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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 20, 2003 as doi:10.1096/fj.03-0501fje. |
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,,2
OASI Institute for Research and Care on Mental Retardation and Brain Aging (IRCCS), Neuropharmacology Section, 94018 Troina, Italy;
Department of Pharmacology, Faculty of Medicine, University of Sassari, Italy;
° Department of Pharmacology, Faculty of Pharmacy, University of Catania, Italy; and
# CHUL Research Centre and Department of Anatomy and Physiology, Laval University, Quebec G1V 4G2 Canada
2Correspondence: Neuropharmacology, IRCCS, Via Conte Ruggero 73, 94018 Troina (EN), Italy. E-mail: bianca.marchetti{at}oasi.en.it
SPECIFIC AIMS
Emerging evidence indicates that an inflammatory process is involved in dopaminergic nigrostriatal neuron loss in Parkinson’s disease (PD). We have investigated the effect of lifelong glucocorticoid receptor (GR) deficiency of transgenic (Tg) mice expressing GR antisense RNA from early embryonic life in the vulnerability of the nigrostriatal dopaminergic system to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) -induced experimental PD.
PRINCIPAL FINDINGS
1. Lifelong GR deficiency exacerbates MPTP-induced impairment of dopaminergic neuronal functioning and increases striatal oxidative status
To determine the causal relationship between a dysfunctional GR and vulnerability of the nigrostriatal dopaminergic system, B6C3F1 (C57BL female x C3H male) [H-2b] wild-type (Wt) and Tg mice in which a transgene driven by a neurofilament promotor was inserted in the genome constitutively expressing antisense RNA against the GRs were treated with MPTP, a neurotoxin that selectively destroys dopaminergic neurons in the substantia nigra, pars compacta (SN). Endogenous levels of plasma corticosterone were significantly increased in Wt and Tg mice in response to MPTP treatment; the peak occurred on day 7, and corticosterone levels returned to nearly saline group values by 21 days. Striatal dopaminergic functioning was significantly impaired in Wt and Tg mice, but the decrease in striatal dopamine and its metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) observed in Tg mice 1, 7, and 11 days after MPTP discontinuation was far greater than in Wt counterparts (Fig. 1
A–C). The depletion of striatal dopamine and 3-methoxytyramine (which can be assumed to be an index of dopamine release levels) measured as early as 1 day after MPTP discontinuation, coupled with the sharp, long-lasting increase in DOPAC+HVA/dopamine ratio (a reliable index of dopamine turnover) (Fig. 1E)
, indicates increased oxidative stress in GR-deficient striatum. In fact, dopamine oxidative metabolism represents a known mechanism of ROS generation, since enzymatic oxidation of dopamine generates H2O2. The sharp increase in the dehydroascorbic/ascorbic acid ratio, assumed to be a reliable index of the ascorbic acid oxidative status and ROS formation only in GR-deficient mice, supported increased ROS generation as one potential factor responsible for the increased vulnerability of the Tg mice to MPTP neurotoxicity.
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2. GR-deficient mice fail to recover from MPTP-induced dopaminergic neurotoxicity
To assess the ability of Wt and GR-deficient mice to recover after MPTP exposure, high-affinity synaptosomal up-take, a reliable quantitative indicator of dopaminergic axonal terminal density, was determined 1, 7, 11, and 21 days after MPTP. Results indicated a sharp and significantly greater decrease of synaptosomal dopamine uptake in GR-deficient mice vs. their Wt counterparts and failure to exhibit a significant recovery at 21 days.
3. GR deficiency exacerbates loss of tyrosine hydroxylase immunoreactive (TH-IR) cell bodies with no recovery
To evaluate MPTP effects on dopaminergic cell bodies, we assessed midbrain sections immunostained with TH antiserum. The quantitative analysis of TH-IR cells indicated a sharp and greater reduction in GR-deficient mice as early as after 1 day (60% depletion) and a further reduction (80% depletion) at 7 days, with no recovery 11 and 21 days after MPTP discontinuance, as opposed to Wt mice, which exhibited a 30% and 60% depletion at 1 and 7 days, respectively. From day 11, a trend toward a slight recovery was observed and by 21 days a modest return of TH expression was observed, again supporting the idea that the GR deficiency of Tg mice impaired the ability of TH-IR neurons to recover.
4. GR deficiency increases glial hypertrophy and glial-inducible nitric oxide synthase (iNOS) immunoreactivity in response to MPTP
To investigate whether differences in glial response to MPTP might contribute to the increased vulnerability of GR-deficient mice, striatal and nigral sections were reacted at various times after the lesion with antibodies against astrocyte marker glial fibrillary acidic protein (GFAP), the cytokine-inducible nitric oxide synthase isoform (iNOS) responsible for generation of high levels of NO (a key molecule in the inflammatory response), the peroxynitrite footprint, nitrotyrosine (NT), and the prototypic marker of activated macrophage/microglia, the membranolytic attack complex of complement (Mac-1/CD11b). This analysis indicated an earlier and greater reactivity of GFAP-IR astrocytes and Mac-1-IR microglia, exhibiting a strong iNOS immunofluorescent signal as opposed to Wt counterparts, suggesting increased nitrosative stress as candidate mediator of early TH-IR neuron loss.
