Noradrenergic depletion increases inflammatory responses in brain: effects on IkappaB and HSP70 expression

The inflammatory responses in many cell types are reduced by noradrenaline (NA) binding to beta-adrenergic receptors. We previously demonstrated that cortical inflammatory responses to aggregated amyloid beta (Abeta) are increased if NA levels were first depleted by lesioning locus ceruleus (LC) noradrenergic neurons, which replicates the loss of LC occurring in Alzheimer's disease. To examine the molecular basis for increased responses, we used the selective neurotoxin DSP4 to lesion the LC, and then examined levels of putative anti-inflammatory molecules. Inflammatory responses were achieved by injection of aggregated Abeta1-42 peptide and IL-1beta into frontal cortex, which induced neuronal inducible nitric oxide synthase (iNOS) and microglial IL-1beta expression. DSP4-treatment reduced basal levels of nuclear factor kappa B (NF-kappaB) inhibitory IkappaB proteins, and of heat shock protein (HSP)70. Inflammatory responses were prevented by co-injection (ibuprofen or ciglitzaone) or oral administration (pioglitazone) of peroxisome proliferator-activated receptor gamma (PPARgamma) agonists. Treatment with PPARgamma agonists restored IkappaBalpha, IkappaBbeta, and HSP70 levels to values equal or above those observed in control animals, and reduced activation of cortical NF-kappaB. These results suggest that noradrenergic depletion reduces levels of anti-inflammatory molecules which normally limit cortical responses to Abeta, and that PPARgamma agonists can reverse that effect. These findings suggest one mechanism by which PPARgamma agonists could provide benefit in neurological diseases having an inflammatory component.     

Identification of complement 5a-like receptor (C5L2) from astrocytes: characterization of anti-inflammatory properties

Brain inflammation is regulated by endogenous substances, including neurotransmitters such as noradrenaline (NA), which can increase anti-inflammatory genes. To identify NA-regulated, anti-inflammatory genes, we used TOGA (total gene expression analysis) to screen rat astrocyte-derived RNA. NA-inducible cDNA clone DST11 encodes an isoform of the complement C5a receptor (C5aR), with 39% identity at the amino acid level to the rat C5aR, and 56% identity to a recently described human C5aR variant termed C5L2 (complement 5a-like receptor). Quantitative PCR confirmed that in astrocytes, DST11 mRNA expression is increased by NA, whereas in vivo depletion of cortical NA reduced DST11 levels. Western blot analysis demonstrated basal and NA-induced expression of DST11 as a 45 kDa protein in primary astrocytes cultures. Immunocytochemical staining of adult rat brain revealed DST11-immunoreactivity throughout brain, co-localized to neurons and astrocytes. In astrocytes, induction of nitric oxide synthase type 2 was increased by treatment with antisense oligonucleotides to DST11. Reducing DST11 expression also increased nuclear factor kappaB reporter gene, and decreased cAMP response element reporter gene activation. These results demonstrate that DST11 is a C5aR isoform expressed by glia and neurons, which is regulated by NA, and exerts anti-inflammatory functions. Changes in DST11 levels in diseased brain could therefore contribute to the progression of inflammatory damage.     

Noradrenergic stimulation of BDNF synthesis in astrocytes: mediation via alpha1- and beta1/beta2-adrenergic receptors

