C-reactive protein is increased in schizophrenia but is not altered by antipsychotics: meta-analysis and implications.

The inflammatory hypothesis of schizophrenia (SZ) posits that inflammatory processes and neural-immune interactions are involved in its pathogenesis, and may underpin some of its neurobiological correlates. SZ is the psychiatric disorder causing the most severe burden of illness, not just owing to its psychiatric impairment, but also owing to its significant medical comorbidity. C-reactive protein (CRP) is a commonly used biomarker of systemic inflammation worldwide. There are some conflicting results regarding the behaviour of CRP in SZ. The aims of this study were to verify whether peripheral CRP levels are indeed increased in SZ, whether different classes of antipsychotics divergently modulate CRP levels and whether its levels are correlated with positive and negative symptomatology. With that in mind, we performed a meta-analysis of all cross-sectional studies of serum and plasma CRP levels in SZ compared to healthy subjects. In addition, we evaluated longitudinal studies on CRP levels before and after antipsychotic use. Our meta-analyses of CRP in SZ included a total of 26 cross-sectional or longitudinal studies comprising 85 000 participants. CRP levels were moderately increased in persons with SZ regardless of the use of antipsychotics and did not change between the first episode of psychosis and with progression of SZ (g=0.66, 95% confidence interval (95% CI) 0.43 to 0.88, P<0.001, 24 between-group comparisons, n=82 962). The extent of the increase in peripheral CRP levels paralleled the increase in severity of positive symptoms, but was unrelated to the severity of negative symptoms. CRP levels were also aligned with an increased body mass index. Conversely, higher age correlated with a smaller difference in CRP levels between persons with SZ and controls. Furthermore, CRP levels did not increase after initiation of antipsychotic medication notwithstanding whether these were typical or atypical antipsychotics (g=0.01, 95% CI -0.20 to 0.22, P=0.803, 8 within-group comparisons, n=713). In summary, our study provides further evidence of the inflammatory hypothesis of SZ. Whether there is a causal relationship between higher CRP levels and the development of SZ and aggravation of psychotic symptoms, or whether they are solely a marker of systemic low-grade inflammation in SZ, remains to be clarified.

Nardilysin in human brain diseases: both friend and foe.

Nardilysin is a metalloprotease that cleaves peptides, such as dynorphin-A, α-neoendorphin, and glucagon, at the N-terminus of arginine and lysine residues in dibasic moieties. It has various functionally important molecular interaction partners (heparin-binding epidermal growth factor-like growth factor, tumour necrosis factor-α-converting enzyme, neuregulin 1, beta-secretase 1, malate dehydrogenase, P42(IP4)/centaurin-α1, the histone H3 dimethyl Lys4, and others) and is involved in a plethora of normal brain functions. Less is known about possible implications of nardilysin for brain diseases. This review, which includes some of our own recent findings, attempts to summarize the current knowledge on possible roles of nardilysin in Alzheimer disease, Down syndrome, schizophrenia, mood disorders, alcohol abuse, heroin addiction, and cancer. We herein show that nardilysin is a Janus-faced enzyme with regard to brain pathology, being probably neuropathogenic in some diseases, but neuroprotective in others.

Differential topochemistry of three cationic amino acid transporter proteins, hCAT1, hCAT2 and hCAT3, in the adult human brain.

The cellular uptake of L-arginine and other cationic amino acids (such as L-lysine and L-ornithine) is mainly mediated by cationic amino acid transporter (CAT) proteins. Despite the important roles of cationic amino acid transporters for normal brain functioning and various brain diseases there is currently only fragmentary knowledge about their cellular and regional distribution patterns in the human brain. We mapped the immunohistochemical localization of human cationic amino acid transporters 1, 2 and 3 (hCAT1, 2, and 3) throughout five adult human brains and found a wide but uneven distribution of these transporters. All three hCAT1s were mainly localized in neurons, but were also found in numerous astrocytes, oligodendrocytes, plexus choroideus epithelial cells, and small blood vessels. The highest density of hCAT expressing neurons was observed in the hypothalamus, in some areas of the cerebral cortex, the thalamic reticular nucleus and the caudate nucleus, whereas weak to moderate expression was detected in the hippocampus, the prefrontal cortex (hCAT1 only), pons, brain stem and cerebellum. In contrast to what has been found in rodent brain, we detected hCAT2 and hCAT3 also in astrocytes. Overall, each hCAT has its characteristic, individual cerebral expression patterns, which, however, overlap with the others.

The agmatine-degrading enzyme agmatinase: a key to agmatine signaling in rat and human brain?

