Bioenergetic and proteolytic defects in fibroblasts from patients with sporadic Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is a complex disease and the current interest and focus of scientific research is both investigating the variety of causes that underlie PD pathogenesis, and identifying reliable biomarkers to diagnose and monitor the progression of pathology. Investigation on pathogenic mechanisms in peripheral cells, such as fibroblasts derived from patients with sporadic PD and age/gender matched controls, might generate deeper understanding of the deficits affecting dopaminergic neurons and, possibly, new tools applicable to clinical practice. METHODS: Primary fibroblast cultures were established from skin biopsies. Increased susceptibility to the PD-related toxin rotenone was determined with apoptosis- and necrosis-specific cell death assays. Protein quality control was evaluated assessing the efficiency of the Ubiquitin Proteasome System (UPS) and protein levels of autophagic markers. Changes in cellular bioenergetics were monitored by measuring oxygen consumption and glycolysis-dependent medium acidification. The oxido-reductive status was determined by detecting mitochondrial superoxide production and oxidation levels in proteins and lipids. RESULTS: PD fibroblasts showed higher vulnerability to necrotic cell death induced by complex I inhibitor rotenone, reduced UPS function and decreased maximal and rotenone-sensitive mitochondrial respiration. No changes in autophagy and redox markers were detected. CONCLUSIONS: Our study shows that increased susceptibility to rotenone and the presence of proteolytic and bioenergetic deficits that typically sustain the neurodegenerative process of PD can be detected in fibroblasts from idiopathic PD patients. Fibroblasts might therefore represent a powerful and minimally invasive tool to investigate PD pathogenic mechanisms, which might translate into considerable advances in clinical management of the disease.

Dual target strategy: combining distinct non-dopaminergic treatments reduces neuronal cell loss and synergistically modulates L-DOPA-induced rotational behavior in a rodent model of Parkinson's disease.

The glutamate metabotropic receptor 5 (mGluR5) and the adenosine A2A receptor (A2A R) represent major non-dopaminergic therapeutic targets in Parkinson's disease (PD) to improve motor symptoms and slow down/revert disease progression. The 6-hydroxydopamine rat model of PD was used to determine/compare the neuroprotective and behavioral impacts of single and combined administration of one mGluR5 antagonist, 2-methyl-6-(phenylethynyl)pyridine (MPEP), and two A2A R antagonists, (E)-phosphoric acid mono-[3-[8-[2-(3-methoxyphenyl)vinyl]-7-methyl-2,6-dioxo-1-prop-2-ynyl-1,2,6,7-tetrahydropurin-3-yl]propyl] (MSX-3) and 8-ethoxy-9-ethyladenine (ANR 94). Chronic treatment with MPEP or MSX-3 alone, but not with ANR 94, reduced the toxin-induced loss of dopaminergic neurons in the substantia nigra pars compacta. Combining MSX-3 and MPEP further improved the neuroprotective effect of either antagonists. At the behavioral level, ANR 94 and MSX-3 given alone significantly potentiated L-DOPA-induced turning behavior. Combination of either A2A R antagonists with MPEP synergistically increased L-DOPA-induced turning. This effect was dose-dependent and required subthreshold drug concentration, which per se had no motor stimulating effect. Our findings suggest that co-treatment with A2A R and mGluR5 antagonists provides better therapeutic benefits than those produced by either drug alone. Our study sheds some light on the efficacy and advantages of combined non-dopaminergic PD treatment using low drug concentration and establishes the basis for in-depth studies to identify optimal doses at which these drugs reach highest efficacy. Combined treatment with low concentrations of known adenosine A2A receptor (A2A R) and metabotropic glutamate receptor (mGluR5) antagonists results in a therapeutic benefit and provides better results than those produced by either drug given alone, both in terms of motor performance and neuroprotection. Future trials should involve careful optimization of drug combinations and concentrations that may avoid the emergence of debilitating side effects and slow-down/revert disease progression.

