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Expression of some genes in the brain of ascitic hepatoma of Zajdela bearing rats was compared with that of control animals using Northern blot hybridization technique. The differences revealed were: an increased expression of actin gene and decreased expression of hsp70 gene in the brain of tumor-bearing animals.
Infection of newborn rats with Borna disease virus (BDV) leads to viral persistence in the central nervous system without overt signs of inflammation. Nevertheless, these rats display distinct behavioral and neurodevelopmental abnormalities. The molecular basis of the latter is still unknown. Using a cDNA array representing 1200 genes, we sought to identify cellular genes which are differentially expressed following perinatal BDV-infection. RNA samples prepared from different brain regions were analysed at various time points before or after BDV-induced defects become evident. In infected brains, we found upregulated expression of genes encoding brain fatty acid binding protein (B-FABP), beta2-microglobulin (beta2m) and, as described previously, the chemokine IP-10. Kinetic studies revealed sustained increased expression of B-FABP in infected frontal cortices beginning about three weeks p.i. Moreover, a slight transient increase of B-FABP expression in infected hippocampi was observed 3-5 weeks p.i. In situ hybridization studies combined with immunohistochemistry suggested that expression of beta2m was predominantly upregulated in glial cells and possibly also in some neurons. Employing cultured infected hippocampus slices and infected genetically modified mice, we provide evidence, that the observed upregulation of beta2m expression is not triggered by IFN-gamma, but rather by IFN-alpha/beta.
Metallothionein (MT) gene expression in the brain has been most thoroughly studied using rodents. Although MT is considered to be a 'housekeeping' protein even in the brain, the basal MT mRNA expression level is not always high. Differences in the responses of rats and mice have made it difficult to interpret the data. Moreover, the response to inducers is not always apparent, probably because the brain is protected by the blood-brain barrier and initial responses to inducers in peripheral tissues modulate their accumulation in the brain. A relatively high content of MT protein in the brain might be sufficient to elicit minute alterations in the level of inducers. Nonetheless, regulation of MT gene expression in the brain seems to be important in e.g. maintaining the levels of trace elements and controlling redox potentials. The localization and utilization of trans elements such as MTF-I and MEP-I in the brain will provide new aspects for study. The high homology among MT isoforms with respect to nucleotide as well as amino acid sequences has made it difficult to obtain cDNA probes or antibodies capable of distinguishing MT isoforms. Thus, their cross-reactivity might make changes in MT mRNAs appear minimal when MT isoforms are differently regulated. The rapid developments in methodology permitting sensitive, rapid, high-resolution analysis could clarify the background of tissue- and cell-specific gene regulation as well as differential induction.
BACKGROUND: Gene silencing in mammalian cells is possible with RNA interference (RNAi) with expression plasmids that encode for short hairpin RNAs (shRNA) that hybridize to a specific sequence within a target mRNA. The limiting factor in developing RNAi therapeutics in mammals is the gene delivery system. METHODS: The present studies describe the production of anti-luciferase shRNA expression plasmids, which are encapsulated in the interior of 85 nm pegylated immunoliposomes (PILs). C6 rat glioma cells, permanently transfected with the luciferase gene, are implanted in the caudate-putamen nucleus of adult rats, which produces luciferase-expressing intracranial brain cancer. The PILs are targeted across the blood-brain barrier and across the tumor cell membrane in vivo with a monoclonal antibody (MAb) to the rat transferrin receptor (TfR). The TfRMAb is tethered to the tips of 1-2% of the poly(ethylene glycol) strands conjugated to the surface of the liposome. RESULTS: The TfRMAb-targeted PILs inhibit luciferase gene expression in the brain cancer by 90%, and this effect persists for at least 5 days after a single intravenous injection of 10 micro g/rat of plasmid DNA. RNAi therapy directed against the luciferase gene caused no change in expression of tumor gamma-glutamyl transpeptidase. Targeting the empty expression plasmid with the TfRMAb-PIL resulted in no change in luciferase activity in the brain cancer in vivo. CONCLUSIONS: In vivo RNAi is enabled with a new form of gene delivery system that encapsulates expression plasmids in PILs, which are targeted to distant sites based on the specificity of a receptor-specific monoclonal antibody. The combined application of the PIL gene delivery system and RNAi expression plasmids enables gene silencing in remote sites such as brain cancer in mammals after intravenous administration.
1. Gene expression studies of human brain tissues in both health and disease require the use of intact, high spectral quality messenger RNA (mRNA). Short post-mortem tissues, when adequately controlled, provide a valuable primary source of brain mRNA. 2. Typical mRNA isolation methodologies rely on total RNA extraction from whole cells or tissues. Here we report that as an alternative approach, nuclei can be isolated from the same tissues and programmed for run-on gene transcription to generate enriched mRNA fractions. 3. This novel technique represents a significant improvement over previous methods and provides high quality, high yield mRNA samples suitable for downstream applications that include cloning, sequencing and gene expression studies using DNA array technologies.
