Arginase and autoimmune inflammation in the central nervous system

Using a high throughput gene microarray technology that detects approximately 22 000 genes, we found that arginase I was the most significantly up-regulated gene in the murine spinal cord during experimental autoimmune encephalomyelitis (EAE). By Northern blot and arginase enzyme assay, we detected high levels of arginase I mRNA and protein, respectively, in the spinal cord of EAE mice, but not in the spinal cord of normal mice or mice that had recovered from EAE. In vitro, both microglia and astrocytes produced arginase and nitric oxide synthase, two enzymes that are involved in arginine metabolism. To explore the roles of arginase in EAE, we injected the arginase inhibitor amino-6-boronohexanoic acid (ABH) into mice during the inductive and effector phases of the disease. Compared with mice that received vehicle control, mice treated with ABH developed milder EAE with delayed onset, reduced disease score and expedited recovery. Spleen mononuclear cells from ABH-treated mice produced more nitric oxide and secreted less interferon-gamma and tumour necrosis factor-alpha as compared to control mice. These results indicate that arginase plays important roles in autoimmune inflammation in the central nervous system.     

The expression and localization of Prune2 mRNA in the central nervous system

A family of Bcl-2/adenovirus E1B 19 kDa-interacting proteins (BNIPs) plays critical roles in several cellular processes such as cellular transformation, apoptosis, neuronal differentiation, and synaptic function, which are mediated by the BNIP2 and Cdc42GAP homology (BCH) domain. Prune homolog 2 (Drosophila) (PRUNE2) and its isoforms - C9orf65, BCH motif-containing molecule at the carboxyl terminal region 1 (BMCC1), and BNIP2 Extra Long (BNIPXL) - have been shown to be a susceptibility gene for Alzheimer's disease, a biomarker for leiomyosarcomas, a proapoptotic protein in neuronal cells, and an antagonist of cellular transformation, respectively. However, precise localization of PRUNE2 in the brain remains unclear. Here, we identified the distribution of Prune2 mRNA in the adult mouse brain. Prune2 mRNA is predominantly expressed in the neurons of the cranial nerve motor nuclei and the motor neurons of the spinal cord. The expression in the dorsal root ganglia (DRG) is consistent with the previously described reports. In addition, we observed the expression in another sensory neuron in the mesencephalic trigeminal nucleus. These results suggest that Prune2 may be functional in these restricted brain regions.     

Time course analysis of tyrosine hydroxylase and angiotensinogen mRNA expression in central nervous system of rats submitted to experimental hypertension

Catecholaminergic and angiotensinergic systems are involved in the neural control of blood pressure. The present study analysed the expression of tyrosine hydroxylase (TH), a key enzyme for catecholamine synthesis and of angiotensinogen (AGT), the precursor of angiotensin II (Ang II), in areas of the central nervous system (CNS) involved with cardiovascular regulation such as nucleus tractus solitarius (NTS), ventrolateral medulla (VLM), locus coeruleus (LC) and hypothalamic paraventricular nucleus (PVN) 2 h, 3 and 7 days after aortic coarctated hypertensive rats. In situ hybridization, was employed for the analysis of messenger RNA (mRNA) expression with anatomical resolution. No changes were seen in TH and AGT mRNA expression in the analysed areas 2 h and 3 days after aortic coarctation when compared to the respective sham group. TH mRNA expression was increased in the NTS and LC of rats 7 days after coarctation hypertension when compared to sham rats. Time course analysis, showed an increase in TH mRNA expression in the NTS 7 days after aortic coarctation when compared to 2 h and 3 days groups, as well as an increase in LC 3 days and 7 days following coarctation hypertension in comparison with the 2 h group. Analysis of AGT mRNA in the NTS expression revealed a decrease at 3 days, followed by an increase in mRNA expression 7 days following coarctation hypertension when compared to the sham group. Time course analysis, showed an increase in AGT mRNA expression in the NTS 7 days after coarctation when compared to 2 h and 3 days groups. The results show that TH and AGT mRNA expression changes during the different phases of experimental hypertension, suggesting that the noradrenaline (NOR) and angiotensin II (Ang II) might participate in the modulation/maintenance of coarctation hypertension.     

