Jin H. Son

INDUCTION OF INTERFERON-GAMMA INDUCING FACTOR IN THE ADRENAL CORTEX

Interferon-γ inducing factor (IGIF) is a recently identified cytokine also called interleukin-1γ (IL-1γ) or interleukin-18 (IL-18). Its biological activity is pleiotropic, and, so far, it has been shown to induce interferon-γ production in Th1 cells, to augment the production of granulocyte-macrophage-CSF, and to decrease that of interleukin-10 (IL-10). We first detected newly synthesized IGIF mRNA by differential display in the adrenal gland of reserpine-treated rats and then isolated two transcripts by reverse transcription polymerase chain reaction. They were identified as rat IGIF on the basis of the high homology with mouse: 91% at both the nucleotide and the amino acid level. Subsequently, we investigated the effects of stress on IGIF mRNA levels and found that acute cold stress strongly induced IGIF gene expression. In situ hybridization analysis showed that IGIF is synthesized in the adrenal cortex, specifically in the zona reticularis and fasciculata that produce glucocorticoids. The presence of IGIF mRNA was also detected in the neurohypophysis although induction by stress was not significant. Our results call for more attention to the role of the adrenal gland as a potential effector of immunomodulation and suggest that IGIF itself might be a secreted neuroimmunomodulator and play an important role in orchestrating the immune system following a stressful experience.

Oxidative stress regulated genes in nigral dopaminergic neuronal cells: correlation with the known pathology in Parkinson’s disease

Oxidative stress (OS) is a primary pathogenic mechanism of nigral dopaminergic (DA) cell death in Parkinsons disease (PD). Oxidative damage, Lewy body formation and decreased mitochondrial complex I activity are the consistent pathological findings in PD. In nigral DA neurons, however, it is unknown whether any gene expressional changes induced by OS contribute to the typical PD pathology. Here, using microarray analysis, we identified several groups of genes in the nigral DA cell line, SN4741 [J. Neurosci. 19 (1999) 10; J. Neurochem. 76 (2001) 1010], that were regulated by OS. Approximately 36 significantly regulated genes that encode functional molecules of nuclear subunits of mitochondrial complex I, exocytosis and membrane trafficking proteins, markers for OS and oxidoreductases, regulatory molecules of apoptosis and unidentified EST clones were further analysed. OS modulated the expression of specific genes, of which physiological dysfunctions have been implicated in PD. For instance, the expression of the nuclear-encoded subunits of mitochondrial complex I, B8 and B17, were significantly down-regulated by OS, possibly contributing to selective defect in mitochondrial complex I activity in PD. Furthermore, syntaxin 8 and heme oxygenase-1 (HO-1) are most dramatically up-regulated by OS in DA cells. Syntaxin 8 is a SNARE protein, regulating lipid vesicle docking and fusion as well as early endosome membrane recycling. Lipid membranes are significantly oxidative-damaged in PD. HO-1 is an important cytoplasmic constituent of Lewy bodies, a pathological hallmark of idiopathic PD. Thus, our findings provide novel molecular probes that may be useful in unraveling the molecular mechanism(s) of OS-induced pathogenesis in PD. Further functional characterization of the affected genes including ESTs can help elucidate the underlying molecular pathology as well as develop biomarkers for monitoring degenerating DA neurons in PD.

Neuropeptide Y mRNA and serotonin innervation in the arcuate nucleus of anorexia mutant mice

The anorexia (anx) mutation causes reduced food intake in preweanling mice, resulting in death from starvation within 3-4 weeks. In wild-type rodents, starvation induces increased neuropeptide Y (NPY) mRNA levels in the arcuate nucleus that promotes compensatory hyperphagia. Despite severely decreased body weight and food intake at 3-weeks age, anx/anx mice do not show elevated NPY mRNA levels in the hypothalamic arcuate nucleus compared to wild-type/heterozygous littermates. The NPY mRNA levels can be upregulated in normal mice at this chronological age, because 24-h food deprivation increased arcuate NPY mRNA in wild-type littermates. The unresponsiveness of NPY expression in the arcuate of anx/anx mice was paralleled by serotonergic hyperinnervation of the arcuate nucleus, comparable to the serotonergic hyperinnervation previously reported in the rest of the anx/anx brain. This result is consistent with the hypothesis that wasting disorders are accompanied by disregulation of NPY mRNA expression in the arcuate nucleus, and suggests that reduced food intake, the primary behavioral phenotype of the anx/anx mouse, may be the result of altered hypothalamic mechanisms that normally regulate feeding.