Gene expression profiling of three human temporal lobe brain tissue samples (normal) and four primary glioblastoma multiforme (GBM) tumors using oligonucleotide microarrays was done. Moreover, confirmation of altered expression was performed by whole cell patch clamp, immunohistochemical staining, and RT-PCR. Our results identified several ion and solute transport-related genes, such as N-methyl-d-aspartate (NMDA) receptors, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-2 receptors, GABA(A) receptor subunits alpha3, beta1, beta2, and beta3, the glutamate transporter, the glutamate/aspartate transporter II, the potassium channel K(V)2.1, hK(V)beta3, and the sodium/proton exchanger 1 (NHE-1), that are all downregulated in the tumors compared with the normal tissues. In contrast, aquaporin-1, possibly aquaporins-3 and -5, and GLUT-3 message appeared upregulated in the tumors. Our results also confirmed previous work showing that osteopontin, nicotinamide N-methyltransferase, murine double minute 2 (MDM2), and epithelin (granulin) are upregulated in GBMs. We also demonstrate for the first time that the cytokine and p53 binding protein, macrophage migration inhibitory factor (MIF), appears upregulated in GBMs. These results indicate that the modulation of ion and solute transport genes and heretofore unsuspected cytokines (i.e., MIF) may have profound implications for brain tumor cell biology and thus may identify potential useful therapeutic targets in GBMs.
Ischemia is one of the strongest stimuli for gene induction in the brain. More than 80 different mRNAs have been found to be induced by brain ischemia so far. Many of these genes encode protein products that are involved directly or indirectly in neuronal survival. These include genes that promote recovery by enhanced gene expression (for example, heat shock proteins or growth factors) or attempt to protect them from delayed neuronal death (for example anti-apoptosis genes). Neuronal degeneration can be promoted by induction of apoptosis genes or genes that cause a stress to the cells, such as free radical production by nNOS or iNOS. Even though so many ischemia-inducible genes have been identified, the general reduction of gene transcription and inhibition of protein translation affect neuronal survival the most. The lack of protein synthesis is especially significant when the cells are challenged by ischemia followed by the attack of free radicals during the subsequent recirculation. Even though the ischemia-induced gene expression has a dichotomy to beneficial and harmful genes, several genes such as those encoding transcription factors may participate in both cellular responses. Therefore, pinpointing the receptors and signal transduction mechanisms responsible for the induction of different genes is of interest. So far, only NMDA (Fig. 1) and possibly KA/ AMPA receptor and to some extent alpha 2-adrenoreceptor have proved to be involved in the regulation of perifocal gene induction. Nevertheless, interfering with gene expression offers a potential opportunity for the development of a novel stroke therapy.
Brain opioid peptide systems are known to play an important role in motivation, emotion, attachment behaviour, the response to stress and pain, and the control of food intake. Opioid peptides within the ventral striatum are thought to play a key role in the latter function, regulating the affective response to highly palatable, energy-dense foods such as those containing fat and sugar. It has been shown previously that stimulation of mu opiate receptors within the ventral striatum increases intake of palatable food. In the present study, we examined enkephalin peptide gene expression within the striatum in rats that had been given restricted daily access to an energy-dense, palatable liquid food, chocolate Ensure(R). Rats maintained on an ad libitum diet of rat chow and water were given 3-h access to Ensure(R) daily for two weeks. One day following the end of this period, preproenkephalin gene expression was measured with quantitative in situ hybridization. Compared with control animals, rats that had been exposed to Ensure(R) had significantly reduced enkephalin gene expression in several striatal regions including the ventral striatum (nucleus accumbens), a finding that was confirmed in a different group with Northern blot analysis. Rats fed this regimen of Ensure(R) did not differ in weight from controls. In contrast to chronic Ensure(R), acute ingestion of Ensure(R) did not appear to affect enkephalin peptide gene expression. These results suggest that repeated consumption of a highly rewarding, energy-dense food induces neuroadaptations in cognitive-motivational circuits.
BACKGROUND: Transmissible spongiform encephalopathies (TSEs) or prion diseases are fatal neurodegenerative disorders which occur in humans and various animal species. Examples include Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in deer and elk, and scrapie in sheep, and experimental mice. To gain insights into TSE pathogenesis, we made and used cDNA microarrays to identify disease-associated alterations in gene expression. Brain gene expression in scrapie-infected mice was compared to mock-infected mice at pre-symptomatic and symptomatic time points. Three strains of mouse scrapie that show striking differences in neuropathology were studied: ME7, 22L, and Chandler/RML. RESULTS: In symptomatic mice, over 400 significant gene expression alterations were identified. In contrast, only 22 genes showed significant alteration in the pre-symptomatic animals. We also identified genes that showed significant differences in alterations in gene expression between strains. Genes identified in this study encode proteins that are involved in many cellular processes including protein folding, endosome/lysosome function, immunity, synapse function, metal ion binding, calcium regulation and cytoskeletal function. CONCLUSION: These studies shed light on the complex molecular events that occur during prion disease, and identify genes whose further study may yield new insights into strain specific neuropathogenesis and ante-mortem tests for TSEs.
