High Expression of Nicotinamide N-Methyltransferase in Patients with Sporadic Alzheimer's Disease.

We have previously shown that the expression of nicotinamide N-methyltransferase (NNMT) is significantly increased in the brains of patients who have died of Parkinson's disease (PD). In this study, we have compared the expression of NNMT in post-mortem medial temporal lobe, hippocampus and cerebellum of 10 Alzheimer's disease (AD) and 9 non-disease control subjects using a combination of quantitative Western blotting, immunohistochemistry and dual-label confocal microscopy coupled with quantitative analysis of colocalisation. NNMT was detected as a single protein of 29 kDa in both AD and non-disease control brains, which was significantly increased in AD medial temporal lobe compared to non-disease controls (7.5-fold, P < 0.026). There was no significant difference in expression in the cerebellum (P = 0.91). NNMT expression in AD medial temporal lobe and hippocampus was present in cholinergic neurones with no glial localisation. Cell-type expression was identical in both non-disease control and AD tissues. These results are the first to show, in a proof-of-concept study using a small patient cohort, that NNMT protein expression is increased in the AD brain and is present in neurones which degenerate in AD. These results suggest that the elevation of NNMT may be a common feature of many neurodegenerative diseases. Confirmation of this overexpression using a larger AD patient cohort will drive the future development of NNMT-targetting therapeutics which may slow or stop the disease pathogenesis, in contrast to current therapies which solely address AD symptoms.

Na+, K+-ATPase α3 isoform in frontal cortex GABAergic neurons in psychiatric diseases.

Na+, K+-ATPase is an essential membrane transporter. In the brain, the α3 isoform of Na+, K+-ATPase is vital for neuronal function. The enzyme and its regulators, endogenous cardiac steroids (ECS), were implicated in neuropsychiatric disorders. GABAergic neurotransmission was also studied extensively in diseases such as schizophrenia and bipolar disorder (BD). Post mortem brain samples from subjects with depression, schizophrenia or BD and non-psychiatric controls were provided by the Stanley Medical Research Institute. ECS levels were determined by ELISA. Expression levels of the three Na+, K+-ATPase-α isoforms, α1, α2 and α3, were determined by Western blot analysis. The α3 levels in GABAergic neurons in different regions of the brain were quantified by fluorescence immunohistochemistry. The results show that Na+, K+ -ATPase α3 isoform levels were lower in GABAergic neurons in the frontal cortex in BD and schizophrenia as compared with the controls (n = 15 subjects per group). A study on a 'mini-cohort' (n = 3 subjects per group) showed that the α3 isoform levels were also lower in GABAergic neurons in the hippocampus, but not amygdala, of bipolar and schizophrenic subjects. In the temporal cortex, higher Na+, K+ -ATPase α3 protein levels were found in the three psychiatric groups. No significant differences in ECS levels were found in this brain area. This is the first report on the distribution of α3 in specific neurons in the human brain in association with mental illness. These results strengthen the hypothesis for the involvement of Na+, K+ -ATPase in neuropsychiatric diseases.

Purine-related metabolites and their converting enzymes are altered in frontal, parietal and temporal cortex at early stages of Alzheimer's disease pathology.

Adenosine, hypoxanthine, xanthine, guanosine and inosine levels were assessed by HPLC, and the activity of related enzymes 5'-nucleotidase (5'-NT), adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) measured in frontal (FC), parietal (PC) and temporal (TC) cortices at different stages of disease progression in Alzheimer's disease (AD) and in age-matched controls. Significantly decreased levels of adenosine, guanosine, hypoxanthine and xanthine, and apparently less inosine, are found in FC from the early stages of AD; PC and TC show an opposing pattern, as adenosine, guanosine and inosine are significantly increased at least at determinate stages of AD whereas hypoxanthine and xanthine levels remain unaltered. 5'-NT is reduced in membranes and cytosol in FC mainly at early stages but not in PC, and only at advanced stages in cytosol in TC. ADA activity is decreased in AD when considered as a whole but increased at early stages in TC. Finally, PNP activity is increased only in TC at early stages. Purine metabolism alterations occur at early stages of AD independently of neurofibrillary tangles and ß-amyloid plaques. Alterations are stage dependent and region dependent, the latter showing opposite patterns in FC compared with PC and TC. Adenosine is the most affected of the assessed purines.

