plays major part in the purine/adenosine metabolism, which has been hypothesised to play a role in schizophrenia as well. METHODS: We examined the activity of XO in the striato-cortico-limbic system of schizophrenic patients (SP) and controls using a commercially available activity assay. RESULTS: We found decreased activity of XO in the occipital cortex and thalamus of patients with psychosis. Furthermore, XO shows a significant positive correlation with chlorpromazine equivalents in the putamen and the temporal cortex. CONCLUSIONS: Nevertheless, our results might suggest a downregulation of cellular defence mechanisms in schizophrenia in several brain regions, which could account for neuronal alterations which have been described before. This demonstrates that more research is needed to fully understand the role of the complex enzyme XO in the pathophysiology of schizophrenia.

Differential distribution of NADPH-diaphorase histochemistry in human cerebral cortex.

Beta-nicotinamidedinucleotide phosphate diaphorase (NADPH-d) colocalizes with NOS in the central nervous system. Two types of NADPH-d-positive neurons are present in the primate cerebral cortex: type 1, intensely and Golgi-like labeled neurons, a subset of GABAergic interneurons; type 2, lightly labeled neurons (divided into two subclasses, a first one having a lightly stained cell body bearing only one short process, and a second one showing intense NADPH-d staining with short processes extending radially). We have analyzed the distribution of NADPH-d activity in human frontal, temporal, and occipital cortical areas, finding remarkable laminar and interareal differences in cell size and distribution of the different cell types. There was a clear bias for type 1 neurons in infragranular layers in all areas considered; both in supra- and infragranular layers, their density was highest in frontal, and lowest in temporal cortex. The density of type 2 neurons was lower supragranularly in temporal cortex and infragranularly in occipital cortex. The overall density of type 2 cells was remarkably higher in occipital cortex than in the temporal and frontal ones. Type 1 neurons were significantly larger than type 2, and were smaller in the supragranular than in the infragranular subzone in occipital and temporal cortex. Type 1 cells were significantly larger in frontal cortex than in occipital and temporal cortex, and type 2 cells were significantly smaller in occipital than in temporal and frontal cortex. These area-related differences might reflect differences between heterotypic and homotypic cortex in the regulation of cortical blood flow.

Impaired GABA neuronal response to acute benzodiazepine administration in panic disorder.

OBJECTIVE: Disturbances in the metabolism of the brain amino acid transmitter gamma-aminobutyric acid (GABA) may contribute to the pathophysiology of human anxiety disorders. Animal studies indicate that deletions or reductions in the expression of the gene for the GABA synthetic enzyme, glutamate decarboxylase 65 (GAD(65)), reduce basal cortical GABA levels or stress-induced release of GABA in the cerebral cortex and increase fear behaviors. Complementing these findings, the authors recently observed lower than normal cortical GABA levels in patients with panic disorder. In the current study, the authors tested the hypothesis that panic disorder patients have a deficient GABA neuronal response to benzodiazepine (clonazepam) administration. METHOD: In a parallel-group, repeated-measures design, occipital cortex GABA responses to acute oral, open-label benzodiazepine administration were tested in 10 panic disorder patients and nine healthy comparison subjects. Occipital cortex total GABA levels were measured before and after medication administration by means of a novel proton magnetic resonance spectroscopic technique. RESULTS: Panic disorder patients had a deficient GABA neuronal response (blunted reduction of occipital cortex GABA level) to acute benzodiazepine administration, compared to the healthy subjects, who exhibited a significant decrease in occipital cortex GABA levels after this intervention. The patients also appeared to have persistently low occipital cortex GABA after chronic benzodiazepine treatment. CONCLUSIONS: Overall, these data are consistent with the hypothesis that a trait-like abnormality in GABA neuronal function contributes to the pathogenesis of human panic disorder. The data raise the possibility that GAD(65) enzyme dysfunction could be a pathogenic factor in panic disorder.

Timing and plasticity of specification of CaM-Kinase II alpha expression by neocortical neurons.