After our 2002 report on the changes of gene expression in the brain from phencyclidine-treated mouse by using DNA microarray (Toyooka et al., Ann. N.Y. Acad. Sci. 965: 10-20), we decided to apply this DNA microarray method for the brain of the mouse treated with other drugs. We are now examining the effects of clozapine on the brain function. Clozapine is an atypical antipsychotic drug. Clozapine has affinity for neurotransmitter receptors, including D4 dopamine, serotonin, histamine, and adrenergic receptors. MacGibbon et al. (Mol. Brain Res. 23: 21-32) reported that clozapine and haloperidol produced a differential pattern of immediate early gene expression in rat caudate-putamen. Kobayashi et al. (Br. J. Pharmacol. 123: 421-426) have observed the effects of clozapine on the opioid receptors and G-protein-activated inwardly rectifying potassium channel of Xenopus oocytes. Thomas et al. (J. Neurochem. 76: 789-796) found that clozapine increases apolipoprotein D expression in the mouse brain. Therefore, we are going to apply this DNA microarray method to examine the effects of clozapine on mouse brain function. After we injected clozapine into mice for 20 days, we decapitated the mice. We then used the DNA microarray method to examine the gene expression of mouse brain. We found some changes in the mouse brain treated with clozapine. For example, the intensity of a potassium channel spot decreased, and that of a serotonin receptor spot increased.
In order to understand the role of brain localized uncoupling proteins, we have examined the UCP2 and BMCP-1 gene expression in mice brain in two different catabolic states: administration of lipopolysaccharide (LPS) (2.5 mg/kg, i.p.) and tumour burden. Administration of LPS resulted in an increased UCP2 gene expression both in brain (208%) and cerebellum (77%). An increase in UCP2 gene expression was also observed after LPS treatment in double knockout mice for tumour necrosis factor-alpha (TNF) receptors 1 and 2 (75% in brain and 33% in cerebellum). Tumour growth also resulted in increased brain UCP2 gene expression (80%) in mice bearing the Lewis lung carcinoma as compared with the non-tumour-bearing controls. No changes were observed in BMCP-1 mRNA levels of either LPS-injected or tumour-bearing mice. From the results presented it may be suggested that: (a) the brain may contribute significantly to the increase in energy expenditure associated with hypermetabolic states such as fever and tumour burden, and (b) the regulation of UCP2 gene expression in brain does not seem to be influenced by TNF; therefore the action of other cytokines cannot be discarded.
OBJECTIVES: Hepatitis C virus (HCV) infection is often associated with cognitive dysfunction, fatigue and depression. The current study was undertaken to determine whether HCV infection affects gene expression in brain tissue. DESIGN: We analysed the gene expression pattern in brain tissue in a group of HCV-infected patients compared with HCV-negative controls. METHODS: Brain tissue samples were obtained at autopsy from three HCV-positive patients and three HCV-negative control patients. The analysis of gene expression was conducted using differential display and reverse Northern hybridization. Only those genes that were up or downregulated more than 1.8 times were considered to be differentially expressed. RESULTS: Altogether, 29 differentially expressed genes were identified by differential display and subsequently confirmed by reverse Northern hybridization. A prominent finding was the downregulation of mitochondrial oxidative phosphorylation genes in HCV-infected patients. The impairment of brain oxidative/energy metabolism has previously been suggested to be the proximate cause of many disorders that impair mentation. Another finding was the downregulation of some ribosomal protein genes and several genes involved in transcription regulation, perhaps reflecting reduced metabolic activities. CONCLUSION: Our findings suggest for the first time that there may be a biological basis for the neuropsychiatric symptoms and cognitive impairment associated with HCV infection.
Cholecystokinin (CCK) gene expression has been compared in the brain and duodenum of control and 5 days fasted rats. To study transcription, CCK mRNA was quantified using a solution hybridization assay. Large and small molecular weight CCK peptides were separated using a sequential extraction process and subsequently quantified by radioimmunoassay. In the duodenum, a fall in weight was paralleled by a decrease in CCK mRNA and in the large forms of CCK peptides. Small molecular species of CCK peptides did not change. There was no change in weight, CCK transcriptional or translational products in the brain as a whole. These data indicate location-specific differential regulation of the products of CCK gene expression in the fasted rat.
