Birgit Mertens

The role of the central renin-angiotensin system in Parkinson’s disease

Since the discovery of a renin-angiotensin system (RAS) in the brain, several studies have linked this central RAS to neurological disorders such as ischaemia, Alzheimer’s disease and depression. In the last decade, evidence has accumulated that the central RAS might also play a role in Parkinson’s disease. Although the exact cause of this progressive neurodegenerative disorder of the basal ganglia remains unidentified, inflammation and oxidative stress have been suggested to be key factors in the pathogenesis and the progression of the disease. Since angiotensin II is a pro-inflammatory compound that can induce the production of reactive oxygen species due to activation of the NADPH-dependent oxidase complex, this peptide might contribute to dopaminergic cell death. In this review, three different strategies to interfere with the pathogenesis or the progression of Parkinson’s disease are discussed. They include inhibition of the angiotensin-converting enzyme, blockade of the angiotensin II type 1 receptor and stimulation of the angiotensin II type 2 receptor.

Time-dependent changes in striatal xCT protein expression in hemi-Parkinson rats.

Altered glutamate signaling is associated with Parkinsons disease. To study the involvement of the cystine/glutamate antiporter in the pathogenesis of Parkinsons disease, we developed new polyclonal antibodies recognizing xCT, the specific subunit of this antiporter. The striatal xCT protein expression level was investigated in a hemi-Parkinson rat model, using semiquantitative western blotting. We observed time-dependent changes after a unilateral 6-hydroxydopamine lesion of the nigrostriatal pathway with increased expression levels in the deafferented striatum after 3 weeks. Twelve weeks postlesion, expression levels returned to normal. These data suggest, for the first time, an involvement of the cystine/glutamate antiporter in determining the aberrant glutamate neurotransmission in the striatum of a parkinsonian brain.

Reversible neurochemical changes mediated by delayed intrastriatal glial cell line-derived neurotrophic factor gene delivery in a partial Parkinson's disease rat model

Efficient protection of dopaminergic neurons against a subsequent 6‐hydroxydopamine lesion by glial cell line‐derived neurotrophic factor (GDNF) gene delivery has been demonstrated. By contrast, the neurorestorative effects of GDNF administered several weeks after the toxin have been less characterized. In particular, whether these were permanent or dependent on the continuous presence of GDNF remains elusive.

Direct angiotensin II type 2 receptor stimulation decreases dopamine synthesis in the rat striatum

A relationship between the central renin angiotensin system and the dopaminergic system has been described in the striatum. However, the role of the angiotensin II type 2 (AT(2)) receptor in this interaction has not yet been established. The present study examined the outcome of direct AT(2) receptor stimulation on dopamine (DA) release and synthesis by means of the recently developed nonpeptide AT(2) receptor agonist, compound 21 (C21). The effects of AT(2) receptor agonism on the release of DA and its major metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) and on the activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in the catecholamine biosynthesis, were investigated using in vivo microdialysis. Local administration of C21 (0.1 and 1 microM) resulted in a decrease of the extracellular DOPAC levels, whereas extracellular DA concentrations remained unaltered, suggesting a reduced synthesis of DA. This effect was mediated by the AT(2) receptor since it could be blocked by the AT(2) receptor antagonist PD123319 (1 microM). A similar effect was observed after local striatal (10 nM) as well as systemic (0.3 and 3 mg/kg i.p.) administration of the AT(1) receptor antagonist, candesartan. TH activity as assessed by accumulation of extracellular levels of L-DOPA after inhibition of amino acid decarboxylase with NSD1015, was also reduced after local administration of C21 (0.1 and 1 microM) and candesartan (10 nM). Together, these data suggest that AT(1) and AT(2) receptors in the striatum exert an opposite effect on the modulation of DA synthesis rather than DA release.

Microbore liquid chromatography with UV detection to study the in vivo passage of compound 21, a non-peptidergic AT2 receptor agonist, to the striatum in rats

A microbore liquid chromatography method coupled to UV detection was developed and validated in order to monitor the passage of compound 21 (C21), a non-peptide angiotensin II type 2 receptor agonist, to the striatum of rats. For this purpose, sampling from the striatum was performed using the in vivo microdialysis technique. Separations were performed on a C₁₈ microbore (1mm i.d.) column using gradient elution. The retention time for C21 was found to be 6.3 min. The calibration curve was linear between 10 and 200 ng/ml with a correlation coefficient ≥ 0.999. The limit of detection and the limit of quantification were 3 and 10 ng/ml respectively. The intra-day and the inter-day precision (RSD%) ranged between 0.5 and 4.6% with an average recovery of 101.5±10.0% (mean±SD, n=15). In vivo experiments were performed on rats to measure the concentration of C21 in striatal dialysates after intraperitoneal (10 or 50mg/kg) or intravenous injection (10 mg/kg or 20 mg/kg) of C21 and suggest minimal passage of the compound to the striatum.