Selective blockade of mGlu5 metabotropic glutamate receptors is protective against hepatic mitochondrial dysfunction in 6-OHDA lesioned Parkinsonian rats.

Non-motor symptoms including those involving the splanchnic district are present in Parkinson's disease (PD). The authors previously reported that PD-like rats, bearing a lesion of the nigrostriatal pathway induced by the injection of 6-hydroxydopamine (6-OHDA), have impaired hepatic mitochondrial function. Glutamate intervenes at multiple levels in PD and liver pathophysiologies. The metabotropic glutamate receptor 5 (mGluR5) is abundantly expressed in brain and liver and may represent a pharmacological target for PD therapy. This study investigated whether and how chronic treatment with 2-methyl-6-(phenylethynyl)-pyridine (MPEP), a well-characterized mGluR5 antagonist, may influence hepatic function with regard to neuronal cell loss in PD-like rats. Chronic treatment with MPEP was started immediately (Early) or 4 weeks after (Delayed) intrastriatal injection of 6-OHDA and lasted 4 weeks. Early MPEP treatment significantly prevented the decrease in adenosine triphosphate (ATP) production/content and counteracted increased reactive oxygen species (ROS) formation in isolated hepatic mitochondria of PD-like animals. Early MPEP administration also reduced the toxin-induced neurodegenerative process; improved survival of nigral dopaminergic neurons correlated with enhanced mitochondrial ATP content and production. ATP content/production, in turn, negatively correlated with ROS formation suggesting that the MPEP-dependent improvement in hepatic function positively influenced neuronal cell survival. Delayed MPEP treatment had no effect on hepatic mitochondrial function and neuronal cell loss. Antagonizing mGluR5 may synergistically act against neuronal cell loss and PD-related hepatic mitochondrial alterations and may represent an interesting alternative to non-dopaminergic therapeutic strategies for the treatment of PD.

Impaired hepatic function and central dopaminergic denervation in a rodent model of Parkinson's disease: a self-perpetuating crosstalk?

In Parkinson's disease (PD), aside from the central lesion, involvement of visceral organs has been proposed as part of the complex clinical picture of the disease. The issue is still poorly understood and relatively unexplored. In this study we used a classic rodent model of nigrostriatal degeneration, induced by the intrastriatal injection of 6-hydroxydopamine (6-OHDA), to investigate whether and how a PD-like central dopaminergic denervation may influence hepatic functions. Rats received an intrastriatal injection of 6-OHDA or saline (sham), and blood, cerebrospinal fluid, liver and brain samples were obtained for up to 8 weeks after surgery. Specimens were analyzed for changes in cytokine and thyroid hormone levels, as well as liver mitochondrial alterations. Hepatic mitochondria isolated from animals bearing extended nigrostriatal lesion displayed increased ROS production, while membrane potential (ΔΨ) and ATP production were significantly decreased. Reduced ATP production correlated with nigral neuronal loss. Thyroid hormone levels were significantly increased in serum of PD rats compared to sham animals while steady expression of selected cytokines was detected in all groups. Hepatic enzyme functions were comparable in all animals. Our study indicates for the first time that in a rodent model of PD, hepatic mitochondria dysfunctions arise as a consequence of nigrostriatal degeneration, and that thyroid hormone represents a key interface in this CNS-liver interaction. Liver plays a fundamental detoxifying function and a better understanding of PD-related hepatic mitochondrial alterations, which might further promote neurodegeneration, may represent an important step for the development of novel therapeutic strategies.

A reliable indirect cell-labelling protocol for optical imaging allows ex vivo visualisation of mesenchymal stem cells after transplantation.