Immunocytochemical localization of acyl-CoA oxidase in the rat central nervous system

Peroxisomal -oxidation, consisting of four steps catalysed by an acyl-CoA oxidase, a multifunctional protein and a thiolase, is responsible for the shortening of a variety of lipid compounds. The first reaction of this pathway is catalysed by a FAD-containing acyl-CoA oxidase, three isotypes of which have been so far recognised. Among these, straight-chain acyl-CoA oxidase (ACOX) acts on long and very long chain fatty acids, prostaglandins and some xenobiotics. We investigated ACOX localisation by means of a sensitive, tyramide based, immunocytochemical technique, thus obtaining a complete distribution atlas of the enzyme in adult rat CNS. Granular immunoreaction product was found in the cytoplasm of neuronal and glial cells, both in the perikarya and in the cell processes. ACOX immunoreactive neurons were present to variable extent, in either forebrain or hindbrain areas. Specifically, the strongest signal was detected in the pallidum, septum, red nucleus, reticular formation, nuclei of the cranial nerves, and motoneurons of the spinal cord. We then compared the ACOX immunoreactivity pattern with our previous distribution maps of other peroxisomal enzymes in the adult rat brain. While ACOX appeared to colocalise with catalase in the majority of cerebral regions, some differences with respect to d-amino acid oxidase were noted. These observations support the hypothesis of heterogeneous peroxisomal populations in the nervous tissue. The wide distribution of the enzyme in the brain is consistent with the severe and generalised neurological alterations characterising the peroxisomal disorder caused by ACOX deficiency (pseudo-neonatal adrenoleukodystrophy).     

p53 protein expression in central nervous system neoplasms.

AIMS: To demonstrate, immunohistochemically, p53 protein expression in a selection of central nervous system tumours; to investigate the relation between p53 expression and that of the proliferation related antigen, PCNA. METHODS: Surgical specimens from 86 central nervous system tumours were routinely fixed, paraffin wax embedded, and immunostained with a monoclonal (PAb 1801) and a policlonal antibody (CM1) p53 protein and a monoclonal antibody against PCNA (PC10). Normal brain samples obtained at necropsy and 10 surgically obtained samples of gliotic brain parenchyma were also immunostained. RESULTS: p53 protein expression was observed in 35 of 86 brain tumours, suggesting frequent p53 gene mutation. p53 protein alterations were associated with all grades of malignancy in tumours displaying solely astrocytic differentiation, with the exception of pilocytic astrocytomas. In those showing oligodendroglial or ependymal differentiation they appeared to be restricted almost to only high grade lesions. No p53 immunoreactivity was observed in normal or gliotic brain tissue; p53 altered expression was not related to the percentage of PCNA labelled cells. CONCLUSIONS: The use of sophisticated gene amplification techniques or highly sensitive immunohistochemical methods might be useful in distinguishing between reactive and neoplastic astrocytic lesions, and in the identification of malignant progression in other non-astrocytic glial tumours. Tumours with very similar histogenetic differentiation features might actually be a genetically heterogeneous group with possible different clinical courses.     

Dual inhibition of proteasomal and lysosomal proteolysis ameliorates autoimmune central nervous system inflammation

Multiple sclerosis (MS) is a detrimental disease of the central nervous system (CNS) leading to long-term disability. In the course of animal models of multiple sclerosis (experimental autoimmune encephalomyelitis), we find enhanced activity of proteasome subunits beta1i, beta2, beta2i and beta5 in the CNS. We demonstrate that pharmacological inhibition of the proteasome by bortezomib ameliorates experimental autoimmune encephalomyelitis in mice and rats in prophylactic and therapeutic treatment with reduced numbers of T-cells secreting proinflammatory cytokines. The anti-inflammatory effect of proteasome inhibition was accompanied by reduced NF-kappaB activity in the CNS and lymphoid organs. The combined inhibition of proteasomes and lysosomal proteases involved in major histocompatibility complex II antigen presentation further improved therapeutic efficacy. We suggest proteasome inhibition alone or in combination with inhibition of lysosomal proteases as a novel therapeutic strategy against inflammation-induced neurodegeneration in the CNS. We demonstrate the impact of the proteasome and lysosomal proteases on development of autoimmunity.     

Contrasting patterns in the localization of glutamic acid decarboxylase and Ca2+/calmodulin protein kinase gene expression in the rat central nervous system.

The expression of the genes encoding the alpha subunit of type II calcium calmodulin-dependent protein kinase (CaM II kinase alpha) and the 67,000 mol. wt form of glutamic acid decarboxylase was examined throughout the rat central nervous system. In situ hybridization histochemistry, using cRNA probes, revealed a dense population of CaM II kinase alpha-expressing cells throughout the telencephalon and diencephalon. CaM II kinase alpha mRNA was also expressed in the midbrain, cerebellum and medulla oblongata, but at greatly reduced levels. No CaM II kinase alpha gene expression was detected in nuclei producing monoamines or acetylcholine. By contrast, the glutamic acid decarboxylase gene was moderately to highly expressed throughout the central nervous system. In several regions there was a complementarity in the distributions of cells expressing the glutamic acid decarboxylase or CaM II kinase alpha genes. Cells in certain nuclei such as the thalamic reticular nucleus or globus pallidus showed glutamic acid decarboxylase gene expression only; others such as the majority of the dorsal thalamic nuclei showed CaM II kinase alpha gene expression only. Several regions contained both glutamic acid decarboxylase and CaM II kinase alpha expressing cells. However, simultaneous immunostaining for both proteins revealed only two regions where CaM II kinase alpha and glutamic acid decarboxylase immunoreactivity were colocalized: the cerebellar Purkinje cells and the commissural nucleus of the stria terminalis. The results imply that CaM II kinase alpha is primarily expressed in non-GABAergic neurons. In several regions CaM II kinase alpha mRNA is concentrated in nuclei known to contain populations of neurons that use excitatory amino acid transmitters.     