Tissue Expressions of Soluble Human Epoxide Hydrolase-2 Enzyme in Patients with Temporal Lobe Epilepsy.

OBJECTIVE: We sought to simply demonstrate how levels of soluble human epoxide hydrolase-2 show changes in both temporal the cortex and hippocampal complex in patients with temporal lobe epilepsy. METHODS: A total of 20 patients underwent anterior temporal lobe resection due to temporal lobe epilepsy. The control group comprised 15 people who died in traffic accidents or by falling from a height, and their autopsy findings were included. Adequately sized temporal cortex and hippocampal samples were removed from each patient during surgery, and the same anatomic structures were removed from the control subjects during the autopsy procedures. Each sample was stored at -80°C as rapidly as possible until the enzyme assay. RESULTS: The temporal cortex in the epilepsy patients had a significantly higher enzyme level than did the temporal cortex of the control group (P = 0.03). Correlation analysis showed that as the enzyme level increases in the temporal cortex, it also increases in the hippocampal complex (r2 = 0.06, P = 0.00001). More important, enzyme tissue levels showed positive correlations with seizure frequency in both the temporal cortex and hippocampal complex in patients (r2 = 0.7, P = 0.00001 and r2 = 0.4, P = 0.003, respectively). The duration of epilepsy was also positively correlated with the hippocampal enzyme level (r2 = 0.06, P = 0.00001). CONCLUSIONS: Soluble human epoxy hydrolase enzyme-2 is increased in both lateral and medial temporal tissues in temporal lobe epilepsy. Further studies should be conducted as inhibition of this enzyme has resulted in a significant decrease in or stopping of seizures and attenuated neuroinflammation in experimental epilepsy models in the current literature.

Genetic Variation in δ-Opioid Receptor Associates with Increased ß- and γ-Secretase Activity in the Late Stages of Alzheimer's Disease.

The agonist-induced activation of human δ-opioid receptor (δOR) has been shown to increase ß- (BACE1) and γ-secretase activities leading to increased production of amyloid-ß (Aß) peptide. We have recently shown that phenylalanine to cysteine substitution at amino acid 27 in δOR (δOR-Phe27Cys) increases amyloid-ß protein precursor processing through altered endocytic trafficking. Also, a genetic meta-analysis of the δOR-Phe27Cys variation (rs1042114) in two independent Alzheimer's disease (AD) patient cohorts indicated that the heterozygosity of δOR-Phe27Cys increases the risk of AD. Here, we investigated α-, ß-, and γ-secretase activities in human brain with respect to δOR-Phe27Cys variation in the temporal cortex of 71 subjects with varying degree of AD-related neurofibrillary pathology (Braak stages I-VI). As a result, a significant increase in ß- (p = 0.03) and γ- (p = 0.01), but not α-secretase, activities was observed in late stage AD samples (Braak stages V-VI), which were heterozygous for δOR-Phe27Cys as compared to the δOR-Phe27 and δOR-Cys27 homozygotes. The augmented ß-secretase activity was not associated with increased mRNA expression or protein levels of BACE1 in the late stage AD patients, who were heterozygous for the δOR-Phe27Cys variation. These findings suggest that δOR-Phe27Cys variation modulates ß- and γ-secretase activity in the late stages of AD likely via post-translational mechanisms other than alterations in the mRNA or protein levels of BACE1, or, in the expression of γ-secretase complex components.

Increased densities of nitric oxide synthase expressing neurons in the temporal cortex and the hypothalamic paraventricular nucleus of polytoxicomanic heroin overdose victims: possible implications for heroin neurotoxicity.

Heroin is one of the most dangerous drugs of abuse, which may exert various neurotoxic actions on the brain (such as gray matter loss, neuronal apoptosis, mitochondrial dysfunction, synaptic defects, depression of adult neurogenensis, as well as development of spongiform leucoencephalopathy). Some of these toxic effects are probably mediated by the gas nitric oxide (NO). We studied by morphometric analysis the numerical density of neurons expressing neuronal nitric oxide synthase (nNOS) in cortical and hypothalamic areas of eight heroin overdose victims and nine matched controls. Heroin addicts showed significantly increased numerical densities of nNOS immunoreactive cells in the right temporal cortex and the left paraventricular nucleus. Remarkably, in heroin abusers, but not in controls, we observed not only immunostained interneurons, but also cortical pyramidal cells. Given that increased cellular expression of nNOS was accompanied by elevated NO generation in brains of heroin addicts, these elevated levels of NO might have contributed to some of the known toxic effects of heroin (for example, reduced adult neurogenesis, mitochondrial pathology or disturbances in synaptic functioning).