We set out to assess the feasibility of exploiting expression of the mCherry gene, after lentiviral infection, in order visualise bone marrow-derived human mesenchymal stem cells (hMSCs) by optical imaging, and to provide proof of principle of this approach as a method for cell tracking and quantification in pre-clinical models. Commercial hMSCs were infected with a lentiviral vector carrying the mCherry gene under the control of the phosphoglycerate kinase promoter. After extensive in vitro culture, infected hMSCs were analysed for viability, morphology, differentiation capability, and maintenance of fluorescence. Thereafter, mCherry-positive cells were transplanted into unilaterally 6-hydroxy-dopamine lesioned rats (an experimental model of Parkinson's disease). Our analysis showed that hMSCs can be efficiently transduced with the lentiviral vector, retaining their biological features even in the long term. Intrastriatally transplanted mCherry-positive hMSCs can be detected ex vivo by a sensitive cooled CCD camera, both in the whole brain and in serial slices, and relatively quantified. Our protocol was found to be a reliable means of studying the viability of implanted hMSCs. mCherry labelling appears to be readily applicable in the post-transplantation tracking of stem cells and could favour the rapid development of new therapeutic targets for clinical treatments.

Neuroprotective effects of human mesenchymal stem cells on neural cultures exposed to 6-hydroxydopamine: implications for reparative therapy in Parkinson's disease.

Stem cell (SC) transplantation represents a promising tool to treat neurodegenerative disorders, such as Parkinson's disease (PD), but positive therapeutic outcomes require elucidation of the biological mechanisms involved. Therefore, we investigated human Mesenchymal SCs (hMSCs) ability to protect murine differentiated Neural SCs (mdNSCs) against the cytotoxic effects of 6-hydroxydopamine (6-OHDA) in a co-culture model mimicking the in vivo neurovascular niche. The internalization of 6-OHDA mainly relies on its uptake by the dopamine active transporter (DAT), but its toxicity could also involve other pathways. We demonstrated that mdNSCs consistently expressed DAT along the differentiative process. Exposure to 6-OHDA did not affect hMSCs, but induced DAT-independent apoptosis in mdNSCs with generation of reactive oxygen species and caspases 3/7 activation. The potential neuroprotective action of hMSCs on mdNSCs exposed to 6-OHDA was tested in different co-culture conditions, in which hMSCs were added to mdNSCs prior to, simultaneously, or after 6-OHDA treatment. In the presence of the neurotoxin, the majority of mdNSCs acquired an apoptotic phenotype, while co-cultures with hMSCs significantly increased their survival (up to 70%) in all conditions. Multiplex human angiogenic array analysis on the conditioned media demonstrated that cytokine release by hMSCs was finely modulated. Moreover, sole growth factor addition yielded a similar neuroprotective effect on mdNSCs. In conclusion, our findings demonstrate that hMSCs protect mdNSCs against 6-OHDA neurotoxicity, and rescue cells from ongoing neurodegeneration likely through the release of multiple cytokines. Our findings provide novel insights for the development of therapeutic strategies designed to counteract the neurodegenerative processes of PD.

Adhesion molecules as potential targets for neuroprotection in a rodent model of Parkinson's disease.

Cell adhesion molecules might play an important role in the inflammatory mechanisms associated with neurodegeneration. We have previously observed, in rats, that subcutaneous injection of complete Freund's adjuvant (CFA), a pro-inflammatory agent that induces a peripheral inflammatory stimulus, reduces the nigrostriatal degeneration and microglial activation caused by stereotaxic injection of 6-hydroxydopamine (6-OHDA). Here we further investigated the effects of CFA in 6-OHDA-lesioned rats by evaluating the expression of selected adhesion molecules, both at central and peripheral levels. Male, Sprague-Dawley rats received a subcutaneous injection of CFA followed, 10 days later, by intrastriatal injection of 6-OHDA. Animals were sacrificed at various time points and changes affecting intercellular (ICAM-1), vascular (VCAM-1), platelet endothelial (PECAM-1) and neural (NCAM-1) cell adhesion molecules were analyzed in striatum, ventral midbrain (containing the substantia nigra) and sera. Our results confirmed the protective effect of systemic CFA on 6-OHDA-induced nigrostriatal degeneration. Injection of 6-OHDA increased striatal ICAM-1 and PECAM-1 expression, while opposite changes (decreased expression) were detected in the ventral midbrain, particularly for VCAM-1 and NCAM-1. Pretreatment with CFA counteracted these changes. Nigrostriatal degeneration also affected peripheral immune function, with lesioned animals showing increased sPECAM levels with respect to intact animals. Also in this case, CFA pretreatment blocked the 6-OHDA induced increase of sPECAM. Our findings confirm that a pre-existing, peripheral pro-inflammatory condition reduces the neuroinflammatory response and associated neurodegeneration provoked by centrally-administered 6-OHDA, with a mechanism that seems to involve selected adhesion molecules. The link between peripheral and central immune responses may, therefore, represent a target for new therapeutic strategies aimed at reducing the neuroinflammatory component associated with neurodegeneration.

