Alzheimer Disease (AD) is a progressive neurodegenerative disorder characterized by memory loss and dementia. Both energy metabolism and the function of creatine kinase are known to be affected in Alzheimer diseased brain. With synchrotron FTIR microscopy, extensive deposits of crystalline creatine (Cr) had been discovered in TgCRND8 mouse brain tissue by previous students in our lab. In this thesis, regions of hippocampus and caudate of 5 pairs of transgenic mice and their non-transgenic littermate controls were mapped using Raman and IR microspectroscopy to find clues to Cr origin in transgenic mouse brain. Raman spectra obtained at higher spatial resolution (1-2 µm) were used for better delineation of the Cr crystalline deposits and their environs. These results indicate that Cr crystals were formed after snap-freezing and desiccation of brain tissue. Therefore, it can be speculated that Cr might be exist in solution form in vivo.

Alzheimer Disease (AD) is a progressive neurodegenerative disorder characterized by memory loss and dementia. Both energy metabolism and the function of creatine kinase are known to be affected in Alzheimer diseased brain. With synchrotron FTIR microscopy, extensive deposits of crystalline creatine (Cr) had been discovered in TgCRND8 mouse brain tissue by previous students in our lab. In this thesis, regions of hippocampus and caudate of 5 pairs of transgenic mice and their non-transgenic littermate controls were mapped using Raman and IR microspectroscopy to find clues to Cr origin in transgenic mouse brain. Raman spectra obtained at higher spatial resolution (1-2 µm) were used for better delineation of the Cr crystalline deposits and their environs. These results indicate that Cr crystals were formed after snap-freezing and desiccation of brain tissue. Therefore, it can be speculated that Cr might be exist in solution form in vivo.

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No description available.

Schizophrenia is a devastating psychiatric disorder that affects approximately 1% of the population. The main treatments for schizophrenia are antipsychotic drugs that target dopamine receptors, yet the underlying biological mechanisms through which they alleviate the symptoms of schizophrenia remain ill defined. In this study, we used microarray analysis to profile the expression changes of thousands of genes simultaneously, following antipsychotic drug treatment of mice. Mice were treated chronically (28 days), or for a novel intermediate time-point (7 days), with one of three antipsychotic drugs: clozapine, haloperidol or olanzapine. The use of three drugs enabled us to discern antipsychotic-specific effects co-regulated by multiple drugs, rather than the side effects of individual compounds. Transcript profiling and validation by quantitative PCR of whole brain tissue revealed antipsychotic drug regulation of genes in diverse biological pathways, including: dopamine metabolism, neuropeptide and second-messenger signalling, neurogenesis, synaptic plasticity, cell adhesion, myelination, and voltage-gated ion channels. The regulation of voltage-gated channels by antipsychotic drugs has been suggested previously by electrophysiological studies, although thorough analysis has not been undertaken in vivo. Therefore, the second aim of this study was to characterise the regional mRNA and protein expression of two genes altered by multiple APDs, the voltage-gated potassium channel ??-subunit (Kcna1) and voltage-gated potassium channel interacting protein (Kchip3). Regional characterisation and expression analyses were carried out by immunohistochemistry, in situ hybridisation, and Western blot analysis of mouse brain regions of interest to schizophrenia and its treatment. Following 7-day haloperidol treatment we observed up-regulation of Kcna1 in the striatum and dentate gyrus, with increased protein in the striatum, hippocampus and midbrain; and down-regulation of Kchip3 in the striatum, with decreased protein in the cortex, hippocampus and midbrain. These studies implicate voltage-gated potassium channels in the antipsychotic drug regulation of midbrain dopaminergic neuronal activity, adult neurogenesis and/or striatothalamic GABAergic neuronal inhibition. These findings indicate that regulation of potassium channels may underlie some of the mechanisms of action of antipsychotic drugs, and that voltage-gated ion channels may provide alternative drug targets for the treatment of schizophrenia.

