Melanocortin-3 receptor mRNA expression in pro-opiomelanocortin neurones of the rat arcuate nucleus

The melanocortins alpha- and gamma-melanocyte-stimulating hormones (alpha- and gamma-MSH) derive from the pro-opiomelanocortin (POMC) precursor. Melanocortins exert a wide range of biological activities in the brain through activation of at least three distinct melanocortin receptor (MC-R) subtypes. In order to determine whether POMC neurones can modulate their own activity, we looked for the possible expression of the MC3-R gene in POMC-positive cell bodies in the rat hypothalamus. In situ hybridization experiments revealed that the density of MC3-R mRNA is particularly high in the arcuate nucleus which contains the main population of POMC neurones in the brain. The occurrence of MC3-R mRNA in POMC-positive cell bodies was demonstrated using a double-labelling in situ hybridization technique. The proportion of POMC neurones expressing MC3-R mRNA was significantly higher in the most rostral (43.5%) than in the most posterior part of the arcuate nucleus (8.2%). These results indicate that melanocortins likely exert a direct regulatory feedback on POMC neurones through activation of MC3-R receptors. Our data also suggest that MC3-R may be involved in the neuroendocrine responses induced by centrally administered melanocortins.     

BACE1 mRNA expression in Alzheimer's disease postmortem brain tissue

β-site AβPP cleaving enzyme 1 (BACE1) catalyses the rate-limiting step for production of amyloid-β (Aβ) peptides, involved in the pathological cascade underlying Alzheimer's disease (AD). Elevated BACE1 protein levels and activity have been reported in AD postmortem brains. Our study explored whether this was due to elevated BACE1 mRNA expression. RNA was prepared from five brain regions in three study groups: controls, individuals with AD, and another neurodegenerative disease group affected by either Parkinson's disease (PD) or dementia with Lewy bodies (DLB). BACE1 mRNA levels were measured using quantitative realtime PCR (qPCR) and analyzed by qbasePLUS using validated stably-expressed reference genes. Expression of glial and neuronal markers (glial fibrillary acidic protein (GFAP) and neuron-specific enolase (NSE), respectively) were also analyzed to quantify the changing activities of these cell populations in the tissue. BACE1 mRNA levels were significantly elevated in medial temporal and superior parietal gyri, compared to the PD/DLB and/or control groups. Superior frontal gryus BACE1 mRNA levels were significantly increased in the PD/DLB group, compared to AD and control groups. For the AD group, BACE1 mRNA changes were analyzed in the context of the reduced NSE mRNA, and strongly increased GFAP mRNA levels apparent as AD progressed (indicated by Braak stage). This analysis suggested that increased BACE1 mRNA expression in remaining neuronal cells may contribute to the increased BACE1 protein levels and activity found in brain regions affected by AD.     

Regional distribution of neuropeptide Y mRNA in postmortem human brain

The distribution of messenger RNA encoding neuropeptide Y (NPY) was studied in 11 different postmortem human brain regions using in situ hybridization histochemistry, and RNA blot analysis. In situ hybridization data revealed that the highest numerical density of labeled cells corresponded to neurons in accumbens area, caudate nucleus, putamen, and substantia innominata. Significantly fewer NPY mRNA-containing neurons were found in frontal and parietal cortex, amygdaloid body and dentate gyrus. No NPY mRNA-containing cells were found in substantia nigra. NPY mRNA-positive neurons from all regions studied showed relatively similar labeling, as revealed by computerized image analysis. Blot analysis showed an approximately 0.8 kb NPY mRNA in all brain regions studied, except in substantia nigra and cerebellum. Densitometric scanning of the autoradiograms revealed levels of NPY mRNA in the following order: putamen greater than caudate nucleus greater than frontal cortex (Brodmann areas 4 and 6) greater than temporal cortex (Brodmann area 38) greater than parietal cortex (Brodmann areas 5 and 7) greater than frontal cortex (Brodmann area 11). Hence, although NPY mRNA is widely distributed in neurons of the human brain large regional variation exists, with the highest expression in accumbens area and parts of the basal ganglia.     

