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Dopamine-dependent neurotoxicity of lipopolysaccharide in substantia nigra

Departamento de Bioquímica, Bromatología, Toxicología y Medicina Legal, Facultad de Farmacia, Universidad de Sevilla, Seville, Spain

¹Correspondence: E-mail: machado{at}us.es

SPECIFIC AIMS

The purpose of this study was to discover why dopaminergic neurons of the substantia nigra are especially vulnerable to various insults, particularly lipopolysaccharide (LPS) intranigral injection. Our hypothesis is that endogenous dopamine may be responsible for this vulnerability. We studied the possibility of decreasing dopamine (DA) synthesis by the inhibition of tyrosine hydroxylase (TH) with alpha-methyl-p-tyrosine (-MPT) and then restoring DA synthesis with L-dopa/benserazide that bypasses TH inhibition, producing DA from L-dopa by the dopa decarboxylase enzyme.

PRINCIPAL FINDINGS

1. -MPT (150, 200 mg/kg), an inhibitor of tyrosine hydroxylase activity, prevented the loss of nigral dopaminergic neurons induced by intranigral injection of LPS, a potent inflammogen
It also prevented the decrease in TH and DA transporter (DAT) mRNAs expression induced by LPS.

2. Strong glial activation and the loss of astrocytes induced by LPS were both prevented by -MPT treatment

3. These protective effects were dose dependent and not due to the hypothermic effect induced by the catecholamine depletion produced by -MPT

4. When the inhibition of TH produced for -MPT was bypassed by treatment with L-dopa/benserazide, the intranigral injection of LPS recovered its toxic effects, with loss of dopaminergic neurons and astrocytes to values close to those produced by the injection of LPS alone

CONCLUSIONS AND SIGNIFICANCE

Our results show that a decrease of DA synthesis as a consequence of TH inhibition prevents the degeneration of dopaminergic neurons (seen as loss of TH positive neurons and decrease of TH and DAT mRNA expression in the neurons around the injection (Figs. 1 , 2 ) and the inflammatory process induced by LPS. Treatment of these animals with L-dopa/benserazide, which is able to restore the synthesis of DA, recovers LPS toxicity (Figs. 1 , 2) . These results strongly suggest that the special vulnerability of nigral dopaminergic neurons is produced by its content in endogenous DA, enhancing toxin effects. We suggest that endogenous DA, which is stored in special vesicles, could be released endogenously for toxins or other compounds with the consequent increase in free radical production (Fig. 3 ). LPS induces the production of free radicals (as superoxide) through the activation of NADPH oxidase along with the production of nitric oxide (NO) by the induction of inducible NOS (iNOS). In dopaminergic neurons, endogenous DA enhances free radical production and oxidative damage. These effects are not produced in other neuronal phenotypes. The knowledge of the mechanism involved in this process may prevent this important degenerative process that could trigger Parkinson’s disease.

View larger version (87K): [in this window] [in a new window]   Figure 1. Coronal sections showing the changes in TH immunoreactivity in the SN of rats after different treatments. A) TH immunoreactivity in a LPS-injected animal. Arrow: injection site of 2 µg LPS. The inert tracer is visible in the lesioned area (arrowhead), where TH-immunostaining appears reduced when compared with the right side of the panel, corresponding to a control SN. B) TH immunoreactivity in an -MPT(150 mg/kg)-LPS-treated animal. Arrow: injection site of 2 µg of LPS. TH immunoreactivity is quite similar to the control SN. Monastral trace (arrowhead) is closely surrounded by TH positive cell bodies and fibers. C) TH immunoreactivity in an -MPT-B/D-LPS-treated animal. The recovery of the DA levels after bypassing the -MPT-induced TH inhibition by the benserazide/L-dopa treatment yields a reduction of TH-positive bodies and fibers around the tracer (arrow head). Scale bar: 500 µm. D) Quantification of the TH positive cells in the SN of rats after the treatments shown in panels A–C. Results are mean ± SD of at least 4 independent experiments and are expressed as TH positive cells per section. Statistical significance: 1-way ANOVA followed by the LSD post hoc test for multiple comparisons; = 0.05. a, compared with vehicle; b, compared with LPS-injected animals; P< 0.001.

View larger version (112K): [in this window] [in a new window]   Figure 2. TH mRNA in situ hybridization in the SN of rats after different treatments. A) Vehicle injected animal. Dark field photograph from the emulsion-dipped slides. Arrow: injection site. B) Bright-field high magnification photograph from the area marked with the arrow in panel A, showing the normal expression of TH mRNA (arrow). C) LPS injected animal. Arrow: injection site of 2 µg of LPS. The loss of cells expressing TH mRNA is evident. D) High magnification of the area marked with arrow in C. The damaged area contains spared TH mRNA-expressing cells (arrows) with a decreased expression level. E) -MPT(150 mg/kg)-LPS-treated animal. Arrow: injection site of 2 µg of LPS. TH mRNA expressing cells are clearly visible close to the injection site. F) High magnification of the area marked with arrow in panel E. Cells (arrow) around the Monastral tracer show an expression level similar to vehicle injected animals. G) -MPT-B/D-LPS-treated animal. Arrow: injection site of 2 µg of LPS. The loss of cells expressing TH mRNA resembles the LPS-injected group. H) High magnification of the area marked with arrow in panel G. TH mRNA expression (arrows) is reduced to levels similar to the LPS-injected animals. Scale bars: A, C, E, G) 250 µm; B, D, F, H) 25 µm.

View larger version (36K): [in this window] [in a new window]   Figure 3. Schematic diagram showing the possible influence of DA on the LPS-induced degeneration of the nigral dopaminergic neurons. 1) In a dopaminergic neuron, DA is synthesized in a two step process from tyrosine, where TH is the limiting step. 2) DA is vesiculized and can be released outside, being reuptaken by the DAT. 3) Once in the cytoplasm, DA catabolism produces H2O2 and autoxidation products, DA-quinone and DA-Cys adducts that can damage mitochondria and increase the normal production of reactive oxygen species (ROS), which could induce devesiculation of stored DA to the cytoplasm. 4) The inflammogen LPS strongly activates both microglial (M) and astroglial (A) cells, which become active producers of ROS and NO. After penetrating neurons, these compounds join to the cytoplasmic pool of ROS, which could reinforce DA devesiculation. Such a positive feedback loop can induce damaging effects on neuronal macromolecules and structures, represented here as an effect on the nucleus. 5) When DA synthesis is reduced by the inhibition of TH by -MPT, an input to the damaging ROS loop described in 3 is diminished. Although LPS-induced ROS production is still active, it seems not to be enough to produce neuronal damage. When DA synthesis is restored after bypassing TH inhibition (treatment with L-dopa/benserazide), the ROS loop and neuronal damage are restored.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2153fje;

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This Article Full Text (PDF) All Versions of this Article: 19/3/407 04-2153fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by De Pablos, R. M. Articles by Machado, A. Search for Related Content PubMed PubMed Citation Articles by De Pablos, R. M. Articles by Machado, A.