In this work, the differential expression of a chemical marker, the alpha-isoform of the calcium/calmodulin-dependent protein kinase II (CaM-Kinase II alpha) and the development of the spinal cord projection were used to determine in vivo the embryonic stages at which different aspects of the phenotype of neocortical cells are specified. We first performed a quantitative, immunocytochemical study on the levels of CaM-Kinase II alpha expression in the frontal, parietal and occipital cortical areas of control adult rats. We found that the levels of expression of CaM-Kinase II alpha were larger in the frontal and parietal areas than in the occipital areas. In addition, all layer V neurons identified as projecting to the spinal cord were CaM-Kinase II alpha immunopositive. We then grafted embryonic day (E) 12 or 14 cells from the presumptive frontal or occipital cortex of donor fetuses into the frontal or occipital cortex of newborn hosts. Cortical cells grafted at E12 differentiate neurons with molecular (CaM-Kinase II alpha) and connectivity (spinal cord projection) phenotypes appropriate to the cortical area where they complete their development whereas cells taken at E14 differentiate neurons with molecular and connectivity phenotypes appropriate to their cortical locus of origin. These findings suggest that E12 progenitors destined to generate layer V neurons are multipotent. The final phenotype of their progeny depends on regionalizing signals expressed in the environment. Later in corticogenesis, committed progenitors become unable to respond to regionalizing signals and generate neurons whose phenotype is appropriate to the initial cortical position of the precursor.

Distribution of prolyl endopeptidase activities in rat and human brain.

Prolyl endopeptidase is a proteolytic enzyme which could have a neuropeptide catabolising role in the central nervous system. Although prolyl endopeptidase has been described as a cytosolic enzyme, it has become clear that it can also be found in particulate form. The regional and subcellular distribution of this enzyme was evaluated in rat and human brain. The activity of the enzyme was higher in the human than in the rat brain. In the human brain, the activity levels of both soluble and particulate prolyl endopeptidase were the highest in frontal, parietal and occipital cortices and the lowest in the cerebellum. In the rat brain, the regional distribution of the enzyme was more homogeneous. The activity in all the areas of the central nervous system is higher than in peripheral tissues. Subcellular distribution of the enzyme in the brain indicates that prolyl endopeptidase was higher in the cytosolic fraction than in the particulate fractions. The particulate form was enriched in the synaptosomal and the myelinic membranes. The high activity of prolyl endopeptidase in the human cortex suggests that prolyl endopeptidase could play a role in the functions of this brain area.

Unique morphological features of the proliferative zones and postmitotic compartments of the neural epithelium giving rise to striate and extrastriate cortex in the monkey.

We examined the development of the occipital lobe in fetal monkeys between embryonic day 37 (E37) and E108 in Nissl-stained and acetylcholine esterase (AChE)-reacted sections. We paid particular attention to features that distinguish the development of presumptive area 17. At E46 the neuroepithelium consists of a ventricular zone and a monolayer cortical plate sandwiched between a thin marginal zone and a minimal presubplate. Between E55 and E65 an augmented subplate emerges and continues to expand up to E94 to become a major compartment of the developing cortex. A mitotic subventricular zone is established by E55. Peaking in depth at E72, it constitutes the principal germinal zone. By E78 an invading fibre tract divides it into an outer radially organized zone and a more conventional inner zone. AChE staining reveals the future area 17/18 border from E86 onwards. Proceeding from presumptive area 17 to area 18 there is a progressive thinning of the radially structured subventricular zone. Comparison of these results with corticogenesis in rodents suggests a number of potentially unique primate features: (i) a minimal preplate stage; (ii) a radially augmented germinal zone not previously described in non-primates; (iii) a fibre tract dividing the subventricular zone into two laminae; (iv) late generation and expansion of the subplate.

Low GSK-3 activity in frontal cortex of schizophrenic patients.

Glycogen synthase kinase-3 (GSK-3) (EC 2.7.1.37) is a protein kinase highly abundant in brain and involved in signal transduction cascades of multiple cellular processes, particularly neurodevelopment. Two forms of the enzyme, GSK-3alpha and -3beta have been previously identified. We have previously reported reduced GSK-3beta protein levels in postmortem frontal cortex of schizophrenic patients. In an attempt to explore whether reduction of GSK-3beta levels is brain region specific we examined it in occipital cortex. In order to find out if the reduction in frontal cortex is reflected in altered activity we measured GSK-3 enzymatic activity in this brain region. Western-blot analysis of GSK-3beta was carried out in postmortem occipital cortex of 15 schizophrenic, 15 bipolar, and 15 unipolar patients, and 15 normal controls. GSK-3 activity was measured by quantitating the phosphorylation of the specific substrate phospho-CREB in the frontal cortex specimens. GSK-3beta levels in occipital cortex did not differ between the four diagnostic groups. GSK-3 activity in the frontal cortex of schizophrenic patients was 45% lower than that of normal controls (0.196+/-0.082 and 0.357+/-0.084 pmol/mg proteinxmin, respectively; Kruskal-Wallis analysis: chi-square=8.27, df=3, p=0.04). The other two diagnostic groups showed no difference from the control group. Our results are consistent with the notion that schizophrenia involves neurodevelopmental pathology.