Sex differences in the regulation of tyrosine hydroxylase gene transcription by estrogen in the locus coeruleus of TH9-LacZ transgenic mice

Although estrogen is recognized increasingly as having an important role in modulating extrahypothalamic brain function, the mechanisms through which this occur are not well established. The norepinephrine (NE) neurons of the locus coeruleus provide an important neuromodulatory influence upon multiple neural networks throughout the brain and estrogen has been implicated in their regulation. Using a tyrosine hydroxylase (TH) promoter-LacZ transgenic mouse model, which enables rates of TH gene transcription to be examined in vivo, we have examined here whether estrogen regulates expression of the TH gene in the locus coeruleus of males and females. Optical area measurements of Xgal reaction product in the locus coeruleus revealed that gonadectomy exerted opposite effects on TH gene transcription in males and females; transgene expression was increased in males (P<0.01) but reduced in females (P<0.05). Estrogen reversed these effects in both sexes by suppressing gene expression in males (P<0.05) but elevating it in the female (P<0.05). These studies reveal a marked and unexpected sex difference in the regulation of TH gene activity in the mouse. While estrogen in the male, synthesized from circulating testosterone, suppresses TH gene transcription, estrogen in the female enhances TH promoter activity. The present results indicate that estrogen may exert very different sex-dependent effects upon the biosynthesis of NE within the locus coeruleus.

Neurotoxicity and behavioral deficits associated with Septin 5 accumulation in dopaminergic neurons

Septin 5, a parkin substrate, is a vesicle‐ and membrane‐associated protein that plays a significant role in inhibiting exocytosis. The regulatory function of Septin 5 in dopaminergic (DAergic) neurons of substantia nigra (SN), maintained at relatively low levels, has not yet been delineated. As loss of function mutations of parkin are the principal cause of a familial Parkinsons disease, a prevailing hypothesis is that the loss of parkin activity results in accumulation of Septin 5 which confers neuron‐specific toxicity in SN‐DAergic neurons. In vitro and in vivo models were used to support this hypothesis. In our well‐characterized DAergic SN4741 cell model, acute accumulation of elevated levels of Septin 5, but not synphilin‐1 (another parkin substrate), resulted in cytotoxic cell death that was markedly reduced by parkin co‐transfection. A transgenic mouse model expressing a dominant negative parkin mutant accumulated moderate levels of Septin 5 in SN‐DAergic neurons. These mice acquired a progressive l‐DOPA responsive motor dysfunction that developed despite a 25% higher than normal level of striatal dopamine (DA) and no apparent loss of DAergic neurons. The phenotype of this animal, increased striatal dopamine and reduced motor function, was similar to that observed in parkin knockout animals, suggesting a common DAergic alteration. These data suggest that a threshold level of Septin 5 accumulation is required for DAergic cell loss and that l‐DOPA‐responsive motor deficits can occur even in the presence of elevated DA.

Drastic and selective hyperinnervation of central serotonergic neurons in a lethal neurodevelopmental mouse mutant, Anorexia (anx)

The autosomal recessive lethal anorexia mutation in mice (anx/anx) causes starvation in preweanlings. In addition, this murine neurodevelopmental mutant shows other distinct phenotypic characteristics and dysfunctional behaviors. Previous studies strongly suggested that the mutation results in elevated serotonergic stimulation, because these traits are characteristic of such overstimulation and because brain serotonin is believed to have an inhibitory effect on feeding behavior. In this report, we show extensive serotonergic hyperinnervation in normal target fields (hippocampus, cortex, olfactory bulb and cerebellum) of mutant mice. Despite the extensive hyperinnervation, the normal laminar organization of the brain was retained. The specificity of the mutation to the serotonergic system was confirmed by demonstration of normal catecholaminergic innervation in the central nervous system (CNS), and this specificity was especially striking in a common target field, the cerebellum. Serotonergic hyperinnervation in these mutant preweanling mice may represent the underlying etiology of increased serotonergic stimulation which leads to anorexic starvation, abnormal behavior, and premature death.

Differential expression of monoamine oxidase A, serotonin transporter, tyrosine hydroxylase and norepinephrine transporter mRNA by anorexia mutation and food deprivation