Agmatinase, an ureohydrolase belonging to the arginase family, is widely expressed in mammalian tissues including the brain. Here, it may serve two different functions, the inactivation of the arginine derivative agmatine, a putative neurotransmitter, and the formation of the diamine putrescine. In order to identify the cellular sources of agmatinase expression in the brain, we generated a polyclonal monospecific antibody against recombinant rat agmatinase. With immunocytochemistry, selected areas of rat and human brain were screened. Clearly, in both species agmatinase-like immunoreactivity was predominantly detected in distinct populations of neurons, especially cortical interneurons. Also, principal neurons in limbic regions like the habenula and in the cerebellum robustly expressed agmatinase protein. When comparing the overall agmatinase expression with immunocytochemical data available for agmatine and polyamine biosynthetic enzymes, the observed pattern may argue in favor of an agmatine inactivating function rather than fueling the alternative pathway of polyamine synthesis. The putative neurotransmitter agmatine is seemingly involved with mental disorders. Therefore, agmatinase may be similarly important for pathogenesis. The normal expression profile of the protein as described here may therefore be altered under pathological conditions.

Haloperidol and clozapine decrease S100B release from glial cells.

Recent meta-analyses showed consistently elevated levels of S100B in serum and cerebrospinal fluid of schizophrenic patients. This finding has been attributed to glial pathology because S100B is produced by astrocytes and oligodendrocytes. However, S100B may be likewise associated with schizophrenia-related disturbances in glial cell as well as adipocyte energy supply and glucose metabolism. The influence of antipsychotic drugs on S100B levels remains unclear, and some studies have suggested that treatment with these drugs may actually contribute to the elevated S100B levels observed in schizophrenic patients. In this study, we explored the effects of the typical antipsychotic haloperidol and the atypical prototype drug clozapine on the release of S100B by astrocytic C6 cells and oligodendrocytic OLN-93 cells. Because of the association between schizophrenia and disturbances in energy metabolism, we assessed the effects of these drugs under basal condition (BC) compared to serum and glucose deprivation (SGD). We found that treatment of C6 and OLN-93 cells with haloperidol and clozapine reduced the release of S100B from C6 and OLN-93 cells under BC and SGD in vitro at a tissue concentration corresponding to the assumed therapeutic dose range of these drugs. These data suggest that elevated levels of S100B in bodily fluids of schizophrenic patients are normalized rather than increased by the effects of antipsychotic drugs on glial cells.

Evidence for structural abnormalities of the human habenular complex in affective disorders but not in schizophrenia.

BACKGROUND: The habenular complex is composed of important relay nuclei linking the limbic forebrain to the midbrain and brain stem nuclei. Based on clinical observations, experiments with animals and theoretical considerations, it has been speculated that this brain area might be involved in psychiatric diseases (i.e. schizophrenia and depression). However, evidence in favour of this hypothesis is still lacking because the human habenular complex has rarely been studied with regard to mental illness. METHOD: We examined habenular volumes in post-mortem brains of 17 schizophrenia patients, 14 patients with depression (six patients with major depression and eight patients with bipolar depression) and 13 matched controls. We further determined the neuronal density, cell number and cell area of the medial habenular nuclei of the same cohorts using a counting box and a computer-assisted instrument. RESULTS: Significantly reduced habenular volumes of the medial and lateral habenula were estimated in depressive patients in comparison to normal controls and schizophrenia patients. We also found a reduction in neuronal cell number and cell area in depressive patients for the right side compared to controls and schizophrenia patients. No such changes were seen in schizophrenia. CONCLUSIONS: Our anatomical data argue against prominent structural alterations of the habenular nuclei in schizophrenia but demonstrate robust alterations in depressive patients. We are currently applying immunohistochemical markers to better characterize neuronal subpopulations of this brain region in schizophrenia and depression.

A comparison of the synaptic proteome in human chronic schizophrenia and rat ketamine psychosis suggest that prohibitin is involved in the synaptic pathology of schizophrenia.

Many studies in recent years suggest that schizophrenia is a synaptic disease that crucially involves a hypofunction of N-methyl-D-aspartate receptor-mediated signaling. However, at present it is unclear how these pathological processes are reflected in the protein content of the synapse. We have employed two-dimensional gel electrophoresis in conjunction with mass spectrometry to characterize and compare the synaptic proteomes of the human left dorsolateral prefrontal cortex in chronic schizophrenia and of the cerebral cortex of rats treated subchronically with ketamine. We found consistent changes in the synaptic proteomes of human schizophrenics and in rats with induced ketamine psychosis compared to controls. However, commonly regulated proteins between both groups were very limited and only prohibitin was found upregulated in both chronic schizophrenia and the rat ketamine model. Prohibitin, however, could be a new potential marker for the synaptic pathology of schizophrenia and might be causally involved in the disease process.