Long-term culture and differentiation of CNS precursors derived from anterior human neural rosettes following exposure to ventralizing factors.

In this study we demonstrated that neural rosettes derived from human ES cells can give rise either to neural crest precursors, following expansion in presence of bFGF and EGF, or to dopaminergic precursors after exposure to ventralizing factors Shh and FGF8. Both regionalised precursors are capable of extensive proliferation and differentiation towards the corresponding terminally differentiated cell types. In particular, peripheral neurons, cartilage, bone, smooth muscle cells and also pigmented cells were obtained from neural crest precursors while tyrosine hydroxylase and Nurr1 positive dopaminergic neurons were derived from FGF8 and Shh primed rosette cells. Gene expression and immunocytochemistry analyses confirmed the expression of dorsal and neural crest genes such as Sox10, Slug, p75, FoxD3, Pax7 in neural precursors from bFGF-EGF exposed rosettes. By contrast, priming of rosettes with FGF8 and Shh induced the expression of dopaminergic markers Engrailed1, Pax2, Pitx3, floor plate marker FoxA2 and radial glia markers Blbp and Glast, the latter in agreement with the origin of dopaminergic precursors from floor plate radial glia. Moreover, in vivo transplant of proliferating Shh/FGF8 primed precursors in parkinsonian rats demonstrated engraftment and terminal dopaminergic differentiation. In conclusion, we demonstrated the derivation of long-term self-renewing precursors of selected regional identity as potential cell reservoirs for cell therapy applications, such as CNS degenerative diseases, or for the development of toxicological tests.

Peripheral-Central Neuroimmune Crosstalk in Parkinson's Disease: What Do Patients and Animal Models Tell Us?

The brain is no longer considered an immune privileged organ and neuroinflammation has long been associated with Parkinson's disease. Accumulating evidence demonstrates that innate and adaptive responses take place in the CNS. The extent to which peripheral immune alterations impacts on the CNS, or vice and versa, is, however, still a matter of debate. Gaining a better knowledge of the molecular and cellular immune dysfunctions present in these two compartments and clarifying their mutual interactions is a fundamental step in understanding and preventing Parkinson's disease (PD) pathogenesis. This review provides an overview of the current knowledge on inflammatory processes evidenced both in PD patients and in toxin-induced animal models of the disease. It discusses differences and similarities between human and animal studies in the context of neuroinflammation and immune responses and how they have guided therapeutic strategies to slow down disease progression. Future longitudinal studies are necessary and can help gain a better understanding on peripheral-central nervous system crosstalk to improve therapeutic strategies for PD.

Proteasomal inhibition and apoptosis regulatory changes in human isolated lymphocytes: the synergistic role of dopamine.