Effect of pre- and postmortem variables on specific mRNA levels in human brain.

The method of polymerase chain reaction was used to investigate the pre- and postmortem factors which affect the stability of specific mRNAs in the C1 region of human autopsy brain. Eight premortem and 4 postmortem factors were correlated to levels of phenylethanolamine N-methyltransferase (PNMT), three splice forms of amyloid precursor protein (APP) and actin mRNAs in 10 control brains using Pearson's correlation coefficient. Significant negative correlations were found between hypoxia and PNMT mRNA, and between postmortem and storage intervals and APP751 and beta-actin mRNAs. A positive correlation was found between death-refrigeration interval and total APP and APP695 mRNAs. There was also a positive correlation between seizure activity and APP770 mRNA. The results indicate that a variety of pre- and postmortem factors can affect mRNA levels. The possible effect of pre- and postmortem factors on specific mRNA levels should be investigated prior to comparing mRNA levels in different disease states.     

Glutamate receptor subtype expression in human postmortem brain

Antibodies to functional glutamate receptor subunits were utilized as probes to characterize glutamatergic receptors in human postmortem brain tissue. Crude membranes from rat, monkey, and various dissected human postmortem brain regions were fractionated by SDS-PAGE and electrotransferred to nitrocellulose. Using antisera raised against rat antigens for AMPA/kainate (GluR1-3) and kainate (GluR5) glutamate receptor subunits, we have been able to detect specific bands on Western blots in rat, monkey, and human postmortem brain tissue. These antisera recognized bands at approx 105 kDa for the GluR1-3 and 115 kDa for GluR5 in humans, monkeys, and rats. All of these glutamate receptor subtypes appear to be glycosylated. We observed varying levels of expression in the human brain areas examined, with the highest degree of expression in the hippocampus and temporal cortex for AMPA/kainate receptor subunits, and in the cortex and cerebellum for the kainate receptor subunits. In addition, considerable heterogeneity in expression was observed between patient samples with these antisera, as well as with antisera to the structural protein, NCAM. Our studies indicate that glutamatergic receptor protein changes related to various human disease states may be examined in human postmortem tissue by Western blotting techniques utilizing these antibodies raised to the rat protein.     

Effect of agonal and postmortem factors on gene expression profile: quality control in microarray analyses of postmortem human brain.

There are major concerns that specific agonal conditions, including coma and hypoxia, might affect ribonucleic acid (RNA) integrity in postmortem brain studies. We report that agonal factors significantly affect RNA integrity and have a major impact on gene expression profiles in microarrays. In contrast to agonal factors, gender, age, and postmortem factors have less effect on gene expression profiles. The Average Correlation Index is proposed as a method for evaluating RNA integrity on the basis of similarity of microarray profiles. Reducing the variance due to agonal factors is critical in investigating small but validated gene expression differences in messenger RNA levels between psychiatric patients and control subjects.     

Gene expression profiles derived from single cells in human postmortem brain

The study of postmortem human brain tissue remains the basis for the understanding of many CNS disorders and to verify data obtained in experimental studies. So far, however, gene expression profiling in cellular sub-populations derived from human postmortem brain was hampered by several technical drawbacks. Here, we describe a method that allows the generation of mRNA expression profiles from single neurons. Dopaminergic neurons from different midbrain areas including substantia nigra, central gray substance and ventral tegmental area were identified and isolated by immuno-laser capture microscopy (LCM). Expression profiles were generated from microdissected cells using a modified RNA fingerprinting protocol. Using this approach, we were able to generate specific RNA fingerprints at a high resolution from phenotype-specific single neurons. Polymorphic fragments were isolated from gels and differential gene expression was confirmed by real-time PCR using gene-specific primer pairs and hybridization probes. The method described here is easy to use and reliable for profiling gene expression at the single cell level in human postmortem brain. It could therefore be valuable to open new insights into the molecular pathogenesis of CNS disorders.     