Inositol monophosphatase activity in brain and lymphocyte-derived cell lines of bipolar patients.

BACKGROUND: Inositol monophosphatase (IMPase) activity was reported to be low in lymphocyte-derived cell lines of bipolar patients. METHODS: IMPase activity was measured spectrophotometrically as inorganic phosphate liberated from inositol-1-phosphate. RESULTS: The previously reported reduction was replicated in a new, small group of bipolar patients. The reduction is not present in cell lines of unipolar or schizophrenic patients. IMPase activity in postmortem frontal and occipital cortical samples of unipolar, bipolar and schizophrenic patients was not different from controls. CONCLUSIONS: A reduction in lymphocyte-derived IMPase activity without a parallel reduction in cortical IMPase activity could be due to the fact that most leukocyte IMPase activity is the product of the IMPA-2 gene.

Brain matrix metalloproteinase 1 levels are elevated in Alzheimer's disease.

Several lines of evidence indicate that there may be an inflammatory component to the pathology of Alzheimer's disease (AD), the major form of degenerative dementia in the elderly. Activity of inflammatory cells, and the elaboration of toxic molecules by such cells may be a significant factor in disease progression. In peripheral inflammatory states, the increased activity of matrix metalloproteinase (MMP) enzymes are a major cause of tissue breakdown and secondary damage in diseases such as rheumatoid arthritis. The activity of such enzymes in the normal or diseased central nervous system is, however, not well characterized. We have therefore determined the levels of MMP 1 (collagenase) in the normal human brain and in AD. MMP1 levels were relatively low though were significantly elevated by approximately 50% in AD in all cortical areas examined. Given the activity towards collagen of MMP1, it is possible that enhanced MMP1 activity in AD, may contribute to the blood-brain barrier dysfunction seen in AD.

Altered phosphofructokinase mRNA levels but unchanged isoenzyme pattern in brains from patients with Alzheimer's disease.

In order to find out whether the increased phosphofructokinase (PFK) activities observed in brains from Alzheimer's disease (AD) patients are associated with alterations in PFK mRNA levels, we determined total PFK mRNA and the three different PFK isoenzyme mRNAs in AD and control patients by ribonuclease protection assay (RPA) and quantitative RT-PCR. PFK mRNA levels were found increased in some brain areas in AD patients. While all three PFK isoenzyme mRNAs were detectable in every studied brain sample, no changes of the PFK isoenzyme pattern were observed in patients with AD.

Abnormal protein phosphorylation in post-mortem brain tissue from bipolar patients.

Abnormal phosphorylation has been proposed to be involved in the pathogenesis of affective disorders. The present study investigated basal and cAMP-stimulated endogenous protein phosphorylation in human post-mortem brain tissue from bipolar and schizophrenic patients. Furthermore, basal kinase activity and stimulated protein kinase A activity were measured. The frontal and occipital cortex were analysed. Using [gamma-32P]ATP as phosphate donor, basal and cAMP-stimulated phosphorylation of endogenous proteins was measured in the absence or presence of 8-Br-cAMP, respectively. The proteins were separated on SDS-gels and the radioactivity in the individual bands was measured. We observed a significant reduction of 32P incorporation in three protein substrates (15, 16 and 21 kD) in frontal cortex of bipolar patients. However, there were no differences in the PKA activity between any of the groups. The present study demonstrates abnormal phosphorylation of specific proteins in brain tissue obtained from bipolar patients in comparison to schizophrenics and controls.

Deficient platelet-activating factor and related enzymes in hemimegalencephaly.

Platelet-activating factor (PAF) may influence neuronal migration, and gyral anomaly in hemimegalencephaly is believed to result from a neuronal disorder. A 7-month-old girl with hemimegalencephaly presented with intractable seizures, for which graded hemispherectomy was performed. In the resected specimen, we could not detect PAF, and related enzyme activities were low. These results suggest a role of PAF in neuronal migration.