Brain-derived neurotrophic factor (BDNF) synthesis in astrocytes induced by noradrenaline (NA) is a receptor-mediated process utilizing two parallel adrenergic pathways: beta1/beta2-adrenergic/cAMP and the novel alpha1-adrenergic/PKC pathway. BDNF is produced by astrocytes, in addition to neurons, and the noradrenergic system plays a role in controlling BDNF synthesis. Since astrocytes express various subtypes of alpha- and beta-adrenergic receptors that have the potential to be activated by synaptically released NA, we focused our present study on the mediatory role of adrenergic receptors in the noradrenergic up-regulation of BDNF synthesis in cultured neonatal rat cortical astrocytes. NA (1 microM) elevates BDNF levels by four-fold after 6 h of incubation. Its stimulation was partly inhibited by either the beta1-adrenergic antagonist atenolol, the beta2-adrenergic antagonist ICI 118,551, or by the alpha1-adrenergic antagonist prazosin, while the alpha2-adrenergic antagonist yohimbine showed no effect. BDNF levels in astrocytes were increased by the specific beta1-adrenergic agonist dobutamine and the beta2-adrenergic agonist salbutamol, as well as by adenylate cyclase activation (by forskolin) and PKA activation (by dBcAMP). However, none of the tested agonists or mediators of the intracellular beta-adrenergic pathways were able to reach the level of NA's stimulatory effect. BDNF cellular levels were also elevated by the alpha1-adrenergic agonist methoxamine, but not by the alpha2-adrenergic agonist clonidine. The increase in intracellular Ca2+ by ionophore A23187 showed no effect, whereas PKC activation by phorbol 12-myristate 13-acetate (TPA) potently stimulated BDNF levels in the cells. The methoxamine-stimulated BDNF synthesis was inhibited by desensitizing pretreatment with TPA, indicating that the alpha1-stimulation was mediated via PKC activation. In conclusion, the synthesis of astrocytic BDNF stimulated by noradrenergic neuronal activity is an adaptable process using multiple types (alpha1 and beta1/beta2) of adrenergic receptor activation.     

Involvement of Dopamine Neurons in the Regulation of β-Adrenergic Receptor Sensitivity in Rat Prefrontal Cortex

The contribution of dopamine (DA) afferents to the regulation of beta-adrenergic receptor sensitivity (isoproterenol-stimulated adenylate cyclase activity) in the rat prefrontal cortex was investigated by comparing the effects of lesions affecting either both DA and noradrenaline (NA) or NA fibers alone. Bilateral 6-hydroxydopamine (6-OHDA) lesions made in the ventral tegmental area destroyed ascending DA and to a variable extent ascending NA fibers innervating the prefrontal cortex. Two opposite effects were observed depending on the extent of cortical NA denervation: (a) When NA denervation was complete (less than 4% of controls), a marked increase in the isoproterenol-sensitive adenylate cyclase activity (+78%) was found. The amplitude of this denervation supersensitivity was similar to that occurring following complete and selective destruction of NA innervation induced by bilateral 6-OHDA injections made into the pedunculus cerebellaris superior. (b) When 6-OHDA injections into the ventral tegmental area led to a partial destruction of cortical NA afferents (10-40% of control values), a hyposensitivity of the isoproterenol-induced adenylate cyclase activity (-30%) was observed. This effect contrasted with the moderate supersensitivity seen in rats with partial, but selective, destruction of NA innervation (pedunculus cerebellaris superior lesions). The hyposensitivity of beta-adrenergic receptors obtained in rats with partial lesions of cortical NA fibers, but devoid of cortical DA innervation, suggests that DA neurons may regulate, under certain conditions, the denervation supersensitivity of beta-adrenergic receptors.     

Inhibition of locus coeruleus neuronal activity by beta-phenylethylamine

The effect of beta-phenylethylamine (PEA) on brain noradrenaline (NA) neurons in the rat locus coeruleus (LC) was analyzed using single unit recording techniques including microiontophoretic methodology. Systemic injection of low doses of PEA consistently produced an instantaneous and dose-dependent inhibition of firing rate of the LC neurons. The effect was strongly antagonized by administration of the alpha 2-receptor antagonist yohimbine (1 mg/kg, i.v.) or by depletion of endogenous stores of NA by pretreatment with reserpine (10 mg/kg, i.p., 6 h), but unaffected by inhibition of tyrosine hydroxylase (alpha-met-hyl-p-tyrosine (alpha-MT), 250 mg/kg, i.p., 30 min). In contrast, the inhibitory effect of PEA on the LC neurons was strongly potentiated by pretreatment with the selective monoamine oxidase (MAO) - B inhibitor pargyline (2 mg/kg, i.p., 1 h), but, unexpectedly, also by pretreatment with the MAO-A selective inhibitors clorgyline (2 mg/kg, i.p., 1 h) or FLA 336 (2 mg/kg, i.p., 1 h). When microiontophoretically applied directly onto the LC neurons, PEA produced inhibition of a majority of the NA neurons. This action was prevented by intravenous injection of yohimbine (2.5 mg/kg). The results suggests that the action of PEA on NA neurons in the LC is an indirect effect, requiring availability of a reserpine-sensitive storage pool of NA, and mediated via activation of central alpha 2-receptors within the LC.     