Quantifying mRNA in postmortem human brain: influence of gender, age at death, postmortem interval, brain pH, agonal state and inter-lobe mRNA variance

The quantification of mRNA in postmortem human brain is often made complicated by confounding factors. To assess the importance of potential confounders TaqMan real-time RT-PCR was used to measure seven mRNAs (beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin, microtubule-associated protein (MAP) 2, neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP), amyloid precursor protein (APP) isoform 770) in cortical samples taken from 90 Alzheimer's disease (AD) and 81 control brains. Demographic data for the brain samples were assessed for interaction between factors and amounts of mRNA. Gender was found to play a role in that females had lower levels of mRNA relative to males; this was consistent in both the AD and control brains. Age at death had inconsistent but significant correlations to amounts of mRNA; male and female controls both had negative correlations, female AD a positive correlation and male AD no correlation. Positive correlations were found between brain pH and amount of mRNA in all genes except glial fibrillary acidic protein (GFAP); correlations were consistent across all groupings of pathology and gender. Mean brain pH was significantly lower in AD (6.4) than in control subjects (6.5, ANOVA, p<0.01), though there was no difference between male and females of either group. No correlation was found between brain pH and age at death. Postmortem interval was correlated with brain pH in Alzheimer's disease brains but not controls. Agonal state was generally a poor predictor of mRNA levels whilst inter-lobe variance of mRNA was found to be non-significant in control brains. Given that gender, age at death and brain pH all have significant effects upon mRNA levels it is recommended that these factors be taken into account when quantifying gene expression in postmortem human brain.     

Distribution of glucocorticoid and mineralocorticoid receptor messenger RNA expression in human postmortem hippocampus

Corticosteroids bind to hippocampal glucocorticoid (GR) and mineralocorticoid (MR) receptors, thereby affecting behaviour and neurochemical transmission. Rat hippocampus has high levels of both receptors and their messenger RNAs (mRNA), but there is little information on receptors in human brain. We used in situ hybridization to determine the distribution of GR and MR mRNA expression in human hippocampus. Frozen sections of human postmortem hippocampus (5 patients, 58-88 years old, without cerebral pathology) were postfixed in paraformaldehyde and hybridized with 35S-UTP-labelled cRNA probes (transcribed in vitro from human cDNA subclones) under stringent conditions. Control included hybridization with sense probes and heterologous cRNA competition studies. GR mRNA was highly expressed in dentate gyrus, CA3 and CA4, but levels were significantly lower in CA1 and CA2. MR mRNA was also very highly expressed in hippocampus, with significantly higher levels in dentate gyrus and CA2, CA3 and CA4 than CA1. Controls confirmed the specificity of hybridization and there was little hybridization of sense probes. High GR and MR mRNA expression is found in both rat and human hippocampus but the subregional distributions clearly differ between the species.     

Regional expression of RGS4 mRNA in human brain

Regulators of G-protein signalling (RGS) proteins are a recently discovered class of proteins that modulate G-protein activity. More than 20 RGS proteins have been identified and are expressed throughout the body and brain. In particular, RGS4 appears to regulate dopamine receptor function and has been implicated in several dopamine related diseases, including schizophrenia. This study presents an extensive examination of the regional distribution of RGS4 mRNA in postmortem human brain. Using in situ hybridization, the expression levels of RGS4 mRNA were determined in human hemicoronal (Talairach sections +8 and -20) brain sections. In the rostral slice (Talairach +8) highest levels were found in the inferior frontal cortex, the superior frontal, and the cingulate cortex. Slightly lower levels were found in the insular cortex and inferior temporal cortex. The caudate, putamen and nucleus accumbens had lower levels. In the caudal slice (-20), the cortical layers showed the highest levels, with moderate levels observed in the parahippocampal gyrus, low levels in the CA-pyramidal region, and almost undetectable levels in the thalamus. In the frontal cortex a dense band was apparent near one of the inner layers of the cortex. In conclusion, RGS4 mRNA distribution in human postmortem tissue from normal persons was very dense in most cortical layers examined, with lower density in the basal ganglia and thalamus.     

