Elaine Del-Bel

Nitric oxide synthase inhibition attenuates l-DOPA-induced dyskinesias in a rodent model of Parkinson's disease

Chronic L-DOPA pharmacotherapy in Parkinsons disease is often accompanied by the development of abnormal and excessive movements known as L-DOPA-induced dyskinesia. Rats with 6-hydroxydopamine lesion of dopaminergic neurons chronically treated with L-DOPA develop a rodent analog of this dyskinesia characterized by severe axial, limb, locomotor and orofacial abnormal involuntary movements. While the mechanisms by which these effects occur are not clear, they may involve the nitric oxide system. In the present study we investigate if nitric oxide synthase inhibitors can prevent dyskinesias induced by repeated administration of L-DOPA in rats with unilateral 6-hydroxydopamine lesion. Chronic L-DOPA (high fixed dose, 100 mg/kg; low escalating dose, 10-30 mg/kg) treatment induced progressive dyskinesia changes. Two nitric oxide synthase inhibitors, 7-nitroindazole (1-30 mg/kg) and NG-nitro-L-arginine (50 mg/kg), given 30 min before L-DOPA, attenuate dyskinesia. 7-Nitroindazolee also improved motor performance of these animals in the rota-rod test. These results suggest the possibility that nitric oxide synthase inhibitors may be useful to treat L-DOPA-induced dyskinesia.

Nitric oxide synthase inhibition decreases l-DOPA-induced dyskinesia and the expression of striatal molecular markers in Pitx3−/− aphakia mice

Nitric oxide (NO), a gaseous messenger molecule synthesized by nitric oxide synthase (NOS), plays a pivotal role in integrating dopamine transmission in the basal ganglia and has been implicated in the pathogenesis of Parkinson disease (PD). To study the role of the nitrergic system in l-DOPA-induced dyskinesia (LID), we assessed the effect of the pharmacological manipulation of NO levels and NO/cyclic guanosine monophosphate (cGMP) signaling on LID in the Pitx3(-/-) aphakia mouse, a genetic model of PD. To evaluate the effect of decreased NO signaling on the development of LID, Pitx3(-/-) mice were chronically treated with l-DOPA and 7-nitroindazole (7-NI, a neuronal NOS inhibitor). To evaluate its effect on the expression of established LID, 7-NI was administered acutely to dyskinetic mice. The chronic 7-NI treatment attenuated the development of LID in the Pitx3(-/-) mice, and the sub-acute 7-NI treatment attenuated established dyskinesia without affecting the beneficial therapeutic effect of l-DOPA. Moreover, 7-NI significantly reduced FosB and pAcH3 expression in the acutely and chronically l-DOPA-treated mice. We also examined how increasing NO/cGMP signaling affects LID expression by acutely administering molsidomine (an NO donor) or zaprinast (a cGMP phosphodiesterase 5-PDE5 inhibitor) before l-DOPA in mice with established dyskinesia. Paradoxically, the administration of either of these drugs also significantly diminished the expression of established LID; however, the effect occurred at the expense of the antiparkinsonian l-DOPA properties. We demonstrate that targeting the NO/cGMP signaling pathway reduces dyskinetic behaviors and molecular markers, but only the 7-NI treatment preserved the antiparkinsonian effect of l-DOPA, indicating that NOS inhibitors represent a potential therapy to reduce LID.

Novel tactics for neuroprotection in Parkinson's disease: Role of antibiotics, polyphenols and neuropeptides