Traumatic brain injury (TBI) is the most common cause of mortality and disability in the younger (<50 years) Swedish population with an incidence rate of 20,000 cases per year. This thesis aims to increase the understanding of brain injury mechanisms, especially in a molecular and cellular context. Bone morphogenetic protein (BMP) signalling was examined in three genetically modified mice (two “loss-of-function”, one “gain-of-function”) exposed to TBI (controlled cortical impact, CCI) with CaMKII used as promoter for Cre-driven recombination in postnatal forebrain neurons. The mice survived, developed normally and did not show any obvious phenotypes except for an upregulation in Mtap2 mRNA in mice with impaired BMP signalling. Reactive Gfap and Timp1 mRNA expression measured using quantitative RT-PCR (qRT-PCR) was reduced in the mice overexpressing BMP signals. The BMP signalling pathway was further studied in cultured PC12 cells with BMP4 and NGF added. Egr3 expression was substantially increased by these growth factors. Blocking Egr or Junb functions reduced neurite outgrowth. TBI-induced mRNA expression changes in 100 selected genes in C57BL/6J mouse neocortex and hippocampus were measured using qRT-PCR at different time points post-injury. Several distinct gene clusters with similar expression patterns were identified. GeneChip analysis (Affymetrix) of the injured mouse neocortex at three days revealed 146 transcripts significantly upregulated, confirming and extending the qRT-PCR results. The findings demonstrate marked increases after injury among chemokine transcripts and activation of many genes involved in inflammation. In conclusion, the present study has revealed transcriptional changes in specific signalling pathways after brain injury. The results may help to identify novel targets for neuroprotective interventions after traumatic brain injury.