Abnormal deposition of protein aggregates and increased susceptibility to apoptotic cell death may result from defects in the activity of the ubiquitin-proteasome system (UPS); neurotoxicity related to UPS defects seems to require dopamine to be fully expressed. The aim of this study was to investigate the pro-apoptotic effects caused by proteasomal activity inhibition, as well as the synergistic effect of dopaminergic stimulation in human lymphocytes isolated from healthy volunteers. Cells were incubated 20 h at 37 degrees C, with: (1) lactacystin, (2) increasing concentrations of dopamine or (3) mixture of dopamine and lactacystin. Activities of proteasome 20S and pro-apoptotic caspases-3 and -9 and levels of anti-apoptotic Bcl-2 were measured with fluorimetric or immunochemical assays, while a "DNA diffusion" assay was used to determine the apoptosis. Incubation of lymphocytes with lactacystin, which caused reduction of proteasomal activity, was associated with activation of caspases. A clear, dose-dependent reduction of proteasomal activity was also seen in the presence of increasing doses of dopamine, which was accompanied by a slight dose-dependent increase of caspases activities and Bcl-2 levels. Both effects on proteasome and caspase activities were enhanced when cells were simultaneously exposed to lactacystin and elevated concentrations of dopamine. Apoptosis was detected in all treated samples, but not in controls, without significant differences among the treatment groups; however, the association of dopamine and lactacystin induced a clear reduction in the number of cells being analyzed, pointing to marked cytotoxicity. Our data confirm the potentiation of cytotoxicity related to proteasome inhibition, in the presence of dopaminergic stimulation.

Activation of brain metabolism and fos during limbic seizures: the role of locus coeruleus.

The noradrenergic nucleus Locus Coeruleus (LC) densely innervates limbic structures. In rats, the damage to LC by the neurotoxin DSP-4, converts episodic limbic seizures induced by bicuculline infusion in the anterior piriform cortex (APC) into self-sustaining status epilepticus (SE). SE induced by this approach is similar to SE induced by co-infusing cyclothiazide and bicuculline into APC in rats bearing an intact LC. As opposed to other commonly used rat SE models (e.g. systemic kainate or pilocarpine), this approach allows one to analyze the effects of SE on brain regions which are solely due to spreading of seizure activity, rather than to direct effect of systemic chemoconvulsant. We evaluated the expression of Fos protein (an immediate early gene product), and the local cerebral metabolic rates for [14C] 2-deoxyglucose (lCMRglc), in rats following SE induced either by cyclothiazide+bicuculline or by DSP-4+bicuculline. We demonstrated that regional Fos expression after SE does not parallel the increase in lCMRglc, in LC-lesioned rats. In DSP-4+bicuculline rats there is an overall lower expression of the protein as compared with the cyclothiazide+bicuculline or bicuculline alone groups; even more, such a difference co-exists with an higher lCMRglc in the DSP-4+bicuculline-treated rats in some regions, as compared with the other groups. These data show that LC neurons play an important role in determining immediate early genes expression even in conditions of strong pathological activation, such as limbic SE. This might have relevant effects in the plastic mechanisms related with epileptogenesis.

The 6-hydroxydopamine model: news from the past.

The investigation of pathogenic and pathophysiological mechanisms of Parkinson's disease relies on experimental models reproducing, in the animal, the pathological and behavioural features of the disease. Despite the availability of innovative models, 6-hydroxydopamine (6-OHDA) remains the most widely used tool to induce a nigrostriatal lesion in the animal (rat). This is due to (1) the relatively low complexity and cost of the procedure, (2) the fact that the 6-OHDA-induced lesion is highly reproducible, and (3) the versatility of the procedure, which can yield varying degrees of nigrostriatal lesions that develop with different temporal profiles, depending on the site chosen for the toxin injection.

Activation of the CREB/c-Fos Pathway during Long-Term Synaptic Plasticity in the Cerebellum Granular Layer.