Parkinsons disease is a progressive neurodegenerative disorder characterized by the degeneration of midbrain nigral dopaminergic neurons. Although its etiology remains unknown, the pathological role of several factors has been highlighted, namely oxidative stress, neuroinflammation, protein misfolding, and mitochondrial dysfunction, in addition to genetic predispositions. The current therapy is mainly symptomatic with l-DOPA aiming to replace dopamine. Novel therapeutic approaches are being investigated with the intention of influencing pathways leading to neuronal death and dysfunction. The present review summarizes three novel approaches, the use of which is promising in pre-clinical studies. Polyphenols have been shown to possess neuroprotective properties on account of their well-established antioxidative and anti-inflammatory actions but also due to their influence on protein misfolding and mitochondrial homeostasis. Within the amazing ancillary effects of antibiotics, their neuroprotective properties against neurodegenerative and neuroinflammatory processes are of great interest for the development of effective therapies against Parkinsons disease. Experimental evidence supports the potential of antibiotics as neuroprotective agents, being useful not only to prevent the formation of toxic α-synuclein oligomers but also to ameliorate mitochondrial dysfunction and neuroinflammation. Neuropeptides offer another approach with their diverse effects in the nervous system. Among them, pituitary adenylate cyclase-activating polypeptide, a member of the secretin/glucagon superfamily, has several advantageous effects in models of neurodegeneration, namely anti-apoptotic, anti-inflammatory and antioxidant actions, the combination of which offers a potent protective effect in dopaminergic neurons. Owing to their pleiotropic modes of action, these novel therapeutic candidates have potential in tackling the multidimensional features of Parkinsons disease.

Role of nitric oxide in motor control: implications for Parkinson's disease pathophysiology and treatment.

According to classical thinking about Parkinsons disease, loss of dopaminergic input from the substantia nigra pars compacta leads to overactivity and underactivity of the indirect and direct output pathways, respectively, in the basal ganglia. Administration of the dopamine precursor L-DOPA (l-3, 4-dihydroxyphenylalanine) is proposed to induce changes in the opposite directions. L-DOPA is the most used drug to treat Parkinson`s disease symptoms. After repeated treatment with this compound, however, disabling secondary effects such as the abnormal involuntary movements usually appear. Nitric oxide is a free radical that can also act as an atypical neurotransmitter and influences dopamine-mediated neurotransmission. In this paper we will briefly review the role of nitric oxide on motor control and in Parkinsons disease, particularly a possible role of nitric oxide in L-DOPA induced dyskinesia in rodents. Recent results show that nitric oxide synthase inhibition reduces L-DOPA-induced dyskinesia in rats and mice. The effect is dose-dependent, does not suffer tolerance nor interferes with L-DOPA positive motor effects. These preclinical findings suggest that nitric oxide is a promising therapeutic target for the reduction of L-DOPA-induced dyskinesia.

Glial activation is associated with l-DOPA induced dyskinesia and blocked by a nitric oxide synthase inhibitor in a rat model of Parkinson's disease.

l-3, 4-dihydroxyphenylalanine (L-DOPA) is the most effective treatment for Parkinsons disease but can induce debilitating abnormal involuntary movements (dyskinesia). Here we show that the development of L-DOPA-induced dyskinesia in the rat is accompanied by upregulation of an inflammatory cascade involving nitric oxide. Male Wistar rats sustained unilateral injections of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle. After three weeks animals started to receive daily treatment with L-DOPA (30 mg/kg plus benserazide 7.5 mg/kg, for 21 days), combined with an inhibitor of neuronal NOS (7-nitroindazole, 7-NI, 30 mg/kg/day) or vehicle (saline-PEG 50%). All animals treated with L-DOPA and vehicle developed abnormal involuntary movements, and this effect was prevented by 7-NI. L-DOPA-treated dyskinetic animals exhibited an increased striatal and pallidal expression of glial fibrillary acidic protein (GFAP) in reactive astrocytes, an increased number of CD11b-positive microglial cells with activated morphology, and the rise of cells positive for inducible nitric oxide-synthase immunoreactivity (iNOS). All these indexes of glial activation were prevented by 7-NI co-administration. These findings provide evidence that the development of L-DOPA-induced dyskinesia in the rat is associated with activation of glial cells that promote inflammatory responses. The dramatic effect of 7-NI in preventing this glial response points to an involvement of nitric oxide. Moreover, the results suggest that the NOS inhibitor prevents dyskinesia at least in part via inhibition of glial cell activation and iNOS expression. Our observations indicate nitric oxide synthase inhibitors as a therapeutic strategy for preventing neuroinflammatory and glial components of dyskinesia pathogenesis in Parkinsons disease.