PPARgamma activation primes human monocytes into alternative M2 macrophages with anti-inflammatory properties

Th1 cytokines promote monocyte differentiation into proatherogenic M1 macrophages, while Th2 cytokines lead to an "alternative" anti-inflammatory M2 macrophage phenotype. Here we show that in human atherosclerotic lesions, the expression of M2 markers and PPARgamma, a nuclear receptor controlling macrophage inflammation, correlate positively. Moreover, PPARgamma activation primes primary human monocytes into M2 differentiation, resulting in a more pronounced anti-inflammatory activity in M1 macrophages. However, PPARgamma activation does not influence M2 marker expression in resting or M1 macrophages, nor does PPARgamma agonist treatment influence the expression of M2 markers in atherosclerotic lesions, indicating that only native monocytes can be primed by PPARgamma activation to an enhanced M2 phenotype. Furthermore, PPARgamma activation significantly increases expression of the M2 marker MR in circulating peripheral blood mononuclear cells. These data demonstrate that PPARgamma activation skews human monocytes toward an anti-inflammatory M2 phenotype.     

Noradrenergic regulation of inflammatory gene expression in brain

It is now well accepted that inflammatory events contribute to the pathogenesis of numerous neurological disorders, including multiple sclerosis (MS), Alzheimer's disease (AD), Parkinson's disease, and AID's dementia. Whereas inflammation in the periphery is subject to rapid down regulation by increases in anti-inflammatory molecules and the presence of scavenging soluble cytokine receptors, the presence of an intact blood-brain barrier may limit a similar autoregulation from occurring in brain. Mechanisms intrinsic to the brain may provide additional immunomodulatory functions, and whose dysregulation could contribute to increased inflammation in disease. The findings that noradrenaline (NA) reduces cytokine expression in microglial, astroglial, and brain endothelial cells in vitro, and that modification of the noradrenergic signaling system occurs in some brain diseases having an inflammatory component, suggests that NA could act as an endogenous immunomodulator in brain. Furthermore, accumulating studies indicate that modification of the noradrenergic signaling system occurs in some neurodiseases. In this article, we will briefly review the evidence that NA can modulate inflammatory gene expression in vitro, summarize data supporting a similar immunomodulatory role in brain, and present recent data implicating a role for NA in attenuating the cortical inflammatory response to beta amyloid protein.     

Peroxisome proliferator-activated receptor gamma up-regulates the Bcl-2 anti-apoptotic protein in neurons and induces mitochondrial stabilization and protection against oxidative stress and apoptosis

Peroxisome proliferator-activated receptor gamma (PPARgamma) has been proposed as a therapeutic target for neurodegenerative diseases because of its anti-inflammatory action in glial cells. However, PPARgamma agonists preventbeta-amyloid (Abeta)-induced neurodegeneration in hippocampal neurons, and PPARgamma is activated by the nerve growth factor (NGF) survival pathway, suggesting a neuroprotective anti-inflammatory independent action. Here we show that the PPARgamma agonist rosiglitazone (RGZ) protects hippocampal and dorsal root ganglion neurons against Abeta-induced mitochondrial damage and NGF deprivation-induced apoptosis, respectively, and promotes PC12 cell survival. In neurons and in PC12 cells RGZ protective effects are associated with increased expression of the Bcl-2 anti-apoptotic protein. NGF-differentiated PC12 neuronal cells constitutively overexpressing PPARgamma are resistant to Abeta-induced apoptosis and morphological changes and show functionally intact mitochondria and no increase in reactive oxygen species when challenged with up to 50 microM H2O2. Conversely, cells expressing a dominant negative mutant of PPARgamma show increased Abeta-induced apoptosis and disruption of neuronal-like morphology and are highly sensitive to oxidative stress-induced impairment of mitochondrial function. Cells overexpressing PPARgamma present a 4- to 5-fold increase in Bcl-2 protein content, whereas in dominant negative PPARgamma-expressing cells, Bcl-2 is barely detected. Bcl-2 knockdown by small interfering RNA in cells overexpressing PPARgamma results in increased sensitivity to Abeta and oxidative stress, further suggesting that Bcl-2 up-regulation mediates PPARgamma protective effects. PPARgamma prosurvival action is independent of the signal-regulated MAPK or the Akt prosurvival pathways. Altogether, these data suggest that PPARgamma supports survival in neurons in part through a mechanism involving increased expression of Bcl-2.     