The effects of different antioxidants on the activity of cerebrocortical MnSOD and Na,K-ATPase from post mortem Alzheimer's disease and age-matched normal brains.

Among the markers and targets of the early phase of Alzheimer's disease (AD) pathogenesis MnSOD (mitochondrial dysfunction) and Na-pump (disturbances in function/regulation) are often highlighted. This paper focused on comparison of the effects of three antioxidants on the activity of cerebrocortical MnSOD and Na,K-ATPase from post mortem Alzheimer's disease and age-matched normal brains. Antioxidant compounds with different origins: natural glutathione, synthetic UPF peptides (glutathione analogues) and phytoestrogen genistein were investigated. Firstly, MnSOD and Na,K-ATPase activities were found to be decreased in the post mortem AD brains compared with age-matched controls. Secondly, GSH had no effect on MnSOD activity, but decreased Na,K-ATPase activity both in the control and AD brains. Thirdly, UPF1 and UPF17 increased MnSOD activity, and UPF17 suppressed Na,K-ATPase activity. Further studies are needed to clarify, if the inhibitory effect of UPF17 on Na,K-ATPase could abolish the beneficial effect gained from MnSOD activation. Both the antioxidative potential of genistein and its potency to up-regulate Na,K-ATPase activity make it an attractive candidate substance to suppress the early phase of the pathogenesis of AD.

Aluminum and ethanol induce alterations in superoxide and peroxide handling capacity (SPHC) in frontal and temporal cortex.

Aluminum is an omnipresent neurotoxicant and has been associated with several neuropathological disorders. Cerebrum and cerebellum have been shown to face augmented oxidative stress when animals are exposed to aluminum and high doses of ethanol. To establish the link between oxidative stress and neurobehavioral alterations, the present study was conducted to determine the extent of oxidative stress in low levels of pro-oxidant (ethanol exposure) status of the functionally discrete regions of the cerebrum. Male Wistar rats were exposed to aluminum (10 mg/kg body wt) and ethanol (0.2-0.6 g/kg body wt) for 4 weeks. Spontaneous motor activity (SMA) and Rota-Rod performances (RRP) were recorded weekly during the period of exposure. At the end of 4th week, oxidative stress parameters were determined from the homogenized cerebral tissue. GSH-independent superoxide peroxide handling capacity (GI-SPHC) and GSH-dependent superoxide peroxide handling capacity (GD-SPHC) were determined for FC and TC upon exposure to ethanol in the absence and presence of aluminum exposure. Aluminum was found to augment the oxidative stress at higher doses (0.6 g Ethanol/kg body wt) of ethanol, particularly in FC. The SPHC of FC was also found to be compromised significantly in aluminum-ethanol co-exposed animals. It was concluded that even though the manifestation of oxidative stress was not observed as revealed by assaying the widely used oxidative stress biochemical markers (indices), aluminum and ethanol (low doses) exposure induced alterations in the handling capacity of oxidant imbalance that could be recognized by studying the SPHC of FC. Comparison of GD-SPHC and GI-SPHC offered a possible mechanism of compromised SPHC in FC. This observation is likely to offer insights into the mechanism of association between aluminium exposure and behavioral changes in neurodegenerative disorders towards therapeutic strategies for these disorders.

Differential BDNF signaling in dentate gyrus and perirhinal cortex during consolidation of recognition memory in the rat.

