Annarosa Carta

Dynamic changes in pro- and anti-inflammatory cytokines in microglia after PPAR-γ agonist neuroprotective treatment in the MPTPp mouse model of progressive Parkinson's disease

Neuroinflammatory changes play a pivotal role in the progression of Parkinsons disease (PD) pathogenesis. Recent findings have suggested that activated microglia may polarize similarly to peripheral macrophages in the central nervous system (CNS), assuming a pro-inflammatory M1 phenotype or the alternative anti-inflammatory M2 phenotype via cytokine production. A skewed M1 activation over M2 has been related to disease progression in Alzheimer disease, and modulation of microglia polarization may be a therapeutic target for neuroprotection. By using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-probenecid (MPTPp) mouse model of progressive PD, we investigated dynamic changes in the production of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β, and anti-inflammatory cytokines, such as transforming growth factor (TGF)-β and IL-10, within Iba-1-positive cells in the substantia nigra compacta (SNc). In addition, to further characterize changes in the M2 phenotype, we measured CD206 in microglia. Moreover, in order to target microglia polarization, we evaluated the effect of the peroxisome-proliferator-activated receptor (PPAR)-γ agonist rosiglitazone, which has been shown to exert neuroprotective effects on nigral dopaminergic neurons in PD models, and acts as a modulator of cytokine production and phenotype in peripheral macrophages. Chronic treatment with MPTPp induced a progressive degeneration of SNc neurons. The neurotoxin treatment was associated with a gradual increase in both TNF-α and IL-1β colocalization with Iba-1-positive cells, suggesting an increase in pro-inflammatory microglia. In contrast, TGF-β colocalization was reduced by the neurotoxin treatment, while IL-10 was mostly unchanged. Administration of rosiglitazone during the full duration of MPTPp treatment reverted both TNF-α and IL-1β colocalization with Iba-1 to control levels. Moreover, rosiglitazone induced an increase in TGF-β and IL-10 colocalization compared with the MPTPp treatment. CD206 was gradually reduced by the chronic MPTPp treatment, while rosiglitazone restored control levels, suggesting that M2 anti-inflammatory microglia were stimulated and inflammatory microglia were inhibited by the neuroprotective treatment. The results show that the dopaminergic degeneration was associated with a gradual microglia polarization to the inflammatory over the anti-inflammatory phenotype in a chronic mouse model of PD. Neuroprotective treatment with rosiglitazone modulated microglia polarization, boosting the M2 over the pro-inflammatory phenotype. PPAR-γ agonists may offer a novel approach to neuroprotection, acting as disease-modifying drugs through an immunomodulatory action in the CNS.

Rosiglitazone decreases peroxisome proliferator receptor-gamma levels in microglia and inhibits TNF-alpha production: new evidences on neuroprotection in a progressive Parkinson's disease model

Thiazolidinedione (TZD) class of peroxisome proliferator receptor gamma (PPAR-γ) agonists display neuroprotective effects in experimental Parkinsons disease (PD) models. Neurons and microglia express PPAR-γ, therefore both of them are potential targets for neuroprotection, although the role of each cell type is not clear. Moreover, receptor-dependent as well as receptor-independent mechanisms have been involved. This study further investigated mechanisms of TZD-mediated neuroprotection in PD. We investigated the rosiglitazone effect in the progressive MPTP/probenecid (MPTPp) model of PD. C57BL/6J mice received MPTP (25 mg/kg) plus probenecid (100 mg/kg) twice per week for 5 weeks. Rosiglitazone (10 mg/kg) was given daily until sacrifice, starting on the fourth week of MPTPp treatment, in presence of an ongoing neurodegeneration with microgliosis. Changes in PPAR-γ levels were measured by immunofluorescence and confocal microscopy in tyrosine hydroxylase (TH)-positive neurons and CD11b-positive microglia of the substantia nigra pars compacta (SNc). Chronic MPTPp treatment induced a PPAR-γ overexpression in both TH-positive neurons and microglia (139.9% and 121.7% over vehicle, respectively). Rosiglitazone administration to MPTPp-treated mice, reverted PPAR-γ overexpression in microglia without affecting TH-positive neurons. Thereafter, changes in CD11b and tumor necrosis factor α (TNF-α) immunoreactivity in microglia were evaluated in the SNc. MPTPp progressively increased CD11b immunoreactivity, conferring to microglia a highly activated morphology. Moreover, TNF-α levels were increased (457.38% over vehicle) after MPTPp. Rosiglitazone administration counteracted the increase in CD11b immunoreactivity caused by MPTPp. Moreover, rosiglitazone reverted TNF-α expression to control levels. Nigrostriatal degeneration was assessed by high pressure liquid chromatography (HPLC) measurement of striatal dopamine, and counting of TH-positive neurons in the SNc. MPTPp treatment caused a severe decline of striatal dopamine and a partial degeneration of the SNc. Rosiglitazone arrested the degenerative process in both areas. Results suggest that PPAR-γ expression in microglia and TNF-α production by these cells are crucial changes by which rosiglitazone exerts neuroprotection in PD.