One of the key events in a virus-infected host cell is the activation and repression of a large number of host genes. In recent years, such differentially expressed host genes have been identified for several viruses, bacteria and parasites. Such studies indicate that reprogramming of host transcriptome during infection by a pathogen is a major component of host response and many of the reprogrammed genes may promote or prevent pathogen infection or may contribute to pathogen-induced pathological changes. Host gene expression changes have been studied for a number of viruses in cell lines. However, in case of neurotropic viruses which infect nonrenewable populations of central nervous system (CNS), changes in the host gene expression need to be studied in the intact host rather than cells grown in culture. Since such studies are reported only for a few neurotropic viruses, an attempt has been made in this thesis to identify and characterize genes that are differentially expressed in the mouse brain during infection by Japanese encephalitis virus (JEV) and rabies virus. Using subtraction hybridization technique, subtraction cDNA libraries were generated representing mRNAs that are induced or repressed in the mouse brain during JEV infection. Sequence analysis of the 350 isolated clones resulted in the identification of 73 unique genes. Out of these, 66 were of forward library clones (upregulated genes) and 7 of reverse library clones (downregulated genes). The forward library clones was clustered in different functional groups such as, proteins involved in immune response and interferon-inducible proteins, GTPase and GTP binding proteins, transcriptional regulators, enzymes, ribosomal proteins, neuronal proteins, carrier proteins, DNA-binding proteins, miscellaneous and proteins of unknown function. The differential expression of all these genes was further validated by northern blot analysis of brain RNA isolated from normal and JEV-infected mice, which indicate that out of 66 forward library clones 33 were genuinely upregulated in JEV-infected mouse brain, whereas all 7 reverse library clones were repressed in JEV infection. Since vaccination is known to prevent virus replication in the brain, host gene expression changes in mice immunized with BIKEN JE vaccine was also examined. There was a good correlation between inhibition of JEV replication and reduced expression of JEV-inducible CNS genes in the vaccinated mice. To check whether JEV-induced CNS genes identified in this study are specific to JEV or can be induced by any other neurotropic virus, expression patterns of 15 randomly chosen genes were checked in RV infected mouse brain. Results indicated that all the chosen genes are modulated in the same way during RV infection as well. Comparison of JEV-induced gene expression changes with those induced by other neurotropic viruses indicated that 83% of the JEV-inducible mouse CNS genes are also induced by Sindbis virus, a neurotropic virus of the family alphaviridae, indicating that despite diverse life cycles, these two viruses may activate common host signaling pathways. This study also led to the identification of 9 unique JEV-inducible genes (LRG-21, VHSV induced gene1, Tpt1, SLC25A3, Olfm1, Ina/NF-66, Dst/Bpag1, Mdm2 and Gbp5) which are not reported to be activated by any other neurotropic virus. Since it is beyond the scope of this study to characterize all the JEV-induced and repressed genes, two genes were chosen for a detailed analysis. These are: JEV-inducible gene encoding GARG-39 protein which is a member of the glucocorticoid attenuated response gene family and an unannotated, JEV-repressible gene designated in this study as clone # 137. The gene encoding GARG-39 identified as a JEV-inducible gene in this study was originally discovered as lipopolysaccharide- and interferon-inducible gene in macrophages. This protein contains tetratricopeptide repeat (TPR) motifs that are known to be involved in protein-protein interactions. However, the function of this protein remains unknown till date. Therefore the gene was cloned and over-expressed in E. coli and antibodies were raised against the recombinant protein. Western blot analysis revealed that GARG-39 protein is detectable only in JEV-infected but not in the normal mouse brain. Surprisingly, immunoflourescence studies carried out in NIH3T3 cells revealed that GARG-39 is localized in the cytosol of normal cells and it colocalizes with α-tubulin in the mitotic spindle in a small fraction of cells which are in the mitotic stage. Further, in an in vitro assay, GARG-39 was found to interact with taxol-stabilized tubulin polymers. Since microtubules are known to play an important role in virus assembly, it is possible that GARG-39 may have a role in virus assembly and maturation. Alternatively, microtubule-associated proteins are implicated in several neurodegenerative disorders including Parkinson’s, Alzheimer’s and mental retardation and therefore, a role for GARG-39 in virus-induced neuropathogenesis cannot be ruled out. In addition, the expression of GARG-39 in normal dividing cells in the culture indicates a role for this protein in mitosis. In a normal mouse brain, mitotically active cells are very low in number and hence GARG-39 expression (both at the RNA and protein levels) is below the detection limits. JEV infection may trigger mitotic activity in brain leading to increased expression of GARG-39. One of the cDNA clones identified in this study, designated as clone # 137, hybridized to a ~2.6 kb transcript which was found to be down regulated in the mouse brain by JEV as well as rabies virus. A series of investigations led to the conclusion that clone #137 corresponds to the 3′ end of a ~2.6 kb transcript encoding mouse calcium calmodulin kinase inhibitor II α (mCaMKIINα). Interestingly, only the α isoform but not the β isoform of mCaMKIINα mRNA is down regulated in the mouse brain during JEV infection. Since the physiological function of mCaMKIINα is not known, the gene encoding 8 kDa mouse mCaMKIINα open reading frame was cloned into an E. coli expression vector and antibodies were raised against the purified recombinant protein. Surprisingly, antibodies raised against the ~8 kDa recombinant mouse CaMKIINα reacted with a ∼37 kDa mouse brain protein. This protein designated as CaMKIINα-immunoreactive protein (CaMKIINα-IRP) is also down regulated during JEV infection and is localized in the post synaptic density (PSD) of normal mouse brain. In addition, distinct changes are also observed in the subcellular localization and phosphorylation of CaMKIIα leading to an increase in cytosolic CaMKII activity in JEV-infected mouse brain. The differential regulation of CaMKIIα and CaMKIINα during JEV infection suggests a possible role for CaMKII signaling pathway in JEV infection and/or JEV-induced neuropathogenesis in the CNS. Conclusions: • A number of host genes whose expression is modulated in the mouse brain during JEV and/or rabies virus infection have been identified. • One of the JEV-inducible genes encoding the GARG-39 protein was shown to be a microtubule-associated protein with a possible role in mitosis. • One of the JEV-repressible genes was found to encode the mouse CaMKIINα mRNA. • A novel JEV-repressible ∼37 kDa protein immunoreactive to antibodies raised against the recombinant CaMKIINα was identified in the post synaptic density of the mouse brain.