Consolidation of long-term memory is dependent on synthesis of new proteins in the hippocampus and associated cortical regions. The neurotrophin brain-derived neurotrophic factor (BDNF) is tightly regulated by activity-dependent cellular processes and is strongly linked with mechanisms underlying learning and memory. BDNF activation of tyrosine receptor kinase (TrkB) stimulates intracellular signaling cascades implicated in plasticity, including the extracellular-signal related kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway and the phosphatidylinositide-3-kinase (PI3K)/Akt pathway. Here, we investigate the role of BDNF, ERK/MAPK, and PI3K/AKT signaling cascade in recognition memory in the rat. We report that recognition memory was associated with increased release of BDNF in the dentate gyrus and perirhinal cortex. This was associated with significant increases in p44ERK activation and c-fos expression in the dentate gyrus and PI3K activation and c-fos expression in the perirhinal cortex. Furthermore, both recognition memory and the associated cell signaling events in dentate gyrus and perirhinal cortex were blocked by intraperitoneal injection of the Trk receptor inhibitor tyrphostin AG879. These data are consistent with the hypothesis that BDNF-stimulated intracellular signaling plays a role in consolidation of recognition memory in the rat.

NOX activity is increased in mild cognitive impairment.

This study was undertaken to investigate the profile of NADPH oxidase (NOX) in the clinical progression of Alzheimer's disease (AD). Specifically, NOX activity and expression of the regulatory subunit p47phox and the catalytic subunit gp91phox was evaluated in affected (superior and middle temporal gyri) and unaffected (cerebellum) brain regions from a longitudinally followed group of patients. This group included both control and late-stage AD subjects, and also subjects with preclinical AD and with amnestic mild cognitive impairment (MCI) to evaluate the profile of NOX in the earliest stages of dementia. Data show significant elevations in NOX activity and expression in the temporal gyri of MCI patients as compared with controls, but not in preclinical or late-stage AD samples, and not in the cerebellum. Immunohistochemical evaluations of NOX expression indicate that whereas microglia express high levels of gp91phox, moderate levels of gp91phox also are expressed in neurons. Finally, in vitro experiments showed that NOX inhibition blunted the ability of oligomeric amyloid beta peptides to injure cultured neurons. Collectively, these data show that NOX expression and activity are upregulated specifically in a vulnerable brain region of MCI patients, and suggest that increases in NOX-associated redox pathways in neurons might participate in the early pathogenesis of AD.

Temporal lobe sclerosis associated with hippocampal sclerosis in temporal lobe epilepsy: neuropathological features.

Widespread changes involving neocortical and mesial temporal lobe structures can be present in patients with temporal lobe epilepsy and hippocampal sclerosis. The incidence, pathology, and clinical significance of neocortical temporal lobe sclerosis (TLS) are not well characterized. We identified TLS in 30 of 272 surgically treated cases of hippocampal sclerosis. Temporal lobe sclerosis was defined by variable reduction of neurons from cortical layers II/III and laminar gliosis; it was typically accompanied by additional architectural abnormalities of layer II, that is, abnormal neuronal orientation and aggregation. Quantitative analysis including tessellation methods for the distribution of layer II neurons supported these observations. In 40% of cases, there was a gradient of TLS with more severe involvement toward the temporal pole, possibly signifying involvement of hippocampal projection pathways. There was a history of a febrile seizure as an initial precipitating injury in 73% of patients with TLS compared with 36% without TLS; no other clinical differences between TLS and non-TLS cases were identified. Temporal lobe sclerosis was not evident preoperatively by neuroimaging. No obvious effect of TLS on seizure outcome was noted after temporal lobe resection; 73% became seizure-free at 2-year follow-up. In conclusion, approximately 11% of surgically treated hippocampal sclerosis is accompanied by TLS. Temporal lobe sclerosis is likely an acquired process with accompanying reorganizational dysplasia and an extension of mesial temporal sclerosis rather than a separate pathological entity.

Aromatase distribution in the monkey temporal neocortex and hippocampus.

Numerous studies have shown that neuronal plasticity in the hippocampus and neocortex is regulated by estrogen and that aromatase, the key enzyme for estrogen biosynthesis, is present in cerebral cortex. Although the expression pattern of aromatase mRNA has been described in the monkey brain, its precise cellular distribution has not been determined. In addition, the degree to which neuronal aromatase is affected by gonadal estrogen has not been investigated. In this study, we examined the immunohistochemical distribution of aromatase in young ovariectomized female rhesus monkeys with or without long-term cyclic estradiol treatment. Both experimental groups showed that aromatase is localized in a large population of CA1-3 pyramidal cells, in granule cells of the dentate gyrus and in some interneurons in which it was co-expressed with the calcium-binding proteins calbindin, calretinin, and parvalbumin. Moreover, numerous pyramidal cells were immunoreactive for aromatase in the neocortex, whereas only small subpopulations of neocortical interneurons were immunoreactive for aromatase. The widespread expression of the protein in a large neuronal population suggests that local intraneuroral estrogen synthesis may contribute to estrogen-induced synaptic plasticity in monkey hippocampus and neocortex of female rhesus monkeys. In addition, the apparent absence of obvious differences in aromatase distribution between the two experimental groups suggests that these localization patterns are not dependent on plasma estradiol levels.