Feedback control of cyclooxygenase-2 expression through PPARgamma

Cyclooxygenase-2 (COX-2), a rate-limiting enzyme for prostaglandins (PG), plays a key role in inflammation, tumorigenesis, development, and circulatory homeostasis. The PGD(2) metabolite 15-deoxy-Delta(12, 14) PGJ(2) (15d-PGJ(2)) was identified as a potent natural ligand for the peroxisome proliferator-activated receptor-gamma (PPARgamma). PPARgamma expressed in macrophages has been postulated as a negative regulator of inflammation and a positive regulator of differentiation into foam cell associated with atherogenesis. Here, we show that 15d-PGJ(2) suppresses the lipopolysaccharide (LPS)-induced expression of COX-2 in the macrophage-like differentiated U937 cells but not in vascular endothelial cells. PPARgamma mRNA abundantly expressed in the U937 cells, not in the endothelial cells, is down-regulated by LPS. In contrast, LPS up-regulates mRNA for the glucocorticoid receptor which ligand anti-inflammatory steroid dexamethasone (DEX) strongly suppresses the LPS-induced expression of COX-2, although both 15d-PGJ(2) and DEX suppressed COX-2 promoter activity by interfering with the NF-kappaB signaling pathway. Transfection of a PPARgamma expression vector into the endothelial cells acquires this suppressive regulation of COX-2 gene by 15d-PGJ(2) but not by DEX. A selective COX-2 inhibitor, NS-398, inhibits production of PGD(2) in the U937 cells. Taking these findings together, we propose that expression of COX-2 is regulated by a negative feedback loop mediated through PPARgamma, which makes possible a dynamic production of PG, especially in macrophages, and may be attributed to various expression patterns and physiological functions of COX-2.     

Lysophosphatidic acid receptor-dependent secondary effects via astrocytes promote neuronal differentiation

Lysophosphatidic acid (LPA) is a simple phospholipid derived from cell membranes that has extracellular signaling properties mediated by at least five G protein-coupled receptors referred to as LPA(1)-LPA(5). In the nervous system, receptor-mediated LPA signaling has been demonstrated to influence a range of cellular processes; however, an unaddressed aspect of LPA signaling is its potential to produce specific secondary effects, whereby LPA receptor-expressing cells exposed to, or "primed," by LPA may then act on other cells via distinct, yet LPA-initiated, mechanisms. In the present study, we examined cerebral cortical astrocytes as possible indirect mediators of the effects of LPA on developing cortical neurons. Cultured astrocytes express at least four LPA receptor subtypes, known as LPA(1)-LPA(4). Cerebral cortical astrocytes primed by LPA exposure were found to increase neuronal differentiation of cortical progenitor cells. Treatment of unprimed astrocyte-progenitor cocultures with conditioned medium derived from LPA-primed astrocytes yielded similar results, suggesting the involvement of an astrocyte-derived soluble factor induced by LPA. At least two LPA receptor subtypes are involved in LPA priming, since the priming effect was lost in astrocytes derived from LPA receptor double-null mice (LPA(1)((-/-))/LPA(2)((-/-))). Moreover, the loss of LPA-dependent differentiation in receptor double-null astrocytes could be rescued by retrovirally transduced expression of a single deleted receptor. These data demonstrate that receptor-mediated LPA signaling in astrocytes can induce LPA-dependent, indirect effects on neuronal differentiation.     