Nitric Oxide Synthase Inhibitor Improves De Novo and Long-Term l-DOPA-Induced Dyskinesia in Hemiparkinsonian Rats

Inhibitors of neuronal and endothelial nitric oxide synthase decrease l-3,4-dihidroxifenilalanine (l-DOPA)-induced dyskinesias in rodents. The mechanism of nitric oxide inhibitor action is unknown. The aims of the present study were to investigate the decrease of l-DOPA-induced abnormal involuntary movements (AIMs) in 6-hydroxydopamine (6-OHDA)-lesioned rats by nitric oxide inhibitors following either acute or chronic treatment. The primary findings of this study were that NG-nitro-l-Arginine, an inhibitor of endothelial and neuronal nitric oxide synthase, attenuated AIMs induced by chronic and acute l-DOPA. In contrast, rotational behavior was attenuated only after chronic l-DOPA. The 6-OHDA lesion and the l-DOPA treatment induced a bilateral increase (1.5 times) in the neuronal nitric oxide synthase (nNOS) protein and nNOS mRNA in the striatum and in the frontal cortex. There was a parallel increase, bilaterally, of the FosB/ΔFosB, primarily in the ipsilateral striatum. The exception was in the contralateral striatum and the ipsilateral frontal cortex, where chronic l-DOPA treatment induced an increase of approximately 10 times the nNOS mRNA. Our results provided further evidence of an anti-dyskinetic effect of NOS inhibitor. The effect appeared under l-DOPA acute and chronic treatment. The l-DOPA treatment also revealed an over-expression of the neuronal NOS in the frontal cortex and striatum. Our results corroborated findings that l-DOPA-induced rotation differs between acute and chronic treatment. The effect of the NOS inhibitor conceivably relied on the l-DOPA structural modifications in the Parkinsonian brain. Taken together, these data provided a rationale for further evaluation of NOS inhibitors in the treatment of l-DOPA-induced dyskinesia.

Doxycycline Suppresses Microglial Activation by Inhibiting the p38 MAPK and NF-kB Signaling Pathways

In neurodegenerative diseases, the inflammatory response is mediated by activated glial cells, mainly microglia, which are the resident immune cells of the central nervous system. Activated microglial cells release proinflammatory mediators and neurotoxic factors that are suspected to cause or exacerbate these diseases. We recently demonstrated that doxycycline protects substantia nigra dopaminergic neurons in an animal model of Parkinson’s disease. This effect was associated with a reduction of microglial cell activation, which suggests that doxycycline may operate primarily as an anti-inflammatory drug. In the present study, we assessed the anti-inflammatory potential of doxycycline using lipopolysaccharide (LPS)-activated primary microglial cells in culture as a model of neuroinflammation. Doxycycline attenuated the expression of key activation markers in LPS-treated microglial cultures in a concentration-dependent manner. More specifically, doxycycline treatment lowered the expression of the microglial activation marker IBA-1 as well as the production of ROS, NO, and proinflammatory cytokines (TNF-α and IL-1β). In primary microglial cells, we also found that doxycycline inhibits LPS-induced p38 MAP kinase phosphorylation and NF-kB nuclear translocation. The present results indicate that the effect of doxycycline on LPS-induced microglial activation probably occurs via the modulation of p38 MAP kinase and NF-kB signaling pathways. These results support the idea that doxycycline may be useful in preventing or slowing the progression of PD and other neurodegenerative diseases that exhibit altered glia function.