Differential modulations of striatal tyrosine hydroxylase and dopamine metabolism by cannabinoid agonists as evidence for functional selectivity in vivo

It is generally assumed that cannabinoids induce transient modulations of dopamine transmission through indirect regulation of its release. However, we previously described a direct cannabinoid-mediated control of tyrosine hydroxylase (TH) expression, in vitro. We herein report on the influence of cannabinoid agonists on the expression of this key enzyme in catecholamine synthesis as well as on the modification of dopamine content in adult rats. As expected for cannabinoid agonists, the exposure to either Δ(9)-THC, HU 210 or CP 55,940 induced both catalepsy and hypolocomotion. Supporting a possible long-lasting control on dopaminergic activity, we noticed a significant HU 210-mediated increase in TH expression in the striatum that was concomitant with an increase in striatal dopamine content. Surprisingly, while a similar trend was reported with Δ(9)-THC, CP 55,940 completely failed to modulate TH expression or dopamine content. Nevertheless, the access of CP 55,940 to brain structures was validated by determinations of drug concentrations in the tissue and by ex vivo binding experiments. Furthermore, confirming the central activity of CP 55,940, the analysis of dopamine metabolites revealed a reduction in striatal DOPAC concentrations. Consistent with the involvement of the CB(1) cannabinoid receptor in these different responses, both HU 210- and CP 55,940-mediated effects were prevented by SR 141716A. Therefore, the present data suggest that both HU 210 and CP 55,940 cause a delayed/persistent regulation of the dopamine neurotransmission system. Nevertheless, these commonly used cannabinoid agonists endowed with similar pharmacodynamic properties clearly triggered distinct biochemical responses highlighting the existence of functional selectivity in vivo.

The neuroprotective action of candesartan is related to interference with the early stages of 6-hydroxydopamine-induced dopaminergic cell death.

Several studies have revealed that manipulation of the renin angiotensin system results in reduced progression of nigrostriatal damage in different animal models of Parkinson’s disease. In the present work, the effect of daily treatment of rats with the angiotensin II (Ang II) type 1 (AT1) receptor antagonist candesartan (3 mg/kg per day, s.c.) initiated 7 days before the intrastriatal injection of 6‐hydroxydopamine (6‐OHDA) was investigated by means of tyrosine hydroxylase‐positive cell counts in the substantia nigra, and dopamine and 3,4‐dihydroxyphenylacetic acid measurements in the striatum. In this experimental set‐up, candesartan protected dopaminergic neurons of the nigrostriatal tract against the neurotoxin‐induced cell death. However, the beneficial effects of AT1 receptor blockade were not confirmed when treatment was started 24 h after the lesion, suggesting that candesartan interferes with the early events of the 6‐OHDA‐induced cell death. Stimulation of the AT1 receptor with Ang II increased the formation of hydroxyl radicals in the striatum of intact rats as measured by the in vivo microdialysis salicylate trapping technique. This Ang II‐induced production of reactive oxygen species was suppressed by candesartan perfusion. Furthermore, the Ang II‐induced production of reactive oxygen species was nicotinamide adenine dinucleotide phosphate ‐ oxidase and protein kinase C dependent as it could be blocked in the presence of apocynin, an nicotinamide adenine dinucleotide phosphate ‐ oxidase inhibitor, and chelerythrine, an inhibitor of protein kinase C. Together, these data further support the hypothesis that Ang II might contribute in an early stage to the neurotoxicity of 6‐OHDA by reinforcing the cascade of oxidative stress.

Effect of nigrostriatal damage induced by 6-hydroxydopamine on the expression of glial cell line–derived neurotrophic factor in the striatum of the rat

Several types of brain injuries have been associated with alterations in the striatal expression of neurotrophic factors, including glial cell line-derived neurotrophic factor (GDNF). However, contradictory results on the striatal expression of GDNF have been reported in different animal models of Parkinsons disease. For this reason, we examined the effect of nigrostriatal damage on the mRNA and protein expression levels of GDNF in the striatum as a function of time following a striatal or medial forebrain bundle 6-hydroxydopamine lesion. At different time points after the administration of 6-hydroxydopamine, striatal expression levels of GDNF were analyzed with semi-quantitative Western blotting. No significant changes in GDNF expression levels were observed within the 35-day observation period, either between the denervated and the intact striatum of medial forebrain bundle and striatally lesioned rats or between the striata of lesioned animals and those of control animals. In order to reinforce these results, striata of lesioned rats, sacrificed 18 days after lesioning, were analyzed with enzyme-linked immunosorbent assay and real-time polymerase chain reaction. At this time point, both techniques confirmed the results of the Western blot analysis, detecting no changes in striatal expression of GDNF, either at the protein level, or at the mRNA level. These data show that nigrostriatal damage induced by 6-hydroxydopamine has no effect on the striatal expression of GDNF.

Acute versus long-term effects of 6-hydroxydopamine on oxidative stress and dopamine depletion in the striatum of mice

Oxidative stress is one of the mechanisms which may be important in the pathogenesis of Parkinsons disease. In the current study, the effects of 6-hydroxydopamine (6-OHDA) perfusion on hydroxyl radical formation in the mouse striatum were investigated using the in vivo salicylate trapping microdialysis technique. The latter uses salicylate as a trapping agent for hydroxyl radicals with formation of 2,3-dihydroxybenzoic acid (2,3-DHBA), which is measured by HPLC. Two different approaches of the technique were validated in mice. First, perfusion of the trapping agent salicylate (1 mM) via the probe in combination with 6-OHDA (5 μM) was used to screen for radical scavenging properties of compounds in mice. Alternatively, striatal administration of 6-OHDA in a concentration known to induce nigrostriatal denervation (1mM), without the trapping agent, allowed to maximally challenge the neuronal microenvironment and as such to investigate both its acute and long-term effects. In the first method, as expected, glutathione (GSH) (1.5 mM) prevented the 6-OHDA-induced increase in 2,3-DHBA levels. In the second method, GSH prevented the hydroxyl radical formation, while depletion of GSH with 2-cyclohexen-1-one (CHO) resulted in significantly higher 2,3-DHBA levels than when 6-OHDA was perfused alone. Three weeks after the local 6-OHDA perfusion, the total striatal dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) content were reduced by 30%, compared to the intact striatum, accompanied by a reduction in striatal tyrosine hydroxylase (TH) immunoreactive (ir) nerve terminals. This suggests that the second method can be used to determine the acute as well as the long-term effects of 6-OHDA in the mouse striatum.