Binding of 5'-O-(2-thiodiphosphate) to rat brain membranes is prevented by diadenosine tetraphosphate and correlates with ecto-nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) activity.

The distribution of binding sites for [(35)S]5'-O-(2-thiodiphosphate) ([(35)S]ADPbetaS), a radioligand of P2Y(1,12,13) receptors, and of ecto-nucleotide pyrophosphatase phosphodiesterase activity were analyzed in the rat forebrain. Binding sites for the radilogand are widespreadly distributed in the rat forebrain, showing the highest density in hypothalamus. K(d) values were in the range 1-2 nM. Diadenosine tetraphosphate (Ap(4)A) and diethenoadenosine tetraphosphate, epsilon-(Ap(4)A), displaced the radioligand, indicating dinucleotide binding to ADPbetaS-recognizing P2Y receptors. Activity ecto-nucleotide pyrophosphatase phosphodiesterase 1 (NPP1), able to hydrolyze Ap(4)A and other diadenosine polyphosphates, is also widely distributed through the rat forebrain, with the highest activity in hypothalamus. These results suggests that Ap(4)A signalling mediated by P2Y(1,12,13) receptors and enzymatically regulated by NPP1 activity may be particularly important in hypothalamus and add new support for neurotransmitter/neuromodulatory functions of diadenosine polyphosphates in brain.

Extracellular signal-regulated protein kinase in human intractable epilepsy.

Extracellular signal-regulated kinases (ERK) such as ERK1 [p44 mitogen-activated protein kinase (MAPK)] and ERK2 (p42 MAPK) are activated in the central nervous system under physiological and pathological conditions such as ischemia and epilepsy. Our aim is to investigate ERK1, ERK2, and phosphorylated ERK (p-ERK) (Thr202/Tyr 204) expression in the temporal lobe of patients with intractable epilepsy (IE) and to explore its possible role of ERK in it. Tissue samples from temporal neocortices of 40 patients who had surgery for IE were used to detect ERK1, ERK2, and p-ERK (Thr 202/Tyr 204) expression through immunohistochemistry and western blot. We compared these tissues against 17 histological normal temporal lobes from head-trauma patients. ERK1, ERK2, and p-ERK in IE were significantly higher than those in the controls. They were mainly expressed in the cytoplasm of neurons and glial cells. There was also increased detection of p-ERK in the gliotic cortex of IE compared with the non-gliotic cortex. These findings were consistently observed in western blot and immunohistochemistry techniques. ERK expression in patients with IE was significantly increased compared with the controls. This suggested a probable role of ERK in the pathogenesis of IE.

Akt activity in Alzheimer's disease and other neurodegenerative disorders.

Enzyme activities of the serine/threonine kinase Akt were compared in mid-temporal and mid-frontal cortices from Alzheimer's disease cases and matched controls. Activities (GSK-3alpha/beta fusion protein phosphorylation by immunoprecipitated Akt) were significantly increased in temporal cortex soluble fractions from Alzheimer's disease compared with non-disease controls and positive disease controls with another neurodegenerative disease. Temporal cortex soluble fraction Akt activities positively correlated with Braak staging for neurofibrillary changes. Frontal cortex soluble fraction activities were significantly reduced in positive disease compared with Alzheimer's disease cases and non-disease controls. Strong Ser Akt immunoreactivity was seen in Alzheimer's disease pyramidal neurons likely undergoing degeneration and in reactive astroglia. Non-disease and positive disease controls showed moderate Ser Akt immunostaining of occasional pyramidal neurons.

Apoptotic proteins in the temporal cortex in schizophrenia: high Bax/Bcl-2 ratio without caspase-3 activation.