Different responsiveness of striatonigral and striatopallidal neurons to L-DOPA after a subchronic intermittent L-DOPA treatment.

Early gene induction by L‐DOPA in the striatum of dopamine denervated rats represents a useful way to study long‐term modifications produced by this drug. The effects of acute and subchronic L‐DOPA administration on zif‐268 mRNA expression were compared in 6‐hydroxydopamine‐lesioned rats. Rats received a subchronic intermittent L‐DOPA (6u2003mg/kg) treatment, which produces behavioural sensitization, a correlate of dyskinetic movements. Three days after interruption of subchronic treatment, zif‐268 mRNA was evaluated after an L‐DOPA challenge. Zif‐268 mRNA levels increased in the lesioned dorsolateral striatum after either acute or subchronic L‐DOPA administration. Double labelling of striatal cells with zif‐268 and enkephalin or dynorphin mRNA probes was performed to assess neuronal activation in the indirect and direct output pathway. Single acute L‐DOPA significantly increased zif‐268 in all striatal neurons reflecting a hyperresponsiveness of dopamine‐depleted striatum. After subchronic L‐DOPA, zif‐268 mRNA labelling was still increased in the striatonigral pathway, limited to dynorphin+ neurons, whereas in all other neurons it was similar to the control value. Results suggest that striatal neurons responding to acute L‐DOPA differ from those responding to subchronic L‐DOPA. L‐DOPA‐induced behavioural sensitization was associated to a down‐regulation in the responsiveness of striatopallidal and striatonigral dynorphin– neurons, whereas in striatonigral neurons containing dynorphin a hyperresponsiveness to L‐DOPA was observed. High levels of zif‐268, together with a persistent hyperresponsiveness of striatonigral dymorphinergic neurons and hyporesponsiveness of striatopallidal neurons, by creating an unbalanced state of striatal efferent neurons, may be implicated in dyskinetic movements observed in Parkinsons disease (PD).

Blockade of muscarinic receptors potentiates D1 dependent turning behavior and c-fos expression in 6-hydroxydopamine-lesioned rats but does not influence D2 mediated responses

In rats with a unilateral 6-hydroxydopamine lesion of the dopaminergic nigrostriatal pathway, blockade of muscarinic receptors by scopolamine potentiates the contralateral turning induced by selective dopaminergic D1 agonists (SKF 38393, A 68930), but does not influence the contralateral turning induced by the D2 agonist LY 171555. Studies on the expression of the early gene c-fos as reflected by the immunohistochemical demonstration of the Fos protein, show that administration of scopolamine (5 mg/kg, i.p.) potentiates c-fos expression elicited by SKF 38393 (1.5 mg/kg, s.c.) in the caudate-putamen of the lesioned side. The results indicate that cholinergic transmission is differentially involved in the behavioral expression of D1 versus D2 receptor stimulation in a denervated condition and suggest that blockade of central cholinergic transmission might be useful in improving the antiparkinsonian efficacy of D1 receptor agonists.

Selective modifications in GAD67 mRNA levels in striatonigral and striatopallidal pathways correlate to dopamine agonist priming in 6-hydroxydopamine-lesioned rats.