Schizophrenia is a devastating psychiatric disorder that affects approximately 1% of the population. The main treatments for schizophrenia are antipsychotic drugs that target dopamine receptors, yet the underlying biological mechanisms through which they alleviate the symptoms of schizophrenia remain ill defined. In this study, we used microarray analysis to profile the expression changes of thousands of genes simultaneously, following antipsychotic drug treatment of mice. Mice were treated chronically (28 days), or for a novel intermediate time-point (7 days), with one of three antipsychotic drugs: clozapine, haloperidol or olanzapine. The use of three drugs enabled us to discern antipsychotic-specific effects co-regulated by multiple drugs, rather than the side effects of individual compounds. Transcript profiling and validation by quantitative PCR of whole brain tissue revealed antipsychotic drug regulation of genes in diverse biological pathways, including: dopamine metabolism, neuropeptide and second-messenger signalling, neurogenesis, synaptic plasticity, cell adhesion, myelination, and voltage-gated ion channels. The regulation of voltage-gated channels by antipsychotic drugs has been suggested previously by electrophysiological studies, although thorough analysis has not been undertaken in vivo. Therefore, the second aim of this study was to characterise the regional mRNA and protein expression of two genes altered by multiple APDs, the voltage-gated potassium channel ??-subunit (Kcna1) and voltage-gated potassium channel interacting protein (Kchip3). Regional characterisation and expression analyses were carried out by immunohistochemistry, in situ hybridisation, and Western blot analysis of mouse brain regions of interest to schizophrenia and its treatment. Following 7-day haloperidol treatment we observed up-regulation of Kcna1 in the striatum and dentate gyrus, with increased protein in the striatum, hippocampus and midbrain; and down-regulation of Kchip3 in the striatum, with decreased protein in the cortex, hippocampus and midbrain. These studies implicate voltage-gated potassium channels in the antipsychotic drug regulation of midbrain dopaminergic neuronal activity, adult neurogenesis and/or striatothalamic GABAergic neuronal inhibition. These findings indicate that regulation of potassium channels may underlie some of the mechanisms of action of antipsychotic drugs, and that voltage-gated ion channels may provide alternative drug targets for the treatment of schizophrenia.

This investigation was designed to study the age-related differences in the survival rates of 2-to 3- and 6- to 7-month-old AKR/J mice after continuous treatment with anti-lymphocyte globulins (ALG), anti-thymocyte globulins (ATG), or rabbit anti-mouse brain serum (RAMB).

Les mutations génétiques et les conditions pathologiques affectent la connectivité functionnelle du cerveau. Nous avons combiné la mutagénèse chez la souris et l’analyse de connectivité fonctionnelle (CF) par imagerie en Resonance Magnétique Nucléaire (IRM) pour déterminer l’impact de la délétion du gène codant pour le récepteur orphelin GPR88 sur la CF du cerveau entier. En utilisant une approche non biaisée, nous avons découvert que la délétion génétique chez la souris altère fortement le Default Mode Network, une caractéristique de nombreuses maladies psychiatriques. Nous avons aussi observé des modifications importantes de la connectivité des cortex moteurs et somatosensoriels,et du striatum en accord avec le pattern d’expression du récepteur. Enfin, une analyse par régions d’intérêt montre une perturbation importante du réseau mesocorticolimbic, qui pourrait expliquer la tendance de ces animaux à consommer de fortes quantités d’alcool. La concordance entre les altérations de CF et celles du comportement des animaux GPR88 knockout positionnent ce récepteur comme une cible prometteuse pour le traitement de maladies psychiatriques. / Pathological agitations of the brain and the expression or mutation of single gene affect overall brain connectivity. Here we combined mouse mutagenesis with functional and structural MRI and explored mouse whole brain connectivity maps non-invasively in response to the inactivation of Gpr88 gene. We perceived robust modifications in the default mode network which is considered a hallmark of many psychiatric conditions, followed by sensori-motor network allied to sensorimotor gating deficiency underlying hyperactivity phenotype in Gpr88-/- mice. In addition, hippocampal and dorsal striatum functional connectivity perturbations might underlie learning deficiency and weakened amygdala connectivity with cortex and striatum might suggest triggering of risk-taking behavior previously observed in these animals. Moreover, Gpr88 deletion strongly modifies the reward network leading Gpr88-/- mice vulnerable to alcohol intake. This is the first evidence of Gpr88 involvement in reshaping the mouse brain connectome. The concordance between connectivity alterations and behavior deficits posits Gpr88 as a potential target for psychiatric disorders.