Zinc transporter mRNA levels in Alzheimer's disease postmortem brain

Zinc (Zn2+) is concentrated into pre-synaptic vesicles and co-released with neurotransmitter at some synapses. Zn2+ can accelerate assembly of the amyloid-β peptides (Aβ) and tau protein central to the neuropathological changes found in Alzheimer's disease (AD). Altered protein levels of the membrane Zn2+ transporters ZnT1, ZnT4, and ZnT6 have been reported in AD postmortem brain tissue. The present study analyzed mRNA levels of five established (LIV1, ZIP1, ZnT1, ZnT4, and ZnT6) and one potential (PRNP) Zn2+ transporter in human postmortem brain tissue from Braak-staged individuals with AD and controls using quantitative real-time PCR. Four cortical regions (middle temporal gyrus, superior occipital gyrus, superior parietal gyrus, and superior frontal gyrus) and cerebellum were examined. PRNP mRNA levels were decreased by ～30% in all four cortical regions examined in AD patients, but unchanged in the cerebellum. In contrast, some increases in mRNA levels of the other more established Zn2+ transporters (LIV1, ZIP1, ZnT1, ZnT6) were found in AD cortex. The ratios of the mRNA levels of LIV1, ZIP1, ZnT1, ZnT4, and ZnT6/mRNA level of neuron specific enolase increased significantly as the disease progressed and Braak stage increased. Significant correlations were also identified between mRNA levels of several of the Zn2+ transporters investigated. These expression changes could either reflect or cause the altered cortical Zn2+ distribution in AD, potentially increasing the likelihood of interactions between Zn2+ and Aβ or tau protein.     

Response of pituitary and spleen pro-opiomelanocortin mRNA, and spleen and thymus interleukin-1 beta mRNA to adjuvant arthritis in the rat.

During development of adjuvant-induced arthritis (AA) in the rat, pituitary pro-opiomelanocortin (POMC) mRNA expression was increased. Pituitary POMC mRNA was much higher following adrenalectomy and AA. Spleen POMC mRNA also increased with a similar time kinetics, although the levels in the spleen were much lower than those in the pituitary. In control animals, spleen interleukin-1 beta (IL-1 beta mRNA) was undetectable, whereas AA led to the accumulation of IL-1 beta mRNA and the highest levels were seen in the adrenalectomised AA group. Thymic IL-1 beta expression was also increased in AA animals. These results suggest that AA leads to the activation of both the neuroendocrine and the immune systems and the interaction between these systems may play a role in this disease state.     

Serotonergic stimulation of corticotropin-releasing hormone and pro-opiomelanocortin gene expression