We previously revealed similarity in gene expression patterns between human brain and testis, based on digital differential display analyses of 760 human Unigenes. In the present work, we reanalyzed the gene expression data in many tissues of human and mouse for a large number of genes almost covering the respective whole genomes. The results indicated that both in human and in mouse, the gene expression profiles exhibited by brain, cerebellum and testis are most similar to each other compared with other tissues.
Members of the ligand-gated neuronal nicotinic acetylcholine receptor (nAChR) gene family ( CHRNA4 and CHRNB2, coding for the alpha4 and beta2 subunits, respectively) are involved in autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). However, ADNFLE is genetically heterogeneous and mutations in CHRNA4 and CHRNB2 account for only a minority of ADNFLE cases. Additional nAChR subunits expressed in the brain are candidates for this epilepsy. The involvement of all genes coding for brain-expressed nAChR subunits, with known chromosome localization ( CHRNB2, 1q21; CHRNA2, 8p21; CHRNA6, CHRNB3, 8p11.2; CHRNA7, 15q14; CHRNA5/A3/B4, 15q24 and CHRNA4, 20q13.2) was investigated in four unrelated ADNFLE Italian families for at least three generations. Families were selected on the basis of anamnestic and videopolysomnographic analyses. Individuals were typed for polymorphic markers located in the above mentioned chromosome regions. Linkage and mutation analyses were performed. In none of the families was linkage between ADNFLE and the analysed chromosome regions detected. These findings support the hypothesis that genes different from those coding for alpha2-7 and beta2-4 neuronal nAChR subunits could be responsible for ADNFLE.
Genes escaping X-inactivation are predicted to contribute to differences in gene dosage between the sexes and are the prime candidates for being involved in the phenotype observed in individuals with X chromosome aneuploidies. Of particular interest is ProtocadherinX (PCDH11X or PCDHX), a recently described gene expressed in brain. In humans, PCDH11X has a homologue on the Y chromosome and is predicted to escape from X-inactivation. Employing bisulphite sequencing analysis we found absence of CpG island methylation on both the active and the inactive X chromosomes, providing a strong indication that PCDH11X escapes inactivation in humans. Furthermore, a sexual dimorphism in levels of expression in brain tissue was observed by quantitative real-time PCR, with females presenting an up to 2-fold excess in the abundance of PCDH11X transcripts. We relate these findings to sexually dimorphic traits in the human brain. Interestingly, PCDH11X/Y gene pair is unique to Homo sapiens, since the X-linked gene was transposed to the Y chromosome after the human-chimpanzee lineages split. Although no differences in promoter methylation were found between humans and chimpanzees, evidence of an upregulation of PCDH11X in humans deserves further investigation.
The major issue in the post-genome sequencing era is determination of gene expression patterns in variety of biological systems. A microarray system is a powerful technology for analyzing the expression profile of thousands of genes at one experiment. In this study, we identified highly expressed genes in mouse brain using the cDNA microarray carrying 2304 cDNAs derived from oligo-capped mouse cDNA library. Nine genes were highly expressed in adult mouse brain compared to kidney, liver, and skeletal muscle. Tissue distribution analysis by reverse transcription-coupled polymerase chain reaction (RT-PCR) revealed that consistent with the microarray data, all of the selected 9 genes were predominantly expressed in the brain. A database search showed that 5 of the 9 genes, MBP, SC1, HiAT3, S100 protein-beta, and SNAP25, were previously known to be expressed at high level in the brain and in the nervous system. One gene was highly sequenced similar to rat S-Rex-s/human NSP-C, suggesting that the gene is a mouse homologue. The remaining three genes did not match to known genes in the GenBank/EMBL database, indicating that these are novel genes highly expressed in the brain. Taken together, our cDNA microarray system can be an excellent tool for identifying differentially expressed genes in mouse brain.
Gonadal steroid hormones act in the brain to elicit changes in gene expression that result in profound effects on behavior and physiology. A variety of in vitro studies indicate that nuclear receptor coactivators are required for efficient transcriptional activity of steroid receptors. Two nuclear receptor coactivators, steroid receptor coactivator-1 (SRC-1) and cAMP response element binding protein-binding protein (CBP), have been shown to act in concert to enhance ER activity in vitro. In the present study, we investigated the function of these important nuclear receptor coactivators in estrogen action in rodent brain. Reduction of SRC-1 and CBP protein in brain disrupted ER-mediated activation of the behaviorally relevant progestin receptor gene. Furthermore, we found that SRC-1 and CBP function in brain to modulate the expression of hormone-dependent female sexual behavior. These findings indicate that these nuclear receptor coactivators function in brain to modulate ER transcriptional activity and the expression of hormone-dependent behavior.