The induction of long-term potentiation and depression (LTP and LTD) is thought to trigger gene expression and protein synthesis, leading to consolidation of synaptic and neuronal changes. However, while LTP and LTD have been proposed to play important roles for sensori-motor learning in the cerebellum granular layer, their association with these mechanisms remained unclear. Here, we have investigated phosphorylation of the cAMP-responsive element binding protein (CREB) and activation of the immediate early gene c-Fos pathway following the induction of synaptic plasticity by theta-burst stimulation (TBS) in acute cerebellar slices. LTP and LTD were localized using voltage-sensitive dye imaging (VSDi). At two time points following TBS (15 min and 120 min), corresponding to the early and late phases of plasticity, slices were fixed and processed to evaluate CREB phosphorylation (P-CREB) and c-FOS protein levels, as well as Creb and c-Fos mRNA expression. High levels of P-CREB and Creb/c-Fos were detected before those of c-FOS, as expected if CREB phosphorylation triggered gene expression followed by protein synthesis. No differences between control slices and slices stimulated with TBS were observed in the presence of an N-methyl-D-aspartate receptor (NMDAR) antagonist. Interestingly, activation of the CREB/c-Fos system showed a relevant degree of colocalization with long-term synaptic plasticity. These results show that NMDAR-dependent plasticity at the cerebellum input stage bears about transcriptional and post-transcriptional processes potentially contributing to cerebellar learning and memory consolidation.

Influence of Estrogen Modulation on Glia Activation in a Murine Model of Parkinson's Disease.

Epidemiological data suggest a sexual dimorphism in Parkinson disease (PD), with women showing lower risk of developing PD. Vulnerability of the nigrostriatal pathway may be influenced by exposure to estrogenic stimulation throughout fertile life. To further address this issue, we analyzed the progression of nigrostriatal damage, microglia and astrocyte activation and microglia polarization triggered by intrastriatal injection of dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) in male, female and ovariectomized (OVX) mice, as well as in OVX mice supplemented with 17ßestradiol (OVX+E). Animals were sacrificed at different time points following 6-OHDA injection and brain sections containing striatum and substantia nigra pars compacta (SNc) underwent immunohistochemistry for tyrosine hydroxylase (TH) (dopaminergic marker), immunofluorescence for IBA1 and GFAP (markers of microglia and astrocyte activation, respectively) and triple immunoflorescent to identify polarization of microglia toward the cytotoxic M1 (DAPI/IBA1/TNFα) or cytoprotective M2 (DAPI/IBA1/CD206) phenotype. SNc damage induced by 6-OHDA was significantly higher in OVX mice, as compared to all other experimental groups, at 7 and 14 days after surgery. Astrocyte activation was higher in OVX mice with respect the other experimental groups, at all time points. Microglial activation in the SNc was detected at earlier time points in male, female and OVX+E, while in OVX mice was detected at all time-points. Microglia polarization toward the M2, but not the M1, phenotype was detected in female and OVX+E mice, while the M1 phenotype was observed only in male and OVX mice. Our results support the protective effects of estrogens against nigrostriatal degeneration, suggesting that such effects may be mediated by an interaction with microglia, which tend to polarize preferentially toward an M2, cytoprotective phenotype in the presence of intense estrogenic stimulation.

Oxidative stress and pro-apoptotic conditions in a rodent model of Wilson's disease.

Wilson's disease (WD) is an inherited disorder, characterized by selective copper deposition in liver and brain, chronic hepatitis and extra-pyramidal signs. In this study, we investigated changes of biochemical markers of oxidative stress and apoptosis in liver, striatum and cerebral cortex homogenates from Long-Evans Cinnamon (LEC) rats, a mutant strain isolated from Long Evans (LE) rats, in whom spontaneous hepatitis develops shortly after birth. LEC and control (LE) rats at 11 and 14 weeks of age were used. We determined tissue levels of glutathione (GSH/GSSG ratio), lipid peroxides, protein-thiols (P-SH), nitric oxide metabolites, activities of caspase-3 and total superoxide-dismutase (SOD), striatal levels of monoamines and serum levels of hepatic amino-transferases. We observed a decrease of protein-thiols, GSH/GSSG ratio and nitrogen species associated to increased lipid peroxidation in the liver and striatum - but not in the cerebral cortex - of LEC rats, accompanied by dramatic increase in serum amino-transferases and decrease of striatal catecholamines. Conversely, SOD and caspase-3 activity increased consistently only in the cortex of LEC rats. Hence, we assume that enhanced oxidative stress may play a central role in the cell degeneration in WD, at the main sites of copper deposition, with discrete pro-apoptotic conditions developing in distal areas.