S100B is expressed in, and released from, OLN-93 oligodendrocytes: Influence of serum and glucose deprivation.

S100B (member of a family of proteins that are 100% soluble in ammonium sulfate at neutral pH) has been widely used as astrocyte marker in animal models and in human brain diseases. Recent studies revealed S100B-immunopositivity in oligodendrocytes and O2A oligodendroglial progenitor cells. It is unknown, however, if oligodendrocytes produce S100B themselves, or if the S100B-immunolabeling is caused by binding or absorption of the protein. To address this question, S100B expression and protein release were analyzed in a highly pure oligodendrocytic OLN-93 cell line (from rat), in the astrocytic C6 cell line (from rat) and primary astrocytes. S100B was gene expressed in all cultures, as revealed by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. OLN-93 cells and glial fibrillary acidic protein (GFAP)-negative astrocytes expressed the multiligand receptor for advanced glycation end products (RAGE). S100B protein levels were determined in supernatants and cell homogenates by immunoluminometry under normal conditions and after serum and glucose deprivation (SGD). SGD led to a several-fold increased release of S100B (after 6 and 24 h), which was particularly pronounced in primary astrocytes. Increased S100B in cell homogenates was most notable in OLN-93 cells under SGD, indicating activated S100B synthesis. These cells also showed the highest percentage of dead cells, as determined by propidium iodide-positivity, after SGD. Incubation with 0.5, 2 and 5 microg/l exogenous S100B was not toxic to OLN-93 cells. In conclusion, OLN-93 cells produce more S100B under SGD than astrocytes and are more susceptible to cell death upon SGD, which provokes leakage of S100B. Our data indicate active S100B secretion from astrocytes under SGD since highly elevated levels of S100B were detected in the supernatant despite a low percentage of dead cells. The experimental results provide further evidence for a production/release of S100B in/from oligodendrocytes, e.g. in metabolic stress conditions like cerebral ischemia. Studies on S100B in bodily fluids should be carefully interpreted in order to avoid misleading hypotheses concerning the specific involvement of astrocytes, due to the various cellular sources of S100B.

Histochemical evidence for wide expression of the metalloendopeptidase nardilysin in human brain neurons.

Nardilysin is a metalloendopeptidase that in vitro cleaves peptides such as dynorphin-A, somatostatin-28, alpha-neoendorphin and glucagon at the N-terminus of arginine and lysine residues in dibasic moieties. The enzyme is highly expressed in many endocrine tissues. Nardilysin has also been found in the brain. Previously, we have detected that nardilysin interacts with brain-specific proteins, i.e. p42(IP4)/centaurin-alpha1 [Stricker R, Chow KM, Walther D, Hanck T, Hersh LB, Reiser G (2006) Interaction of the brain specific protein p42(IP4)/centaurin-alpha1 with the peptidase nardilysin is regulated by the cognate ligands of p42(IP4), PtdIns(3,4,5)P(3) and Ins(1,3,4,5)P(4), with stereospecificity. J Neurochem 98:343-354]. However, very little is known about the distribution of nardilysin in the brain. The aim of the present study was to reveal its regional distribution and cellular localization in developing and adult human brain. Using immunohistochemistry and Western blot analysis we demonstrate that the enzyme is widely, but unevenly, expressed in the human brain. We found high staining intensity in the hypothalamus, neocortex and brain stem nuclei. The cellular localization is almost exclusively confined to neurons. In pre- and perinatal human brain cortex, most neurons express the enzyme. In cortical neurons nardilysin protein was found to be partially co-localized with parvalbumin but not calretinin. No co-expression was seen with somatostatin-28 immunoreactivity. A considerable overlap was revealed between p42(IP4) and nardilysin. Our data support the hypothesis that nardilysin might possibly play a role in brain development, whereas its putative function in brain peptide metabolism remains to be clarified further.

Increased cerebrospinal fluid and serum levels of S100B in first-onset schizophrenia are not related to a degenerative release of glial fibrillar acidic protein, myelin basic protein and neurone-specific enolase from glia or neurones.