5. GR deficiency induces aberrant macrophage/microglia iNOS/NO response to MPTP, which precedes dopaminergic neurotoxicity
The role played by macrophage/microglia iNOS/NO response (measured by its decomposition product nitrite) to MPTP neurotoxicity was assessed at intervals after MPTP, and those levels correlated with GR binding capacity. Starting 6 h after the first MPTP injection, GR-deficient microglia produced NO (3.9±0.7 µM/105 cells) threefold that of Wt microglia (0.9±0.3 µM/105 cells) (P<0.01). Another sharp increase in NO production occurred after 12 h and a plateau was reached thereafter in GR-deficient (3.9±0.7 µM), whereas in Wt mice nitrite levels increased to a lesser extent at 12 h (1.5±0.5 µM) and 24 h (2.6±0.6 µM). From 1 to 21 days after MPTP discontinuance, GR-deficient macrophage/microglia produced almost two- to fourfold more nitrites than their Wt counterparts.
6. GR-deficient macrophage/microglia are resistant to NO inhibition by GCs
The ability of exogenous GCs to regulate NO production by macrophages/microglia cultures from brains of Wt and Tg mice 7 days after MPTP discontinuance was assessed next. Thus, in GR-deficient brain phagocytes, the high production of nitrite levels was not suppressed by exogenous application of corticosterone and microglial sensitivity to the corticosterone inhibitory effect was reduced, supporting inefficient GR signaling of Tg microglia.
7. iNOS inhibition in vivo counteracts increased vulnerability of GR-deficient mice to MPTP
The relevance of iNOS/NO activation in the increased vulnerability of GR-deficient mice to MPTP was assessed next. In vivo treatment of GR-deficient mice with L-NIL prevented glial-derived NO generation and counteracted MPTP-induced neurotoxic effects. In L-NIL-treated GR-deficient mice, neurotoxic effects of MPTP were comparable to those observed in L-NIL-treated Wt counterparts, supporting the suggestion that increased vulnerability was linked to excess in NO production as a result of GR deficiency.
CONCLUSION
The key finding of this study is that early embryonic expression of a defective GR in genetically modified mice "programs" a dramatic increase in the vulnerability of the nigrostriatal dopaminergic system to MPTP. Loss of GR expression, binding capacity, and signaling of Tg mice were associated with a sharper, earlier, and long-lasting impairment of some biochemical parameters of nigrostriatal dopaminergic functioning, aberrant oxidative/nitrosative status, and abnormal activation of astroglia as a result of the GR deficiency. We demonstrate that protection from the exacerbation of MPTP-induced loss of dopaminergic neuronal functioning requires a functional GR. These findings, showing for the first time a direct link between GR, endogenous NO production and MPTP vulnerability, suggest that a dysfunctional GR can program an abnormal response of astrocytic and macrophage/microglia compartments to MPTP-induced neuroinflammation, resulting in aberrant production of cytotoxic nitrosyl radicals, which in turn are responsible for long-lasting neuronal damage and loss of dopaminergic neuron functioning (Fig. 2
). The present data are strengthened by our previous studies using the same mice, demonstrating a critical role of macrophage-derived NO production in determining the sensitivity or resistance to induced experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. That the same GR deficiency resulting in a lack of glucocorticoid regulation of NO generation provides resistance to EAE via immunosuppression, but sensitivity to MPTP-induced parkinsonism via innate inflammatory mechanisms, highlights the importance of the HPA immune dialogue via GR-NO cross-talk in programming vulnerability to degenerative diseases of the CNS (Fig. 2)
.
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The inverse correlation between up-regulation of NO and decreased GR transcription and binding capacity of Tg mice would suggest that increased iNOS/NO generation after MPTP injection may contribute to further lowering of GR binding and signaling as a result of oxidative/nitrosative damage (Fig. 2)
. Hence, GR deficiency may prime microglial cells to produce higher amounts of NO via iNOS induction directly or through increased production of proinflammatory cytokines. Our data argue that an excess of NO production can trigger a feedback loop involving loss of GR signaling, thus perpetrating or exacerbating NO output at a critical period of MPTP intoxication, resulting in hyperinduction of reactive nitrogen oxide species and subsequent damage of nigral neuron cell bodies and impairment of dopaminergic functioning (Fig. 2)
. Accordingly, in vivo treatment with the specific iNOS inhibitor L-NIL prevented macrophage/microglia NO production and counteracted MPTP-induced neurotoxicity (identical in Tg and Wt mice), indicating iNOS-derived NO as a potential final common pathway in the HPA axis programming of dopaminergic neuron vulnerability to MPTP. It remains to be determined whether GR-deficient nigrostriatal neurons per se are more vulnerable to oxidative /nitrosative stress.
In summary, this work identifies a direct link between GR and iNOS/NO in MPTP-induced experimental parkinsonism and defines macrophage/microglial cells as the key mediators of increased vulnerability of dopaminergic neurotoxicity in GR-deficient mice. Thus, stress or prenatal experiences known to influence the expression and function of GRs by modulating the response of astroglial cells to inflammatory stimuli may program the vulnerability to degenerative disorders of the CNS. These findings may have clinical implications for the development of neurodegenerative diseases in humans as well as important therapeutic implications for such neurodegenerative diseases (Fig. 2)
.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0501fje 
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