The neurotransmitter serotonin (5-HT) stimulates adrenocorticotropic hormone (ACTH) secretion from the anterior pituitary gland via activation of central 5-HT1 and 5-HT2 receptors. The effect of 5-HT is predominantly indirect and may be mediated via release of hypothalamic corticotropin-releasing hormone (CRH). We therefore investigated the possible involvement of CRH in the serotonergic stimulation of ACTH secretion in male rats. Increased neuronal 5-HT content induced by systemic administration of the precursor 5-hydroxytryptophan (5-HTP) in combination with the 5-HT reuptake inhibitor fluoxetine raised CRH mRNA expression in the paraventricular nucleus (PVN) by 64%, increased pro-opiomelanocortin (POMC) mRNA in the anterior pituitary lobe by 17% and stimulated ACTH secretion five-fold. Central administration of 5-HT agonists specific to 5-HT1A, 5-HT1B, 5-HT2A or 5-HT2C receptors increased CRH mRNA in the PVN by 15-50%, POMC mRNA in the anterior pituitary by 15-27% and ACTH secretion three- to five-fold, whereas a specific 5-HT3 agonist had no effect. Systemic administration of a specific anti-CRH antiserum inhibited the ACTH response to 5-HTP and fluoxetine and prevented the 5-HTP and fluoxetine-induced POMC mRNA response in the anterior pituitary lobe. Central or systemic infusion of 5-HT increased ACTH secretion seven- and eight-fold, respectively. Systemic pretreatment with the anti-CRH antiserum reduced the ACTH responses to 5-HT by 80% and 64%, respectively. It is concluded that 5-HT via activation of 5-HT1A, 5-HT2A, 5-HT2C and possibly also 5-HT1B receptors increases the synthesis of CRH in the PVN and POMC in the anterior pituitary lobe, which results in increased ACTH secretion. Furthermore, the results indicate that CRH is an important mediator of the ACTH response to 5-HT.     

Complement and cytokine gene expression in cultured microglia derived from postmortem human brains

Microglia were successfully cultured from human brain tissue from normal and neurologically diseased cases obtained 3.5–10 hours postmortem. Final cell preparations were more than 99% pure as judged by latex bead phagocytosis, expression of microglial phenotypic markers, and absence of astrocytic markers. The expression of complement genes C1qB, C3, and C4 as well as genes for interleukin-(IL-)1α, IL-1β, IL-6, tumor necrosis factor (TNF)α, IL-1 receptor antagonist, and transforming growth factor β, but not inducible nitric oxide synthase, by these cells was detected by polymerase chain reaction (PCR) analysis. The pattern of gene expression was evaluated following stimulation of the cells with lipopolysaccharide, phorbol myristate acetate, γ interferon, and β amyloid peptide. There was considerable variation in gene response to these activating agents. However, it was of interest that β-amyloid peptide (1–40) increased the expression of IL-1β mRNA in these cells. The number of cases in this study was too small to permit evaluation of microglial response according to the disease state, but the results demonstrate the potential for such studies in the future. © 1995 Wiley-Liss, Inc.     

Quantitative autoradiography of cocaine binding sites in human brain postmortem.

Quantitative autoradiography was used to study cocaine binding sites in the human brain postmortem. Tritiated cocaine was applied to brain sections from three drug- and disease-free subjects at a low (10 nM) concentration and at a high (1 microM) concentration, the latter being in the range of brain concentrations of cocaine found in users of the drug. Nonspecific binding was assessed in the presence of 100 microM unlabeled cocaine. At low (10 nM) concentrations of labeled cocaine, the basal ganglia exhibit the highest density of binding sites, with considerably lower densities in thalamus, cortex, and hippocampus. Cocaine binding at high (1 microM) concentrations displayed a different distribution pattern, more homogeneous with some cortical regions exhibiting binding site densities close to those seen in the basal ganglia. Preliminary competition experiments with several drugs indicate that dopamine uptake inhibitors completely block cocaine binding to the basal ganglia, while serotonin uptake inhibitors were more effective in the hippocampus. These findings suggest that cocaine binds to dopamine uptake sites in the human basal ganglia postmortem but that it also interacts with other classes of binding sites, depending on the concentration and brain region examined.     

Glutamate receptor subtype expression in human postmortem brain tissue from schizophrenics and alcohol abusers