OBJECTIVE: To assess levels of glial fibrillar acidic protein (GFAP), myelin basic protein (MBP), neurone-specific enolase (NSE) and S100B in patients with first-onset schizophrenia. METHOD: We investigated CSF and serum samples from 12 patients with first-onset schizophrenia and from 17 control subjects by ELISA (GFAP, MBP) or immunoluminometric sandwich assays (NSE, S100B). RESULTS: Patients with schizophrenia had significantly higher levels of S100B in CSF (p = 0.004; 2.73 (SD 0.80) v 1.92 (0.58) microg/l) and serum (p = 0.032; 0.09 (0.03) v 0.08 (0.02) microg/l) in comparison with those in the matched control group. No diagnosis-dependent differences of protein concentration were seen for GFAP, MBP and NSE. DISCUSSION: Our finding of increased levels of S100B in patients with schizophrenia without an indication for significant glial (GFAP, MBP) or neuronal (NSE) damage may be interpreted as indirect evidence for increased active secretion of S100B during acute psychosis.

Expression of corticosteroid-binding protein in the human hypothalamus, co-localization with oxytocin and vasopressin.

Corticosteroid-binding globulin, a specific steroid carrier in serum with high binding affinity for glucocorticoids, is expressed in various tissues. In the present study, we describe the immunocytochemical distribution of this protein in neurons and nerve fibers in the human hypothalamus. CBG immunoreactive perikarya and fibers were observed in the paraventricular, supraoptic, and sexual dimorphic nuclei in the perifornical region, as well as in the lateral hypothalamic and medial preoptic areas, the region of the diagonal band, suprachiasmatic and ventromedial nuclei, bed nucleus of the stria terminalis and some epithelial cells from the choroid plexus and ependymal cells. Stained fibers occurred in the median eminence and infundibulum. Double immunostaining revealed a partial co-localization of corticosteroid-binding globulin with oxytocin and, to a lesser extent, with vasopressin in the paraventricular and the supraoptic nuclei. Double immunofluorescence staining showed coexistence of these substances in axonal varicosities in the median eminence. We conclude that neurons of the human hypothalamus are capable of expressing corticosteroid-binding globulin, in part co-localized with the classical neurohypophyseal hormones. The distribution of CBG immunoreactive neurons, which is widespread but limited to specific nuclei, indicates that CBG has many physiological functions that may include neuroendocrine regulation and stress response.

Hypothalamic nitric oxide synthase in affective disorder: focus on the suprachiasmatic nucleus.

Depression is frequently associated with dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, which leads to repeated episodes of hypercortisolemia. Hypothalamic paraventricular neurons are believed to trigger these processes by aberrant generation and/or release of corticotropin releasing hormone, oxytocin, vasopressin, and nitric oxide (NO). Recent findings from two independent laboratories have demonstrated that the suprachiasmatic nucleus, which in part controls the cellular activity of paraventricular neurons (PVN), is also involved in affective disorder. The aim of the present study was to elucidate by stereological analysis, whether suprachiasmatic nucleus (SCN) nitric oxide synthase and neurophysin generating neurons are affected in neuropsychiatric disorders. We show that compared to controls the number of nitric oxide synthase immunoreactive neurons is greatly reduced both in depression and in schizophrenia. In subjects with affective disorder there was a correlation between the number of NOS-expressing cells and duration of treatment with antidepressants. The number of neurophysin-expressing SCN neurons was also fewer in cases with mood disorder. It is concluded that SCN-derived NO may be a relevant pathophysiological factor in neuropsychiatric disorders.

The Alzheimer disease-related calcium-binding protein Calmyrin is present in human forebrain with an altered distribution in Alzheimer's as compared to normal ageing brains.

The EF-hand calcium binding protein Calmyrin (also called CIB-1) was shown to interact with presenilin-2 (PS-2), suggesting that this interaction might play a role in the pathogenesis of Alzheimer's disease (AD). Here we have investigated the distribution of Calmyrin in normal human and AD brain. In normal brain Calmyrin immunoreactivity was unevenly distributed with immunostaining in pyramidal neurones and interneurones of the palaeo-cortex and neocortex, cerebellar granule cells and hypothalamic neurones of the paraventricular, ventromedial and arcuate nuclei. Moderate immunoreactivity was present in hippocampal pyramidal cells and stronger in dentate gyrus neurones. Thalamic and septal neurones were devoid of immunoreactivity. No apparent differences were visible between stainings of brain sections from younger and older nondemented patients. In AD brain a substantial loss of Calmyrin-immunopositive neurones was observed in all regions, especially in cortical areas. Still immunoreactive neurones, however, displayed stronger staining that was especially concentrated in perinuclear regions. Calmyrin immunosignals were in part associated with diffuse and senile plaques. Thus, although protein levels of Calmyrin are low in human forebrain, its cellular localization as well as its altered distribution in AD brain suggest that it may be involved in the pathogenesis of AD.