The Anorexia (anx) mutation causes reduced food intake in preweanling mice, resulting in death from starvation within 3-4 weeks. We have found serotonin (5HT) hyperinnervation in the anx brain; altered noradrenergic (NE) innervation may also mediate eating disorders. We examined the expression of synthetic or catabolic monoamine enzyme genes in brainstem nuclei: serotonin transporter (5HTT) and monoamine oxidase A (MAOA) in the raphe nuclei (RN), and MAOA, norepinephrine transporter (NET), and tyrosine hydroxylase (TH) in the locus ceruleus (LC). We compared 3-week old anx with control and 24-h food-deprived wildtype littermates using in situ hybridization to measure mRNA levels by quantitative autoradiography. The anx mutation was correlated with decreased MAOA mRNA in the LC (but not RN), decreased 5HTT mRNA in the RN, and a trend towards lower NET mRNA in the LC. Food deprivation decreased MAOA mRNA in the LC (but not RN), increased TH mRNA in the LC, and did not alter NET or 5HTT mRNA levels. Thus, the effect of the anx mutation on MAOA expression in the LC paralleled the effect of food-deprivation, but the anx mutation and food-deprivation had differential effects on the expression of TH, NET, and 5HTT genes. Decreased 5HTT expression in the anx RN is consistent with upregulation of serotonergic neurotransmission that may accompany 5HT hyperinnervation. Central NE levels or innervation may be altered in anx mice by decreased expression of NET and MAOA and a lack of TH upregulation induced by food deprivation as in wild-type mice.

A transgenic mouse model to study transsynaptic regulation of tyrosine hydroxylase gene expression

Abstract: Previous studies demonstrated that 9 kb of the rat tyrosine hydroxylase (TH) 5′ flanking sequence directed appropriate spatiotemporal expression of a lacZ reporter gene to catecholaminergic cells in the CNS of transgenic mice. In the present study, specificity of transgene expression was further extended to demonstrate cell type‐specific functional regulation of lacZ expression using manipulations known to alter endogenous TH expression. Alterations in lacZ reporter expression should parallel changes in endogenous TH levels if the DNA elements mediating these functional changes of TH expression in vivo reside within the 9 kb of the TH promoter region. Naris closure induced an activity‐dependent decrease of TH expression in dopaminergic periglomerular cells in the olfactory bulb that was paralleled by down‐regulation of lacZ expression in the transgenic mice. Densitometry and image analysis were used to quantify lacZ expression following acute reserpine administration (5 mg/kg, s.c.), which up‐regulates endogenous TH. At 48 h postinjection, analysis of OD values indicated a significant increase of X‐gal staining in the locus coeruleus and ventral tegmental area but not in the substantia nigra or olfactory bulb of reserpine‐treated transgenic animals. These data showed that the 9‐kb sequence also mediates cell type‐specific transsynaptic regulation of reporter gene expression. Analysis of this transgenic animal offers a useful model system to study in vivo regulation of TH gene expression.

Identification of potential compounds promoting BDNF production in nigral dopaminergic neurons : clinical implication in Parkinson's disease

Parkinsons disease (PD) is characterized by the selective loss of dopamine (DA) neurons in the substantia nigral brain region. Currently, there is no cure or treatment that prevents such neuronal loss. Brain-derived neurotrophic factor (BDNF) has been found to support the survival of DA neurons in animal models and in primary cell cultures. However, the large molecular size of BDNF, coupled with the blood brain barrier, prevents its delivery to DA neurons to promote cell survival in the PD brain. The nigral DA neurons have the ability to produce BDNF for neuroprotection via either autocrine or paracrine mechanisms. Low mol. wt compounds were tested to see whether they could increase the production of BDNF in the DA neurons. The compounds tested include neurotransmitters, neuropeptides, intracellular signaling agents, known neuroprotective agents and growth factors. Our results demonstrate that salicyclic acid, cGMP analog, okadaic acid, IBMX, dipyridamole and glutamate significantly enhance BDNF production in DA neuronal cells.

Alternate Promoters in the Rat Aromatic l‐Amino Acid Decarboxylase Gene for Neuronal and Nonneuronal Expression: An In Situ Hybridization Study

Abstract: Aromatic l‐amino acid decarboxylase (AADC) is found in both neuronal cells and nonneuronal cells, and a single gene encodes rat AADC in both neuronal and nonneuronal tissues. However, two cDNAs for this enzyme have been identified: one from the liver and the other from pheochromocytoma. Exons 1a and 1b are found in the liver cDNA and the pheochromocytoma cDNA, respectively. In the third exon (exon 2), there are two alternatively utilized splicing acceptors specific to these exons, 1a and 1b. Structural analysis of the rat AADC gene showed that both alternative promoter usage and alternative splicing are operative for the differential expression of this gene. To demonstrate whether alternative promoter usage and splicing are tissue specific and whether the exons 1a and 1b are differentially and specifically transcribed in nonneuronal and neuronal cells, respectively, in situ hybridization histochemistry for the rat brain, adrenal gland, liver, and kidney was carried out using these two exon probes. The exon 1a probe specifically identified AADC mRNA only in nonneuronal cells, including the liver and kidney, and the exon 1b probe localized AADC mRNA to monoaminergic neurons in the CNS and the adrenal medulla. Thus, both alternative promoter usage and differential splicing are in fact operative for the tissue‐specific expression of the rat AADC gene.