Antibodies to functional AMPA/kainate (GluR1, GluR2, GluR3), and kainate binding sites (GluR5–7) were used as probes to characterize and quantitate glutamatergic receptor subtypes in human post-mortem brain tissue from schizophrenic subjects and non-psychotic control subjects, which included normal controls and subjects with a previous history of alcohol abuse. Crude membrane fractions from human hippocampi and cingulate cortices were fractionated by SDS-PAGE, electrotransferred to nitrocellulose, and probed for the various glutamate receptor subytpes. Western blots were developed with chemilluminescence and the images analyzed by densitometry. Significant reductions were observed in the hippocampal immunoreactivity of both GluR2 and GluR3 AMPA/kainate receptor subtypes in schizophrenic subjects compared to the entire group of non-psychotic control subjects. No significant changes were observed in schizophrenic hippocampal GluR1 and GluR5 receptor subtypes or in levels of the structural control proteins, NCAM and tau. Significant increases were observed for GluR2 and GluR3 in the hippocampi of subjects with alcohol abuse histories when compared to the non-psychotic normal control group. When subjects with alcohol abuse histories were removed from the non-psychotic control pool, schizophrenics were no longer statistically different from the remaining normal controls. An analysis of GluR2 and GluR3 immunoreactivity in the cingulate cortex revealed no changes in these receptor subtypes among any of the groups. No alterations were observed in the immunoreactivity of these various proteins due to confounding factors such as age, sex, postmortem interval, or smoking history, except in the cingulate cortex were GluR3 receptor subtype levels were significantly reduced in the brains of smokers. These results generally do not support a role for the non-NMDA type glutamatergic receptors in schizophrenia. However, the role of chronic ethanol exposure on the human brain needs to be further investigated, and underscores the importance of defined control tissue for human postmortem studies.     

Elevation of D4 dopamine receptor mRNA in postmortem schizophrenic brain

The D₄ dopamine (DA) receptor has been proposed to be a target for the development of a novel antipsychotic drug based on its pharmacological and distribution profile. There is much interest in whether D₄ DA receptor levels are altered in schizophrenia, but the lack of an available receptor subtype-specific radioligand made this difficult to quantitate. In this study, we examined whether D₄ mRNA levels are altered in different brain regions of schizophrenics compared to controls. Ribonuclease protection assays were carried out on total RNA samples isolated postmortem from frontal cortex and caudate brain regions of schizophrenics and matched controls. ³²P-labelled RNA probes to the D₄ DA receptor and to the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), were hybridised with the RNA samples, digested with ribonucleases to remove unhybridised probe, and separated on 6% sequencing gels. Densitometer analysis on the subsequent autoradiogams was used to calculate the relative optical density of D₄ mRNA compared to G3PDH mRNA. Statistical analysis of the data revealed a 3-fold higher level (P<0.011) of D₄ mRNA in the frontal cortex of schizophrenics compared to controls. No increase was seen in caudate. D₄ receptors could play a role in mediating dopaminergic activity in frontal cortex, an activity which may be malfunctioning in schizophrenia.     

Protein expression profiling of postmortem brain in schizophrenia

Surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) enables the sensitive, high-throughput protein profiling of complex biological mixtures. In combination with bioinformatics, this technology has the potential to identify combinations of spectral peaks that can differentiate individuals with a particular disease from normal controls. SELDI-TOF-MS was used to screen postmortem tissue derived from the dorsolateral prefrontal cortex of individuals with schizophrenia (n = 34) and matched controls (n = 35), obtained from the Stanley Foundation Neuropathology Consortium. Tissue samples were homogenized in urea buffer, applied to four different chip arrays which possess different chromatographic surfaces, and analyzed using the Ciphergen ProteinChip Biomarkers System (Model PBS II). Protein expression profiles of the schizophrenia and control groups were compared and analyzed using the Ciphergen Express (CE) and Biomarker Patterns Software (BPS) package. We detected several protein peaks whose intensities differed between the schizophrenia and control groups to a highly significant degree. A combination of these peaks was capable of distinguishing between schizophrenia and controls with a sensitivity and specificity of about 70%. The classification model that distinguished schizophrenia from controls was complex, suggesting that the biochemical abnormalities underlying schizophrenia are heterogeneous. Our results suggest that SELDI-TOF-MS has the potential for distinguishing individuals with schizophrenia from normal controls and may eventually lead to a better understanding of the classification, diagnosis and pathogenesis of this disorder.