Repeated application of ketamine to rats induces changes in the hippocampal expression of parvalbumin, neuronal nitric oxide synthase and cFOS similar to those found in human schizophrenia.

Treatment with the phencyclidine derivative ketamine, a non-competitive N-methyl-D-aspartate receptor antagonist and a well known anesthetic, has recently been introduced to mimic schizophrenia in animals. Using rats repeatedly treated with sub-anesthetic doses we demonstrate in the hippocampal formation the cellular distribution patterns of proteins being relevant to the pathogenesis of schizophrenia. Compared with controls an increase in the density of reduced nicotinamide adenine dinucleotide phosphate diaphorase-, neuronal nitric oxide synthase- and cFOS-positive hippocampal interneurons was found, whereas the density of parvalbumin expressing cells was decreased. Our experiments show that repeated injections of sub-anesthetic doses of ketamine induce significant changes in the nitrergic and GABAergic system which, in part, resemble those described in postmortem brains of human schizophrenics indicating that sub-chronic treatment with sub-anesthetic doses of ketamine might be a useful animal model to study schizophrenia.

Further immunohistochemical evidence for impaired NO signaling in the hypothalamus of depressed patients.

The cellular expression of nitric oxide synthase (NOS) was studied in neurons of the Nuc. suprachiasmaticus (SCN) of depressed patients and matched controls. The number of NOS-immunoreactive SCN neurons was significantly reduced in depression. We conclude that affective disorders are accompanied by impaired hypothalamic NO signaling.

Circumscribed numerical deficit of dorsal raphe neurons in mood disorders.

BACKGROUND: Neurocircuits comprising limbic, striato-pallidal and thalamo cortical brain areas are assumed to be involved in the pathophysiology of mood disorders. All these brain regions receive serotonergic afferents arising from the rostral raphe, mainly the dorsal raphe. Although serotonergic systems appear to be involved in the pathology of mood disorders, there is uncertainty as to whether structural alterations in raphe nuclei exist alongside a functional dysregulation of the serotonergic system. METHODS: In the brains of 12 patients with mood disorders (major depressive disorder N= 6, bipolar disorder N = 6) and 12 normal subjects we performed a morphometric post-mortem study on neuronal morphology in all subnuclei of the dorsal raphe nucleus using Nissl stained 20 microm axial serial sections of the brainstem. RESULTS: The number of neurones of the ventrolateral subnucleus of the dorsal raphe was reduced by 31 % in patients with mood disorders compared with non-psychiatric control subjects. Ventrally located subnuclei of the rostral dorsal raphe (ventrolateral, ventral, interfascicular) taken together also showed a smaller number of neurones. Neurone numbers of the dorsal and the caudal subnucleus and volumes of all single subnuclei appeared to be unchanged. Analysis of morphological neuronal types revealed a smaller number of triangular neurones in the ventrolateral subnucleus. Numbers of ovoid and round neurones in the ventrolateral subnucleus also showed a trend to reduction. No correlation was found between neurone numbers in any subnucleus of the dorsal raphe and duration of illness. Neurone numbers did not differ in any subnucleus between patients with unipolar and those with bipolar affective disorder. CONCLUSIONS: Results indicate that patients with primary mood disorders have a circumscribed numerical neuronal deficiency in the dorsal raphe. This structural deviation may contribute to impaired serotonergic innervation of brain regions which are involved in the pathology of mood disorders.

Fewer beta-endorphin expressing arcuate nucleus neurons and reduced beta-endorphinergic innervation of paraventricular neurons in schizophrenics and patients with depression.

In order to elucidate whether the hypothalamic expression of beta-endorphin is altered in patients with mental disorders we studied the cellular localization of the peptide in arcuate nucleus neurons as well as the beta-endorphinergic innervation of paraventricular neurons in nine schizophrenics, six subjects with depression, and nine controls. A polyclonal antiserum against beta-endorphin was employed for the immunohistochemical detection of the peptide in sections of postmortem human brains. Quantitative analysis revealed that the number of beta-endorphin-containing arcuate neurons was statistically reduced in schizophrenics and depressives in comparison to controls. Moreover, the number of endorphinoceptive (i.e. beta-endorphin-innervated) paraventricular nerve cells was also lower in psychiatric patients than in control cases. Our results showing an altered endorphinergic system in human hypothalami of schizophrenics and depressives might contribute to a renewal of interest in this peptide as a possible factor of importance in psychiatric disorders.