Investigations into Potential Extrasynaptic Communication between the Dopaminergic and Nitrergic Systems

Nitric oxide is unconstrained by cell membranes and can therefore act along a broad distance as a volume transmitter. Spillover of nitric oxide between neurons may have a major impact on central nervous system diseases and particularly on neurodegeneration. There is evidence whereby communication between nitrergic and dopaminergic systems plays an essential role in the control of the nigrostriatal pathway. However, there is sparse information for either the coexistence or overlap of nitric oxide and dopaminergic structures. The dual localization of immunoreactivity for nitric oxide synthase (NOS) and tyrosine hydroxylase, enzymes responsible for the synthesis of nitric oxide and dopamine, respectively, was examined in neurons of the nigrostriatal pathway in the rat brain by means of a double-immunohistochemical method and confocal laser scanning microscopy, acquired at the resolution limit. After perfusional fixation, the brains were cut and double-immunostained. A proximity analysis of tyrosine hydroxylase and NOS structures was done using binary masks generated from the respective maximum projections, using confocal laser microscopy. Unrevealed regions were determined somatodendritic positive for both NOS and tyrosine hydroxylase, within an image limit resolution at 2 μm-wide margin. The described interconnected localization of nNOS(+) and TH(+) containing neuronal fibers and cells bodies in the nigrostriatal pathway propose a close anatomical link between the two neurotransmitters.

Critical role of nitric oxide in the modulation of prepulse inhibition in Swiss mice

RationaleNitric oxide (NO) modulates the dopamine uptake and release processes and appears to be implicated in dopamine-related pathologies, such as schizophrenia. However, it is unclear whether there is excess or deficient NO synthesis in schizophrenia pathophysiology. Analyses of the intracellular pathways downstream of NO system activation have identified the cyclic nucleotide cyclic guanosine monophosphate (cGMP) as a possible target for drug development. Defects in the sensorimotor gating of the neural mechanism underlying the integration and processing of sensory information have been detected across species through prepulse inhibition (PPI).ObjectivesThe aim of this study was to investigate the effects of NO/cGMP increase on sensorimotor gating modulation during dopamine hyperfunction.MethodsMice were treated with NO donors and subjected to the PPI test. Treatment with the NO donor sodium nitroprusside was preceded by pretreatment with a soluble guanylate cyclase (sGC) inhibitor. Additionally, the mice were treated with NO donors and phosphodiesterases inhibitors prior to amphetamine treatment.ResultsPretreatment with the NO donors enhanced the PPI response and attenuated the amphetamine-disruptive effects on the PPI. The sGC inhibitor did not modify the sodium nitroprusside effects. Additionally, the cGMP increase induced by a specific phosphodiesterase inhibitor did not modify the amphetamine-disruptive effect.ConclusionsThis study provides the first demonstration that an increase in NO can improve the PPI response and block the amphetamine-disruptive effects on the PPI response. Our data are consistent with recent clinical results. However, these effects do not appear to be related to an increase in cGMP levels, and further investigation is thus required.

Are cyclooxygenase-2 and nitric oxide involved in the dyskinesia of Parkinson's disease induced by l-DOPA?

Inflammatory mechanisms are proposed to play a role in l-DOPA-induced dyskinesia. Cyclooxygenase-2 (COX2) contributes to inflammation pathways in the periphery and is constitutively expressed in the central nervous system. Considering that inhibition of nitric oxide (NO) formation attenuates l-DOPA-induced dyskinesia, this study aimed at investigating if a NO synthase (NOS) inhibitor would change COX2 brain expression in animals with l-DOPA-induced dyskinesia. To this aim, male Wistar rats received unilateral 6-hydroxydopamine microinjection into the medial forebrain bundle were treated daily with l-DOPA (21 days) combined with 7-nitroindazole or vehicle. All hemi-Parkinsonian rats receiving l-DOPA showed dyskinesia. They also presented increased neuronal COX2 immunoreactivity in the dopamine-depleted dorsal striatum that was directly correlated with dyskinesia severity. Striatal COX2 co-localized with choline-acetyltransferase, calbindin and DARPP-32 (dopamine-cAMP-regulated phosphoprotein-32), neuronal markers of GABAergic neurons. NOS inhibition prevented l-DOPA-induced dyskinesia and COX2 increased expression in the dorsal striatum. These results suggest that increased COX2 expression after l-DOPA long-term treatment in Parkinsonian-like rats could contribute to the development of dyskinesia.