OBJECTIVE: Neuroimaging findings have identified lower cortical gray matter volume in schizophrenia. Apoptosis (programmed cell death) has been proposed as a contributing pathophysiological mechanism. Levels of antiapoptotic Bcl-2 protein are low in the temporal cortex of schizophrenia patients. Bcl-2 interacts with the proapoptotic Bax protein at an upstream checkpoint to regulate the activation of apoptosis by caspase-3 and other proteolytic caspase proteins. A high Bax/Bcl-2 ratio is associated with greater vulnerability to apoptotic activation, while a high caspase-3 level is often associated with apoptotic activity. It was hypothesized that the Bax/Bcl-2 ratio, but not caspase-3, would be high in the temporal cortex of patients with chronic schizophrenia. METHOD: Bax, Bcl-2, and caspase-3 proteins were measured by semiquantitative Western blot in Brodmann's area 21 (middle temporal gyrus) of postmortem tissue from patients with schizophrenia (N=15), bipolar disorder (N=15), or major depression (N=15) and nonpsychiatric comparison subjects (N=15). RESULTS: The Bax/Bcl-2 ratio was 50% higher in the schizophrenia patients than the nonpsychiatric comparison subjects. The level of caspase-3 (inactive zymogen and activated subunits) was not significantly different. CONCLUSIONS: The higher Bax/Bcl-2 ratio suggests that cortical cells are vulnerable to apoptosis in chronic schizophrenia. However, the normal caspase-3 level suggests that apoptosis is not active in this illness phase. Furthermore, the results appear to distinguish the pathophysiology of schizophrenia from most classic neurodegenerative disorders, in which postmortem caspase-3 levels are high. Further studies are needed to investigate the implications of abnormal apoptotic proteins in schizophrenia.

Characterisation of estrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity in the human brain.

Estrogens play a crucial role in multiple functions of the brain and the proper balance of inactive estrone and active estradiol-17beta might be very important for their cerebral effects. The interconversion of estrone and estradiol-17beta in target tissues is known to be catalysed by a number of human 17beta-hydroxysteroid dehydrogenase (17beta-HSD) isoforms. The present study shows that enzyme catalysed interconversion of estrone and estradiol-17beta occurs in the human temporal lobe. The oxidative cerebral pathway preferred estradiol-17beta to Delta(5)-androstenediol and testosterone, whereas the reductive pathway preferred dehydroepiandrosterone (DHEA) to Delta(4)-androstenedione and estrone. An allosteric Hill kinetic for NAD-dependent oxidation of estradiol-17beta was observed, whereas a typical Michaelis-Menten kinetic was shown for NADPH-dependent reduction of estrone. Investigations of the interconversion of estrogens in cerebral neocortex (CX) and subcortical white matter (SC) preparations of brain tissue from 12 women and 10 men revealed no sex-differences, but provide striking evidence for the presence of at least one oxidative membrane-associated 17beta-HSD and one cytosolic enzyme that catalyses both the reductive and the oxidative pathway. Membrane-associated oxidation of estradiol-17beta was shown to be significantly higher in CX than in SC (P<0.05), whereas the cytosolic enzyme activities were significantly higher in SC than in CX (P<0.0005). Finally, real-time RT-PCR analyses revealed that besides 17beta-HSD types 4 and 5 also the isozymes type 7, 8, 10 and 11 show substantial expression in the human temporal lobe. The characteristics of the isozymes lead us to the conclusion that cytosolic 17beta-HSD type 5 is the best candidate for the observed cytosolic enzyme activities, whereas the data gave no clear answer to the question, which enzyme is responsible for the membrane-associated oxidation of estradiol-17beta. In conclusion, the study strongly suggests that different cell types and different isozymes are involved in the cerebral interconversion of estrogens, which might play a pivotal role in maintaining the functions of the central nervous system.

Allopregnanolone serum levels and expression of 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase isoforms in hippocampal and temporal cortex of patients with epilepsy.