Unilateral lesion of the subthalamic nucleus enhances cortical fos expression associated with focally evoked seizures in the rat.

Reducing subthalamic nucleus (STN) activity has been proposed as an anti-epileptic procedure. Here we show that, on the contrary, a unilateral lesion of the STN causes slight (nonsignificant) increases in the severity of limbic seizures evoked by bicuculline infusion into the piriform cortex, associated with marked Fos expression throughout the cerebral cortex. Abolishing the STN control over the basal ganglia output may therefore play a facilitatory role on cortical activation associated with limbic seizures.

Intracarotid Infusion of Mesenchymal Stem Cells in an Animal Model of Parkinson's Disease, Focusing on Cell Distribution and Neuroprotective and Behavioral Effects.

UNLABELLED: Mesenchymal stem cells (MSCs) have been proposed as a potential therapeutic tool for Parkinson's disease (PD) and systemic administration of these cells has been tested in preclinical and clinical studies. However, no information on survival and actual capacity of MSCs to reach the brain has been provided. In this study, we evaluated homing of intraarterially infused rat MSCs (rMSCs) in the brain of rats bearing a 6-hydroxydopamine (6-OHDA)-induced lesion of the nigrostriatal tract, to establish whether the toxin-induced damage is sufficient to grant MSC passage across the blood-brain barrier (BBB) or if a transient BBB disruption is necessary. The rMSC distribution in peripheral organs and the effects of cell infusion on neurodegenerative process and motor deficits were also investigated. rMSCs were infused 14 days after 6-OHDA injection. A hyperosmolar solution of mannitol was used to transiently permeabilize the BBB. Behavioral impairment was assessed by adjusting step test and response to apomorphine. Animals were sacrificed 7 and 28 days after cell infusion. Our work shows that appreciable delivery of rMSCs to the brain of 6-OHDA-lesioned animals can be obtained only after mannitol pretreatment. A notable percentage of infused cells accumulated in peripheral organs. Infusion of rMSCs did not modify the progression of 6-OHDA-induced damage or the motor impairment at the stepping test, but induced progressive normalization of the pathological response (contralateral turning) to apomorphine administration. These findings suggest that many aspects should be further investigated before considering any translation of MSC systemic administration into the clinical setting for PD treatment. SIGNIFICANCE: This study demonstrates that mesenchymal stem cells infused through the carotid artery do not efficiently cross the blood-brain barrier in rats with a Parkinson's disease-like degeneration of nigrostriatal neurons, unless a permeabilizing agent (e.g., mannitol) is used. The infusion did not reduce the neuronal damage and associated motor impairment, but abolished the motor abnormalities these animals typically show when challenged with a dopaminergic agonist. Therefore, although arterially infused mesenchymal stem cells did not show neurorestorative effects in this study's Parkinson's disease model, they appeared to normalize the pathological responsiveness of striatal neurons to dopaminergic stimulation. This capability should be further explored in future studies.

Dopamine receptor agonists for Parkinson's disease.

INTRODUCTION: Prolonged administration of l-3,4-dihydroxyphenylalanine (l-DOPA) for Parkinson's disease (PD) is hampered by motor complications related to the progressive incapacity of residual nigrostriatal neurons to properly utilize the drug. Direct stimulation of dopaminergic (DAergic) receptors with specific compounds (DA agonists) has, therefore, become an additional therapeutic tool for PD. AREAS COVERED: DA agonists have considerable anti-parkinsonian symptomatic efficacy, although they are less potent than l-DOPA. This review summarizes pre-clinical and clinical data on DA agonists and their role in treating PD. Specific focus was put on second-generation, first-line non-ergolinic DA agonists and their motor, non-motor and putative neuroprotective effects. The anti-parkinsonian potential of recently developed DA agonists that reached Phase II and III clinical trials was also addressed. EXPERT OPINION: DA agonists can be useful along the whole natural course of PD, as monotherapy in the initial phase or combined with l-DOPA in advanced PD. Extended-release formulations have been developed for second-generation DA agonists, which are better appreciated by patients. Neuroprotective properties have been proposed for DA agonists, based on pre-clinical studies, but never convincingly demonstrated in patients. New DA agonists, with better symptomatic efficacy and devoid of the side effects that characterize current compounds, are needed.