IFN-gamma signaling in the central nervous system controls the course of experimental autoimmune encephalomyelitis independently of the localization and composition of inflammatory foci

Background  

Murine experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, presents typically as ascending paralysis. However, in mice in which interferon-gamma (IFNγ) signaling is disrupted by genetic deletion, limb paralysis is accompanied by atypical deficits, including head tilt, postural imbalance, and circling, consistent with cerebellar/vestibular dysfunction. This was previously attributed to intense cerebellar and brainstem infiltration by peripheral immune cells and formation of neutrophil-rich foci within the CNS. However, the exact mechanism by which IFNγ signaling prohibits the development of vestibular deficits, and whether the distribution and composition of inflammatory foci within the CNS affects the course of atypical EAE remains elusive.     

The immunocytochemical localization of somatostatin-containing neurons in the rat central nervous system

Somatostatin-containing neurons in the rat central nervous system were localized by immunocytochemical methods. The detection of somatostatin-like immunoreactivity was facilitated by (1) the use of brains from colchicine-treated rats, (2) the proteolytic pretreatment of sections with pronase and (3) a ‘double-bridge’ immunoperoxidase staining technique. In addition to the known distribution of somatostatin-like immunoreactivity we also observed immunoreactive perikarya in the following regions: the anterior olfactory nucleus, some areas of the preoptic and hypothalamic regions, the claustrum, the periaqueductal gray, the locus ceruleus, the central gray substance, the lateral parabrachial nucleus, the nucleus of the lateral lemniscus, the nucleus ambiguus, the spinal trigeminal nucleus, the nucleus of the solitary tract and various areas of the reticular formation. Immunoreactive neuronal processes were also observed in several major tracts of the brain, including the stria terminalis, the fornix and the medial forebrain bundle.Our results indicate that somatostatin-containing neurons may occur both as interneurons in some areas of the central nervous system and as projection neurons in others. The widespread but selective distribution of these neurons suggests that somatostatin is not only a hypothalamic regulator of neuroendocrine function, but may also function as a major neuromodulator mediating a variety of functions throughout the central nervous system.     

Involvement of Disabled-2 protein in the central nervous system inflammation following experimental cryoinjury of rat brains

Disabled-2 (Dab-2) functions in the mitogenic signal transduction pathway, and is expressed in a variety of tissues. We investigated the roles of Dab-2 expression in the rat brain following experimental cryoinjury in relation to central nervous system (CNS) inflammation. Western blot analysis showed that Dab-2 expression increased significantly (p < 0.001) in the frontal cortex 4-14 days after cryoinjury, and declined slightly thereafter. Immunohistochemistry showed that Dab-2 immunostaining occurred in most of the vessels in the control cerebral cortex. After cryoinjury, Dab-2 was localized in the majority of inflammatory cells (especially in ED1-positive macrophages) in the core and periphery, as well as in vessels. These findings suggest that Dab-2 is involved in the inflammation that follows CNS injury through the migration of activated inflammatory cells in the rat brain.     

The role of CD8 suppressors versus destructors in autoimmune central nervous system inflammation

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) of putative autoimmune origin. Recent evidence indicates that MS autoimmunity is linked to defects in regulatory T-cell function, which normally regulates immune responses to self-antigens and prevents autoimmune diseases. MS and its animal model, experimental autoimmune encephalomyelitis (EAE), have long been regarded as a CD4(+) T-cell-mediated autoimmune disease. Studies addressing the role of CD8(+) T cells, however, have only recently begun to emerge. Pathogenic function was attributed to CD8(+) T cells because of their abundant presence or oligoclonal repertoire within MS lesions. However, CD8(+) T cells appeared to have important regulatory functions, as demonstrated in EAE or human MS studies. We here review the contribution of CD8(+) T cells to inflammation and immune regulation in CNS autoimmunity. The knowledge of distinct CD8(+) T-cell populations exerting destructive versus beneficial functions is summarized. The long-term goal is to delineate the exact phenotypic and functional characteristics of regulatory CD8(+) T-cell populations (natural as well as inducible) in humans. This knowledge may help to further develop concepts of reconstituting or enhancing endogenous mechanisms of immune tolerance in future therapeutic concepts for MS.     