The present study investigated long‐term alterations in striatal gene expression after single exposure of unilaterally 6‐hydroxydopamine‐lesioned rats to different dopamine agonists (priming). Rats were primed with the D1 agonist SKF38393 (10u2003mg/kg), the D2/D3 agonist quinpirole (0.2u2003mg/kg), the dopamine precursor L‐DOPA (50u2003mg/kg) or with vehicle (drug‐naive), and GAD67, dynorphin and enkephalin mRNAs were evaluated in the striatum by in situ hybridization, 3u2003days after priming. To evaluate GAD67 mRNA in striatonigral and striatopallidal neurons, identified as enkephalin (−) and (+) neurons, double‐labelling in situ hybridization was used. Drug‐naive lesioned rats showed an increase in GAD67 mRNA in enkephalin (−) and (+) neurons, an increase in enkephalin and a decrease in dynorphin mRNAs. Priming with either SKF38393 or quinpirole further increased GAD67 mRNA in enkephalin (−) and (+) neurons, however, while SKF38393 produced a high and unbalanced activation toward enkephalin (−) neurons, after quinpirole the increase was of low intensity and similar in the two pathways. Dynorphin mRNA was increased by SKF38393 but not by quinpirole, whereas enkephalin mRNA was not changed by either priming. L‐DOPA produced a high and similar increase in GAD67 mRNA in enkephalin (−) and (+) neurons. Priming differentially affected peptides and GAD67 mRNA in striatopallidal and striatonigral neurons depending on the dopamine agonist used. The degree of enduring overactivity of the striatopallidal and striatonigral pathways may be related to the ability of L‐DOPA and D1 or D2/D3 receptor agonists to prime motor behavioural responses and to produce dyskinetic side‐effects.

Modulating microglia activity with PPAR-γ agonists: a promising therapy for Parkinson's disease?

A dysregulated response of the neuroimmune system is a main contributor to the progression of neurodegeneration in Parkinson’s disease (PD). Recent findings suggest that protracted activating stimuli including α-synuclein, drive microglia to acquire maladaptive functions and to assume a harmful phenotype that prevail over a restorative one. Based on this concept, disease-modifying drugs should be aimed at targeting suppression of harmful-activated microglia and the associated production of neurotoxic molecules as pro-inflammatory cytokines, while sparing or inducing beneficial-activated microglia. In this study, we review current evidence in support of the beneficial effect of targeting peroxisome-proliferator-activated receptor (PPAR)-γ to achieve neuroprotection in PD. PPAR-γ agonists as rosiglitazone and pioglitazone are currently gaining increasing attention as promising disease-modifying drugs in this disorder. Early in vitro studies, followed by studies in in vivo models of PD, have provided convincing evidence that these drugs inhibit neuronal degeneration likely by selectively targeting the expression of neurotoxic factors in reactive microglia. Potential therapeutic application has been corroborated by recent report of pioglitazone neuroprotective activity in a non-human primate model of PD. All together, preclinical evidence have prompted the translation of pioglitazone to a phase II clinical trial in early PD.

Alterations in GAD67, dynorphin and enkephalin mRNA in striatal output neurons following priming in the 6-OHDA model of Parkinson's disease

Abstract In the 6-hydroxydopamine (OHDA) rat model of Parkinsons disease, administration of a dopaminergic agonist sensitizes rats to a subsequent administration of dopaminergic drugs given days apart (priming). In situ hybridization was used to evaluate changes on striatal gene expression of rats primed three days previously with either L-dopa, SKF38393 or quinpirole. Double labeling was used to identify the neuronal population in which such alterations occurred. GAD67 and enkephalin mRNA were increased by the lesion whereas dynorphin mRNA was decreased as compared to the intact striatum. Priming with L-dopa and SKF38393 significantly increased GAD67 mRNA in the lesioned striatum and reversed dynorphin mRNA reduction, as compared to drug-naive rats, whereas quinpirole failed to produce any effect. Enkephalin mRNA was not affected by priming. Results suggest that 6-OHDA lesion-induced adaptive changes on striatal gene expression are modified by priming. Priming brings striatal output neurons to a higher level of activity, which may explain the sensitized behavioral response of observed following a dopaminergic agonist challenge. These changes are in relation to the different types of dopamine agonists utilized and suggest that modifications in gene expression induced by priming might be predictive of the dyskinetic potential of a drug.

Activation of PPAR gamma receptors reduces levodopa-induced dyskinesias in 6-OHDA-lesioned rats.

Long-term administration of l-3,4-dihydroxyphenylalanine (levodopa), the mainstay treatment for Parkinsons disease (PD), is accompanied by fluctuations in its duration of action and motor complications (dyskinesia) that dramatically affect the quality of life of patients. Levodopa-induced dyskinesias (LID) can be modeled in rats with unilateral 6-OHDA lesions via chronic administration of levodopa, which causes increasingly severe axial, limb, and orofacial abnormal involuntary movements (AIMs) over time. In previous studies, we showed that the direct activation of CB1 cannabinoid receptors alleviated rat AIMs. Interestingly, elevation of the endocannabinoid anandamide by URB597 (URB), an inhibitor of endocannabinoid catabolism, produced an anti-dyskinetic response that was only partially mediated via CB1 receptors and required the concomitant blockade of transient receptor potential vanilloid type-1 (TRPV1) channels by capsazepine (CPZ) (Morgese et al., 2007). In this study, we showed that the stimulation of peroxisome proliferator-activated receptors (PPAR), a family of transcription factors activated by anandamide, contributes to the anti-dyskinetic effects of URB+CPZ, and that the direct activation of the PPARγ subtype by rosiglitazone (RGZ) alleviates levodopa-induced AIMs in 6-OHDA rats. AIM reduction was associated with an attenuation of levodopa-induced increase of dynorphin, zif-268, and of ERK phosphorylation in the denervated striatum. RGZ treatment did not decrease striatal levodopa and dopamine bioavailability, nor did it affect levodopa anti-parkinsonian activity. Collectively, these data indicate that PPARγ may represent a new pharmacological target for the treatment of LID.