P2Y2 Nucleotide Receptor-Mediated Responses in Brain Cells

Acute inflammation is important for tissue repair; however, chronic inflammation contributes to neurodegeneration in Alzheimer's disease (AD) and occurs when glial cells undergo prolonged activation. In the brain, stress or damage causes the release of nucleotides and activation of the G_q protein-coupled P2Y₂ nucleotide receptor subtype (P2Y₂R) leading to pro-inflammatory responses that can protect neurons from injury, including the stimulation and recruitment of glial cells. P2Y₂R activation induces the phosphorylation of the epidermal growth factor receptor (EGFR), a response dependent upon the presence of a SH3 binding domain in the intracellular C terminus of the P2Y₂R that promotes Src binding and transactivation of EGFR, a pathway that regulates the proliferation of cortical astrocytes. Other studies indicate that P2Y₂R activation increases astrocyte migration. P2Y₂R activation by UTP increases the expression in astrocytes of α_Vβ_3/5 integrins that bind directly to the P2Y₂R via an Arg-Gly-Asp (RGD) motif in the first extracellular loop of the P2Y₂R, an interaction required for G_o and G₁₂ protein-dependent astrocyte migration. In rat primary cortical neurons (rPCNs) P2Y₂R expression is increased by stimulation with interleukin-1β (IL-1β), a pro-inflammatory cytokine whose levels are elevated in AD, in part due to nucleotide-stimulated release from glial cells. Other results indicate that oligomeric β-amyloid peptide (Aβ_1-42), a contributor to AD, increases nucleotide release from astrocytes, which would serve to activate upregulated P2Y₂Rs in neurons. Data with rPCNs suggest that P2Y₂R upregulation by IL-1β and subsequent activation by UTP are neuroprotective, since this increases the non-amyloidogenic cleavage of amyloid precursor protein. Furthermore, activation of IL-1β-upregulated P2Y₂Rs in rPCNs increases the phosphorylation of cofilin, a cytoskeletal protein that stabilizes neurite outgrowths. Thus, activation of pro-inflammatory P2Y₂Rs in glial cells can promote neuroprotective responses, suggesting that P2Y₂Rs represent a novel pharmacological target in neurodegenerative and other pro-inflammatory diseases.     

On the molecular basis linking Nerve Growth Factor (NGF) to Alzheimer's disease

1. Alzheimer's disease (AD) is pathologically defined by the deposition of amyloid peptide and neurofibrillary tangles and is characterized by a progressive loss of cognition and memory function, due to marked cortical cholinergic depletion. 2. Cholinergic cortical innervation is provided by basal forebrain cholinergic neurons. The neurotrophin Nerve Growth Factor (NGF) promotes survival and differentiation of basal forebrain cholinergic neurons. 3. This assertion has been at the basis of the hypothesis developed in the last 20 years, whereby NGF deprivation would be one of the factor involved in the etiology of sporadic forms of AD. 4. In this review, we shall summarize data that lead to the production and characterization of a mouse model for AD (AD11 anti-NGF mice), based on the expression of transgenic antibodies neutralizing NGF. The AD-like phenotype of AD11 mice will be discussed on the basis of recent studies that have posed NGF and its precursor pro-NGF back to the stage of AD-like neurodegeneration, showing the involvement of the precursor pro-NGF in one of the cascades leading to AD neurodegeneration.     

Generation of reactive oxygen species and activation of NF-kappaB by non-Abeta component of Alzheimer's disease amyloid

Non-amyloid beta (Abeta) component of Alzheimer's disease (AD) amyloid (NAC) coexists with Abeta protein in senile plaques. After exposure to NAC fibrils, cortical neurons of rat brain primary culture became apoptotic, while astrocytes were activated with extension of their processes. NAC fibrils decreased the activity of reducing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in cortical neurons more markedly (IC(50) = 5.6 microm) than in astrocytes (IC(50) approximately 50 microm). The neuron-specific toxicity of NAC fibrils was indicated also by an increased release of lactate dehydrogenase from the cells. Neuronal apoptosis was suppressed by pre-treatment with the antioxidants, propyl gallate (PG) and N-t-butyl-phenylnitrone (BPN), or overexpression of human Bcl-2. Exposure to NAC fibrils enhanced generation of reactive oxygen species (ROS) in neurons and less efficiently in astrocytes, as demonstrated by oxidation of 2',7'-dichlorofluorescin. The site of ROS generation was shown to be mitochondria by oxidation of chloromethyl-tetramethyl rosamine. Exposure to NAC fibrils increased also the nuclear translocation of nuclear factor kappa B (NF-kappaB) and enhanced its DNA-binding activity, which was inhibited by PG and BPN more efficiently in neurons than in astrocytes. These results suggest that NAC fibrils increase mitochondrial ROS generation and activate NF-kappaB, thereby causing a differential change in gene expression between neurons and astrocytes in the AD brain.     