Distribution of ras guanyl releasing protein (RasGRP) mRNA in the adult rat central nervous system

In the nervous system, Ras signal transduction pathways are involved in cellular differentiation, neuronal survival and synaptic plasticity. These pathways can be modulated by Ras guanyl nucleotide exchange factors (Ras GEFs), which activate Ras protein by catalyzing the exchange of GDP for GTP. RasGRP, a recently discovered Ras GEF is expressed in brain as well as in T cells. In addition to the catalytic domain which catalyzes dissociation of Ras-GDP, RasGRP has a pair of calcium-binding EF hands and a diacylglycerol binding domain. The structure of RasGRP suggests that it serves to link calcium and lipid messengers to Ras signaling pathways. We have used an RNase protection assay to detect RasGRP mRNA in various regions of the rat brain and we have determined the cellular distribution of RasGRP mRNA by in situ hybridization. RasGRP mRNA is widely distributed and is present in both interneurons and projection neurons but not confined to any neuronal type or neurotransmitter phenotype. The presence of RasGRP mRNA in archicortical neurons suggests that this pathway may be important in phylogenetically older regions of the CNS. The restriction of RasGRP mRNA to subsets of neurons suggests that activation of Ras by RasGRP has a specific function in certain neuronal types. We did not detect RasGRP in glial cells.     

IL-9 is important for T-cell activation and differentiation in autoimmune inflammation of the central nervous system

Experimental autoimmune encephalomyelitis (EAE) is generally believed to be an autoimmune disease of the central nervous system (CNS) caused by myelin-specific Th1 and/or Th17 effector cells. The underlying cellular and molecular mechanisms, however, are not fully understood. Using mice deficient in IL-9 (IL-9(-/-) ), we showed that IL-9 plays a critical role in EAE. Specifically, IL-9(-/-) mice developed significantly less severe EAE than their WT counterparts following both immunization with myelin proteolipid protein (PLP)(180-199) peptide in the presence of Complete Freund's Adjuvant (CFA), and adoptive transfer of PLP(180-199) peptide-specific effector T cells from WT littermates. EAE-resistant IL-9(-/-) mice exhibited considerably fewer infiltrating immune cells in the CNS, with lower levels of IL-17 and IFN-γ expression, than their WT littermates. Further studies revealed that null mutation of the IL-9 gene resulted in significantly lower levels of PLP(180-199) peptide-specific IL-17 and IFN-γ production. Moreover, IL-9(-/-) memory/activated T cells exhibited decreased C-C chemokine receptors (CCR)2, CCR5, and CCR6 expression. Interestingly, IL-10 was significantly increased in IL-9(-/-) mice compared with WT littermates. Importantly, we found that IL-9-mediated Th17-cell differentiation triggers complex STAT signaling pathways.     

Apoptotic proteins in the course of aging of central nervous system in the rat

Studies were performed on the level of cells with damaged DNA (TUNEL), the level of protein engaged in DNA repair (PARP) and the level of proteins indicating the extent of apoptosis (Bax:Bcl-2) (Western blot). The studies were performed on cerebral cortex (GM), white matter (WM), medulla oblongata (MO), cerebellum (C) of rats, 3.0-3.5-, 12-, 24-months of age. The highest levels of DNA injury in GM of 1-year-old rats and in MO of 2-year-old rats were accompanied by peak levels of PARP. In the remaining structures (WM, C) levels of DNA injury showed no correspondence with levels of PARP. Levels of Bax proteins exceeded levels of Blc-2 protein in all cerebral structures of young rats. In old animals, Bax protein continued to exceed Blc-2 levels both in GM and in MO, in which most pronounced fragmentation of DNA was observed. The data indicated that in spite of high level of TUNEL positive cells in aged brain PARP and Bcl-2 are probably engaged in protection of the cells against death.     

Immunohistochemical studies on the cellular localization of GABAA-receptors in explant cultures of rat central nervous system using a monoclonal antibody

Summary Explant cultures of rat spinal cord, brain stem and cerebellum were used to visualize GABA_A-receptors by means of immunohistochemistry. For these studies we have incubated the cultures with the monoclonal antibody bd 17 against the -subunit of the GABA_A/benzodiazepine/chloride channel complex. In spinal cord cultures, many interneurones were immunoreactive whereas only a small number of large neurones, probably motoneurones was specifically stained. In brain stem cultures, groups of large and medium-sized neurones showed immunoreactivity. In cultures of cerebellum, a great number of neurones was specifically stained. Granule cells showed the strongest immunoreactivity whereas other neurones, presumably Purkinje cells and interneurones, were only moderately stained. The immunoreactivity was mainly confined to the cell bodies of the neurones while their processes were only weakly or not stained. In contrast to neurones, no immunoreactivity could be detected on astrocytes.