Neuroprotective and anti-inflammatory properties of a novel non-thiazolidinedione PPARγ agonist in vitro and in MPTP-treated mice.

Peroxisome proliferator-activated receptor (PPAR)γ is a potential pharmacological target for disease-modification in Parkinsons disease (PD), mainly acting by modulating the neuroinflammatory response. However, currently available agonists thiazolidinediones (TZDs) present limitations due to safety concerns. We evaluated a novel thiobarbituric-like compound MDG548, which acts as a functional PPARγ agonist displaying higher and selective binding affinity as compared to TZDs. Neuroprotection by MDG548 was tested in vitro and in a mouse MPTP model of PD, and neuroinflammation was investigated as a putative underlying mechanism. Viability assay on rat cortical neurons showed lack of cytotoxic effect in the dose-range of 100 nM-10 μM, which was therefore used for testing in vitro protection against H2O2 and MPP+ neurotoxicity. MDG548 dose-dependently increased cell viability of rat cortical neurons co-treated with H2O2 or pre-exposed to MDG548 prior to H2O2. Moreover, MDG548 induced neuroprotection in MPP+-treated PC12 cells. NF-kB activation was investigated to assess anti-inflammatory activity. MDG548 dose-dependently decreased NF-kB activation induced by LPS (100 ng/100ml) in HEK-Blue-hTLR4 cells. Given the supposed cancer risk of other PPARγ agonists, Ames test for genotoxicity was performed in Salmonella typhimurium TA100 and TA98 strains, showing that MDG548 was not genotoxic. In vivo, BL/6J mice were treated with MPTP (20mg/kg i.p. once/day for 4 days) in association with saline or MDG548 (2, 5, 10 mg/kg i.p.). Stereological counting showed that MDG548 prevented the MPTP-induced reduction in TH-positive cells in the substantia nigra compacta (SNc) at all doses tested. Moreover, MDG548 reduced reactive microglia and iNOS induction in the SNc. MDG548, being a non-TZD compound with high PPARγ affinity, void of genotoxicity, and with in vitro as well as in vivo neuroprotective properties, provides a promising alternative in the search for safer PPARγ agonists to be tested as potential disease-modifying drugs in PD.

Effect of MK 801 on priming of D1-dependent contralateral turning and its relationship to c-fos expression in the rat caudate-putamen.

In rats with a unilateral 6-hydroxydopamine lesion of the ascending dopamine neurons, we investigated the relationship between the expression of Fos-like immunoreactivity in the caudate-putamen and contralateral turning behavior in response to dopamine agonists during the induction and expression of sensitization (priming) to D1-dependent turning behavior. Priming was induced by apomorphine (0.1 mg/kg s.c.) or by SKF 38393 (10 mg/kg s.c.) 14 days after 6-hydroxydopamine lesions and was expressed by challenge with SKF 38393 (3 mg/kg s.c.). In the induction phase of priming, administration of MK 801 (0.1 mg/kg s.c.) potentiated contralateral turning but differentially influenced stimulation of Fos expression in the caudate-putamen by apomorphine and by SKF 38393. Thus, MK 801 reduced in the expression phase of priming the stimulation of Fos expression by apomorphine in the dorsolateral caudate-putamen, but did not affect that by SKF 38393. MK 801, while preventing priming of SKF 38393-induced turning by apomorphine, failed to affect priming by SKF 38393. MK 801, given with apomorphine in the induction phase, reduced the stimulation of Fos expression in the dorsolateral caudate-putamen by SKF 38393. No such inhibitory effect of MK 801 on SKF 38393-stimulated Fos expression was observed in rats primed with SKF 38393. These results are consistent with the possibility that MK 801 disrupts sensitization of D1 transduction by reducing the activation of c-fos by the DA agonist during the induction phase of priming.