Somatodendritic α2-Adrenoceptors in the Locus Coeruleus Are Involved in the In Vivo Modulation of Cortical Noradrenaline Release by the Antidepressant Desipramine

Abstract: The effect of the antidepressant and selective noradrenaline reuptake blocker desipramine (DMI) on noradrenergic transmission was evaluated in vivo by dual-probe microdialysis. DMI (1, 3, and 10 mg/kg, i.p.) dose-dependently increased extracellular levels of noradrenaline (NA) in the locus coeruleus (LC) area. In the cingulate cortex (Cg), DMI (3 and 10 mg/kg, i.p.) also increased NA dialysate, but at the lowest dose (1 mg/kg, i.p.) it decreased NA levels. When the α₂-adrenoceptor antagonist RX821002 (1 µ M ) was perfused in the LC, DMI (1 mg/kg, i.p.) no longer decreased but rather increased NA dialysate in the Cg. In electrophysiological experiments, DMI (1 mg/kg, i.p.) inhibited the firing activity of LC neurons by a mechanism reversed by RX821002. Local DMI (0.01–100 µ M ) into the LC increased concentration-dependently NA levels in the LC and simultaneously decreased NA levels in the Cg. This decrease was abolished by local RX821002 administration into the LC. The results demonstrate in vivo that DMI inhibits NA reuptake at somatodendritic and nerve terminal levels of noradrenergic cells. The increased NA dialysate in the LC inhibits noradrenergic activity, which in part counteracts the effects of DMI on the Cg. The modulation of cortical NA release by activity of DMI at the somatodendritic level is mediated through α₂-adrenoceptors located in the LC.     

Anoxic depolarization of hippocampal astrocytes: Possible modulation by P2X7 receptors

Current responses from CA1 neurons and stratum oriens astrocytes were recorded from hippocampal brain slices by means of the whole-cell patch-clamp technique. Anoxic depolarization (AD) was induced by an oxygen/glucose-deprived (OGD) medium also containing sodium iodoacetate and antimycin, in order to block glycolysis and oxidative phosphorylation, respectively. Anoxic depolarization has been reported to be due to the sudden increase of the extracellular K⁺ concentration and the accompanying explosive rise in glutamate concentration. We asked ourselves whether the release of ATP activating P2X7 receptors is also involved in the AD. Although, the AD was evoked in absolute synchrony in neurons and astrocytes, and the NMDA receptor antagonistic AP-5 depressed these responses, neither the non-selective P2 receptor antagonist PPADS, nor the highly selective P2X7 receptor antagonist A438079 interfered with the AD or its delay time in neurons/astrocytes after inducing chemical hypoxia. However, A438079, but not PPADS increased in astrocytes the slow inward current observed in a hypoxic medium. It is concluded that ATP co-released with glutamate by hypoxic stimulation has only a minor function in the present brain slice system.     

The peroxisome-proliferator-activated gamma receptor and chronic inflammatory bowel disease (PPARgamma and IBD)

PPARgamma has been recently described as being a gene of susceptibility for Intestinal Bowel Diseases (IBD) as NOD2/CARD15 gene. IBD are pathologies due to an abnormal immune response, in genetically predisposed patients, to the bacteria of the intestinal flora. PPARgamma, known for its significant role in adipogenesis, is strongly expressed by the epithelial cells of the colon mucosa. PPARgamma is implicated in the regulation of inflammation. Indeed, agonists of this nuclear receptor decrease strongly the intensity of inflammation during experimental colitis induced by chemical agents. A deficit of PPARgamma in patients with ulcerative colitis has been highlighted, that could in part explain the acute inflammation. In addition, bacteria, including those of the commensal flora, are able to regulate PPARgamma. Toll Like Receptor-4 (TLR-4), responsible for the recognition of bacterial motif as lipopolysaccharide (LPS), is implicated in PPARgamma regulation and its anti-inflammatory properties. All these arguments make of PPARgamma a very interesting therapeutic target for the treatment of IBD.     