Nitric oxide synthase neurons in different areas of normal aged and Alzheimer's brains.

This study investigated the distribution of nitric oxide synthase-containing neurons in the cerebral cortex of individuals with Alzheimer's disease, and compared them with age-matched controls. Paraffin-embedded sections of the frontal (area 10), occipital (area 17) and entorhinal cortices (area 28), and hippocampal formation obtained from 13 autopsy cases were used in the study. Neurons expressing nitric oxide synthase messenger RNA and protein were identified, respectively, by in situ hybridization and immunohistochemistry. Optical densities of nitric oxide synthase-positive neurons were assessed in 50 randomly selected fields of each of the above regions of the cortices, in each case by microscopic photometry. In the frontal cortex of the Alzheimer group, while a decrease in the number of nitric oxide synthase-positive neurons was evident, the nitric oxide synthase neurons, on the other hand, showed an increased optical density in layers II-IV when compared with those of normal ageing. In the occipital cortices, no significant differences in optical density were recorded between the normal ageing and Alzheimer specimens. In the entorhinal cortex, the optical densities of nitric oxide synthase neurons were again similar between the Alzheimer and age-matched control groups. In the hippocampar formation itself, there was an increase of nitric oxide synthase staining in the Alzheimer patients. These results show that (i) nitric oxide synthase neurons are abundant in the human cortex, (ii) the distribution of nitric oxide synthase neurons differs between different cortical regions, and (iii) there are differences between normal ageing and Alzheimer patients in the frontal cortex and the hippocampus.

Activity of phosphatidylethanolamine-N-methyltransferase in brain affected by Alzheimer's disease.

To determine whether phospholipid abnormality in Alzheimer's disease is associated with modification of phosphatidylethanolamine-N-methyltransferase, the activity of the enzyme was analysed in the frontal and occipital cortex of the brain from patients with Alzheimer's disease and from aged-matched control. The optimum pH for phosphatidylethanolamine-N-methyltransferase in human brain was 9.0. The enzyme activity was stimulated by detergent TWEEN 20 but inhibited by Triton X-100. Neither magnesium dependence nor chemical methylation was found. A decrease in activity of phosphatidylethanolamine-N-methyltransferase was observed in the frontal cortex of brain affected with Alzheimer's disease. The addition of exogenous phosphatidylethanolamine resulted in no modification in the methylation rate as compared with that of endogenous PE. The addition of phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N-dimethylethanolamine resulted in significantly increased rates of methylation in brain tissues. However, the increased rate of phosphatidylethanolamine-N-methyltransferase activity stimulated by exogenous phospholipids was lower in the frontal cortex of brains with Alzheimer's disease when compared to the normals and there was no difference in the occipital cortex between Alzheimer's disease and the control. It is plausible that the decreased activity of phosphatidylethanolamine-N-methyltransferase and its low compensating ability could relate to the modification of phosphatidylcholine in brain tissues from Alzheimer's disease patients.

Decreased brain protein levels of cytochrome oxidase subunits in Alzheimer's disease and in hereditary spinocerebellar ataxia disorders: a nonspecific change?

Controversy exists as to the clinical importance, cause, and disease specificity of the cytochrome oxidase (CO) activity reduction observed in some patients with Alzheimer's disease (AD). Although it is assumed that the enzyme is present in normal amount in AD, no direct measurements of specific CO protein subunits have been conducted. We measured protein levels of CO subunits encoded by mitochondrial (COX I, COX II) and nuclear (COX IV, COX VIc) DNA in autopsied brain of patients with AD whom we previously reported had decreased cerebral cortical CO activity. To assess disease specificity, groups of patients with spinocerebellar ataxia type I and Friedreich's ataxia were also included. As compared with the controls, mean protein concentrations of all four CO subunits were significantly decreased (-19 to -47%) in temporal and parietal cortices in the AD group but were not significantly reduced (-12 to -17%) in occipital cortex. The magnitude of the reduction in protein levels of the CO subunits encoded by mitochondrial DNA (-42 to -47%) generally exceeded that encoded by nuclear DNA (-19 to -43%). In the spinocerebellar ataxia disorders, COX I and COX II levels were significantly decreased in cerebellar cortex (-22 to -32%) but were normal or close to normal in cerebral cortex, an area relatively unaffected by neurodegeneration. We conclude that protein levels of mitochondrial- and nuclear-encoded CO subunits are moderately reduced in degenerating but not in relatively spared brain areas in AD and that the decrease is not specific to this disorder. The simplest explanation for our findings is that CO is decreased in human brain disorders as a secondary event in brain areas having reduced neuronal activity or neuronal/synaptic elements consequent to the primary neurodegenerative process.