In the human central nervous system, progesterone is rapidly metabolised to 5 alpha-dihydroprogesterone which subsequently is further reduced to allopregnanolone (AP). These conversions are catalysed by 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD). Although different isoforms of both enzymes have been identified in the brain, our knowledge of their expression in the human brain remains limited. The aim of the present study was to investigate the mRNA expression of 5 alpha-reductase 1 as well as 3 alpha-HSD 1, 2, 3 and 20 alpha-HSD in brain tissue from patients with pharmacoresistant temporal lobe epilepsy (TLE). Specimens were derived from either the hippocampus or the temporal lobe cortex and from the tumor-free approach corridor tissue of patients with brain tumors. Quantification of different mRNAs was achieved by real time PCR. In addition, we provide data on simultaneous evaluation of serum AP concentrations. We could demonstrate that 3 alpha-HSD 1 was not expressed in the hippocampus and temporal lobe of patients with TLE. In the hippocampus and temporal lobe, the expression levels of 3 alpha-HSD 2 were about 20% of that in liver tissue, those of 3 alpha-HSD 3 about 7% and those of 20 alpha-HSD about 2%, respectively. In patients with TLE, expression of 3 alpha-HSD 2 was significantly higher in the hippocampus than in temporal lobe cortex tissue (P<0.006). AP concentrations did not correlate significantly with the mRNA expression levels of 5 alpha-reductase 1, 3 alpha-HSD 2 and 3 and 20 alpha-HSD in any of the patient groups under investigation. In conclusion, the present study demonstrates mRNA expression of 5 alpha-reductase 1 and 3 alpha-HSD 2 and 3 and 20 alpha-HSD in the hippocampus and temporal lobe of epileptic patients. These findings provide further molecular biological evidence for the formation and metabolism of neuroactive steroids in the human brain.

Altered cAMP-dependent protein kinase subunit immunolabeling in post-mortem brain from patients with bipolar affective disorder.

Previous findings of reduced [3H]cAMP binding and increased activities of cAMP-dependent protein kinase (PKA) in discrete post-mortem brain regions from patients with bipolar affective disorder (BD) suggest that PKA, the major downstream target of cAMP, is also affected in this illness. As prolonged elevation of intracellular cAMP levels can modify PKA regulatory (R) and catalytic (C) subunit levels, we sought to determine whether these PKA abnormalities are related to changes in the abundance of PKA subunits in BD brain. Using immunoblotting techniques along with PKA subunit isoform-specific polyclonal antisera, levels of PKA RIalpha, RIbeta, RIIalpha, RIIbeta and Calpha subunits were measured in cytosolic and particulate fractions of temporal, frontal and parietal cortices of post-mortem brain from BD patients and matched, non-neurological, non-psychiatric controls. Immunoreactive levels of cytosolic Calpha in temporal and frontal cortices, as well as that of cytosolic RIIbeta in temporal cortex, were significantly higher in the BD compared with the matched control brains. These changes were independent of age, post-mortem interval or pH and unrelated to ante-mortem lithium treatment or suicide. These findings strengthen further the notion that the cAMP/PKA signaling system is up-regulated in discrete cerebral cortical regions in BD.

Caspase-cleaved amyloid precursor protein in Alzheimer's disease.

Caspase-3 mediated cleavage of the amyloid precursor protein (APP) has been proposed as a putative mechanism underlying amyloidosis and neuronal cell death in Alzheimer's disease (AD). We utilized an antibody that selectively recognizes the neo epitope generated by caspase-3 mediated cleavage of APP (alphadeltaC(csp)-APP) to determine if this proteolytic event occurs in senile plaques in the inferior frontal gyrus and superior temporal gyrus of autopsied AD and age-matched control brains. Consistent with a role for caspase-3 activation in AD pathology, alphadeltaC(csp)-APP immunoreactivity colocalized with a subset of TUNEL-positive pyramidal neurons in AD brains. AlphadeltaC(csp)-APP immunoreactivity was found in neurons and glial cells, as well as in small- and medium-size particulate elements, resembling dystrophic terminals and condensed nuclei, respectively, in AD and age-matched control brains. There were a larger number of alphadeltaC(csp)-APP immunoreactive elements in the inferior frontal gyrus and superior temporal gyrus of subjects with AD pathology than age-matched controls. AlphadeltaC(csp)-APP immunoreactivity in small and medium size particulate elements were the main component colocalized with 30% of senile plaques in the inferior frontal gyrus and superior temporal gyrus of AD brains. In some control brains, alphadeltaC(csp)-APP immunoreactivity appeared to be associated with a clinical history of metabolic encephalopathy. Our results suggest that apoptosis contributes to cell death resulting from amyloidosis and plaque deposition in AD.