Neuroprotective potential of adenosine A2A and cannabinoid CB1 receptor antagonists in an animal model of Parkinson disease.

The development of nondopaminergic therapeutic strategies that may improve motor and nonmotor deficits, while possibly slowing down the neurodegenerative process and associated neuroinflammation,is a primary goal of Parkinson disease (PD) research. We investigated the neuroprotective and anti-inflammatory potential of combined and single treatment with adenosine A2A and cannabinoid CB1 receptor antagonists MSX-3 and rimonabant, respectively, in a rodent model of PD. Rats bearing a unilateral intrastriatal 6-hydroxydopamine lesion were treated chronically with MSX-3 (0.5or 1 mg/kg/d) and rimonabant (0.1 mg/kg/d) given as monotherapy or combined. The effects of the treatments to counteract dopaminergic cell death and neuroinflammation were assessed by immunohistochemistry for tyrosine hydroxylase and glial cell markers, respectively. Both rimonabant and MSX-3 (1 mg/kg/d) promoted dopaminergic neuron survival in the substantia nigra pars compacta (SNc) when given alone; this effect was weakened when the compounds were combined. Glial activation was not significantly affected by MSX-3 (1 mg/kg/d), whereas rimonabant seemed to increase astrocyte cell density in the SNc. Our findings demonstrate the neuroprotective potential of single treatments and suggest that glial cells might be involved in this protective effect. The results also indicate that the neuroprotective potential of combined therapy may not necessarily reflect or promote single-drug effects and point out that special care should be taken when considering multidrug therapies in PD.

L-DOPA disrupts adenosine A(2A)-cannabinoid CB(1)-dopamine D(2) receptor heteromer cross-talk in the striatum of hemiparkinsonian rats: biochemical and behavioral studies.

Long-term therapy with L-3,4-dihydroxyphenylalanine (L-DOPA), still the most effective treatment in Parkinson's disease (PD), is associated with severe motor complications such as dyskinesia. Experimental and clinical data have indicated that adenosine A2A receptor antagonists can provide symptomatic improvement by potentiating L-DOPA efficacy and minimizing its side effects. It is known that the G-protein-coupled adenosine A2A, cannabinoid CB1 and dopamine D2 receptors may interact and form functional A2A-CB1-D2 receptor heteromers in co-transfected cells as well as in rat striatum. These data suggest that treatment with a combination of drugs or a single compound selectively acting on A2A-CB1-D2 heteromers may represent an alternative therapeutic treatment of PD. We investigated the expression of A2A-CB1-D2 receptor heteromers in the striatum of both naïve and hemiparkinsonian rats (HPD-rats) bearing a unilateral 6-hydroxydopamine (6-OHDA) lesion, and assessed how receptor heteromer expression and biochemical properties were affected by L-DOPA treatment. Radioligand binding data showed that A2A-CB1-D2 receptor heteromers are present in the striatum of both naïve and HPD-rats. However, behavioral results indicated that the combined administration of A2A (MSX-3 or SCH58261) and CB1 (rimonabant) receptor antagonists, in the presence of L-DOPA does not produce a response different from administration of the A2A receptor antagonist alone. These behavioral results prompted identification of heteromers in L-DOPA-treated animals. Interestingly, the radioligand binding results in samples from lesioned animals suggest that the heteromer is lost following acute or chronic treatment with L-DOPA.