Ornithine decarboxylase in human brain: influence of aging, regional distribution, and Alzheimer's disease.

Although experimental animal data have implicated ornithine decarboxylase, a key regulatory enzyme of polyamine biosynthesis, in brain development and function, little information is available on this enzyme in normal or abnormal human brain. We examined the influence, in autopsied human brain, of postnatal development and aging, regional distribution, and Alzheimer's disease on the activity of ornithine decarboxylase. Consistent with animal data, human brain ornithine decarboxylase activity was highest in the perinatal period, declining sharply (by approximately 60%) during the first year of life to values that remained generally unchanged up to senescence. In adult brain, a moderately heterogeneous regional distribution of enzyme activity was observed, with high levels in the thalamus and occipital cortex and low levels in cerebellar cortex and putamen. In the Alzheimer's disease group, mean ornithine decarboxylase activity was significantly increased in the temporal cortex (+76%), reduced in occipital cortex (-70%), and unchanged in hippocampus and putamen. In contrast, brain enzyme activity was normal in patients with the neurodegenerative disorder spinocerebellar ataxia type I. Our demonstration of ornithine decarboxylase activity in neonatal and adult human brain suggests roles for ornithine decarboxylase in both developing and mature brain function, and we provide further evidence for the involvement of abnormal polyamine system activity in Alzheimer's disease.

Elevated adenosine monophosphate deaminase activity in Alzheimer's disease brain.

Abnormal elevations in ammonia have been implicated in the pathogenesis of Alzheimer's disease. However, the biochemical mechanism(s) leading to increased ammonia in Alzheimer's disease have not yet been identified. A potential source of increased ammonia production is adenosine monophosphate (AMP) deaminase, an important enzyme in the regulation of the purine nucleotide cycle and adenylate energy charge. AMP deaminase activity is expressed in human brain and converts AMP to inosine monophosphate with the release of ammonia. We have investigated AMP deaminase activity in postmortem brain tissue from Alzheimer's disease subjects and age-matched controls. Compared to control brain, Alzheimer's disease brain AMP deaminase activity is 1.6- to 2.4-fold greater in the regions examined--the cerebellum, occipital cortex, and temporal cortex. Similar increases in AMP deaminase protein and mRNA levels are observed in Alzheimer's disease brain. These results suggest that increased AMP deaminase activity may augment ammonia levels in the brain in Alzheimer's disease.

Reduced cholinergic function in normal and Alzheimer's disease brain is associated with apolipoprotein E4 genotype.

Apolipoprotein E (ApoE) is a potent risk factor for Alzheimer's disease. Since the loss of cholinergic function in Alzheimer's disease is known to occur at an early stage in the disease we have examined this function in normal subjects with an Apoepsilon4 allele to see if the deficit occurs in the absence of Alzheimer pathology or symptoms. We report that brain tissue obtained post-mortem from normal subjects and Alzheimer patients with an Apoepsilon4 allele has a lower cholinergic activity than tissue from those subjects without this allele. This has important significance for the interpretation of the cholinergic deficits found in Alzheimer's disease.

Phospholipase A2 activity is selectively decreased in the striatum of chronic cocaine users.

Dopamine-mediated stimulation of arachidonic acid metabolism, via activation of the phospholipid metabolizing enzyme phospholipase A2 (PLA2), has recently been implicated in dopamine neurotransmitter function. We examined the status of PLA2 in autopsied brain of 10 chronic users of cocaine, a dopamine reuptake inhibitor. PLA2 activity, assayed at pH 8.5 in the presence of Ca2+, was significantly (p < 0.01) decreased by 31% in the putamen of cocaine users (n = 10) compared with that in controls (n = 10), whereas activity was normal in the frontal and occipital cortices, subcortical white matter, and cerebellum. In contrast, calcium-independent PLA2 activity, assayed at pH 7.0, was normal in all brain regions examined. Our finding of altered PLA2 activity restricted to a region of high dopamine receptor density suggests that modulation of PLA2 may be involved in mediating some of the dopamine-related behavioral effects of cocaine and could conceivably contribute to dopamine-related processes in the normal brain.