Amphetamine-induced inhibition of central noradrenergic neurons: a pharmacological analysis

The amphetamine-induced inhibition of brain noradrenaline (NA) containing neurons in the rat locus coeruleus (LC) was pharmacologically analyzed utilizing single unit recording techniques. The presynaptic α-receptor blocking agent yohimbine (10 mg/kg i.p., 30 min before) largely prevented the amphetamine-induced depression of LC units in contrast to prazosin (0.6 mg/kg i.p., 30 min) or phenoxybenzamine (20 mg/kg, 30 min) which both slow preference for postsynaptic α-receptors. The β-receptor blocking agent, propranolol (10 mg/kg, 30 min), as well as the peripherally but not centrally active α-receptor blocking drug phentolamine (10 mg/kg, i.p., 30 min), also did not block the amphetamine effect. The LC inhibition by amphetamine was blocked by pretreatment with reserpine (10 mg/kg, i.p., 5 h), which caused almost total depletion of brain catecholamines. However, unlike the amphetamine-induced inhibition of central dopamine (DA) neurons the NA cell inhibition was not blocked by pretreatment with a tyrosine hydroxylase inhibitor (α-MT, 50 or 250 mg/kg i.p., 30 min). These results suggest that the amphetamine-induced inhibition of NA neurons in the LC is an indirect effect, mediated via activation of central α-receptors of presynaptic character. The lack of antagonism by α-MT indicate that the NA release by amphetamine, unlike its effect on brain DA, is not critically dependent on the rate of tyrosine hydroxylation. Thus the euphoriant action of amphetamine, which is blocked by α-MT, may be associated with release of DA rather than NA in brain.     

Protection by pioglitazone in the MPTP model of Parkinson's disease correlates with I kappa B alpha induction and block of NF kappa B and iNOS activation

Inflammation has been implicated in the pathogenesis of Parkinson's disease (PD). In the chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD, inducible NO synthase (iNOS) derived nitric oxide (NO) is an important mediator of dopaminergic cell death. Ligands of the peroxisome proliferator-activated receptor (PPAR) exert anti-inflammatory effects. We here investigated whether pioglitazone, a PPARgamma agonist, protected mice from MPTP-induced dopaminergic cell loss, glial activation, and loss of catecholamines in the striatum. As shown by western blot, PPARgamma was expressed in the striatum and the substantia nigra of vehicle- and MPTP-treated mice. Oral administration of 20 mg/(kg day) of pioglitazone protected tyrosine hydroxylase (TH)-positive substantia nigra neurons from death induced by 5 x 30 mg/kg MPTP. However, the decrease of dopamine in the striatum was only partially prevented. In mice treated with pioglitazone, there were a reduced activation of microglia, reduced induction of iNOS-positive cells and less glial fibrillary acidic protein positive cells in both striatum and substantia nigra pars compacta. In addition, treatment with pioglitazone almost completely blocked staining of TH-positive neurons for nitrotyrosine, a marker of NO-mediated cell damage. Because an increase in inhibitory protein-kappa-Balpha (IkappaBalpha) expression and inhibition of translocation of the nuclear factor kappaB (NFkappaB) subunit p65 to the nucleus in dopaminergic neurons, glial cells and astrocytes correlated with the protective effects of pioglitazone, our results suggest that pioglitazone sequentially acts through PPARgamma activation, IkappaBalpha induction, block of NFkappaB activation, iNOS induction and NO-mediated toxicity. In conclusion, treatment with pioglitazone may offer a treatment opportunity in PD to slow the progression of disease that is mediated by inflammation.     

Glial and neuronal cells express functional chemokine receptor CXCR4 and its natural ligand stromal cell-derived factor 1

Chemokines are a family of proteins that chemoattract and activate cells by interacting with specific receptors on the surface of their targets. The chemokine stromal cell-derived factor 1, (SDF1), binds to the seven-transmembrane G protein-coupled CXCR4 receptor and acts to modulate cell migration, differentiation, and proliferation. CXCR4 and SDF1 are reported to be expressed in various tissues including brain. Here we show that SDF1 and CXCR4 are expressed in cultured cortical type I rat astrocytes, cortical neurons, and cerebellar granule cells. In cortical astrocytes, prolonged treatment with lipopolysaccharide induced an increase of SDF1 expression and a down-regulation of CXCR4, whereas treatment with phorbol esters did not affect SDF1 expression and down-modulated CXCR4 receptor expression. We also demonstrated the ability of human SDF1alpha (hSDF1alpha) to increase the intracellular calcium level in cultured astrocytes and cortical neurons, whereas in the same conditions, cerebellar granule cells did not modify their intracellular calcium concentration. Furthermore, in cortical astrocytes, the simultaneous treatment of hSDF1alpha with the HIV-1 capside glycoprotein gp120 inhibits the cyclic AMP formation induced by forskolin treatment.