Oscar Solís

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.

L-DOPA Oppositely Regulates Synaptic Strength and Spine Morphology in D1 and D2 Striatal Projection Neurons in Dyskinesia

Dopamine depletion in Parkinsons disease (PD) produces dendritic spine loss in striatal medium spiny neurons (MSNs) and increases their excitability. However, the synaptic changes that occur in MSNs in PD, in particular those induced by chronic L-3,4-dihydroxyphenylalanine (L-DOPA) treatment, are still poorly understood. We exposed BAC-transgenic D1-tomato and D2-eGFP mice to PD and dyskinesia model paradigms, enabling cell type-specific assessment of changes in synaptic physiology and morphology. The distinct fluorescence markers allowed us to identify D1 and D2 MSNs for analysis using intracellular sharp electrode recordings, electron microscopy, and 3D reconstructions with single-cell Lucifer Yellow injections. Dopamine depletion induced spine pruning in both types of MSNs, affecting mushroom and thin spines equally. Dopamine depletion also increased firing rate in both D1- and D2-MSNs, but reduced evoked-EPSP amplitude selectively in D2-MSNs. L-DOPA treatment that produced dyskinesia differentially affected synaptic properties in D1- and D2-MSNs. In D1-MSNs, spine density remained reduced but the remaining spines were enlarged, with bigger heads and larger postsynaptic densities. These morphological changes were accompanied by facilitation of action potential firing triggered by synaptic inputs. In contrast, although L-DOPA restored the number of spines in D2-MSNs, it resulted in shortened postsynaptic densities. These changes in D2-MSNs correlated with a decrease in synaptic transmission. Our findings indicate that L-DOPA-induced dyskinesia is associated with abnormal spine morphology, modified synaptic transmission, and altered EPSP-spike coupling, with distinct effects in D1- and D2-MSNs.

Dopamine D3 Receptor Modulates l-DOPA-Induced Dyskinesia by Targeting D1 Receptor-Mediated Striatal Signaling

Abstract The dopamine D3 receptor (D3R) belongs to the dopamine D2‐like receptor family and is principally located in the ventral striatum. However, previous studies reported D3R overexpression in the dorsal striatum following l‐DOPA treatment in parkinsonian animals. This fact has drawn attention in the importance of D3R in l‐DOPA‐induced dyskinesia (LID). Here, we used D3R knockout mice to assess the role of D3R in LID and rotational sensitization in the hemiparkinsonian model. Mice lacking D3R presented a reduction in dyskinesia without interfering with the antiparkinsonian l‐DOPA effect and were accompanied by a reduction in the l‐DOPA‐induced rotations. Interestingly, deleting D3R attenuated important molecular markers in the D1R‐neurons such as FosB, extracellular signal‐regulated kinase, and histone‐3 (H3)‐activation. Colocalization studies in D1R‐tomato and D2R‐green fluorescent protein BAC‐transgenic mice indicated that l‐DOPA‐induced D3R overexpression principally occurs in D1R‐containing neurons although it is also present in the D2R‐neurons. Moreover, D3R pharmacological blockade with PG01037 reduced dyskinesia and the molecular markers expressed in D1R‐neurons. In addition, this antagonist further reduced dyskinetic symptoms in D1R heterozygous mice, indicating a direct interaction between D1R and D3R. Together, our results demonstrate that D3R modulates the development of dyskinesia by targeting D1R‐mediated intracellular signaling and suggest that decreasing D3R activity may help to ameliorate LID.

L-DOPA Reverses the Increased Free Amino Acids Tissue Levels Induced by Dopamine Depletion and Rises GABA and Tyrosine in the Striatum

Perturbations in the cerebral levels of various amino acids are associated with neurological disorders, and previous studies have suggested that such alterations have a role in the motor and non-motor symptoms of Parkinson’s disease. However, the direct effects of chronic L-DOPA treatment, that produces dyskinesia, on neural tissue amino acid concentrations have not been explored in detail. To evaluate whether striatal amino acid concentrations are altered in peak dose dyskinesia, 6-hydroxydopamine (6-OHDA)-lesioned hemiparkinsonian mice were treated chronically with L-DOPA and tissue amino acid concentrations were assessed by HPLC analysis. These experiments revealed that neither 6-OHDA nor L-DOPA treatment are able to alter glutamate in the striatum. However, glutamine increases after 6-OHDA and returns back to normal levels with L-DOPA treatment, suggesting increased striatal glutamatergic transmission with lack of dopamine. In addition, glycine and taurine levels are increased following dopamine denervation and restored to normal levels by L-DOPA. Interestingly, dyskinetic animals showed increased levels of GABA and tyrosine, while aspartate striatal tissue levels are not altered. Overall, our results indicate that chronic L-DOPA treatment, besides normalizing the altered levels of some amino acids after 6-OHDA, robustly increases striatal GABA and tyrosine levels which may in turn contribute to the development of L-DOPA-induced dyskinesia.

Nurr1 blocks the mitogenic effect of FGF-2 and EGF, inducing olfactory bulb neural stem cells to adopt dopaminergic and dopaminergic-GABAergic neuronal phenotypes.

The transcription factor Nurr1 is expressed in the mouse olfactory bulb (OB), although it remains unknown whether it influences the generation of dopaminergic neurons (DA) (DA neurons) in cells isolated from this brain region. We found that expressing Nurr1 in proliferating olfactory bulb stem cells (OBSCs) produces a marked inhibition of cell proliferation and the generation of immature neurons immunoreactive for tyrosine hydroxylase (TH) concomitant with marked upregulations of Th, Dat, Gad, and Fgfr2 transcripts. In long‐term cultures, these cells develop neurochemical and synaptic markers of mature‐like mesencephalic DA neurons, expressing GIRK2, VMAT2, DAT, calretinin, calbindin, synapsin‐I, and SV2. Concurring with the increase in both Th and Gad expression, a subpopulation of induced cells was both TH‐ and GAD‐immunoreactive indicating that they are dopaminergic‐GABAergic neurons. Indeed, these cells could mature to express VGAT, suggesting they can uptake and store GABA in vesicles. Remarkably, the dopamine D1 receptor agonist SKF‐38393 induced c‐Fos in TH+ cells and dopamine release was detected in these cultures under basal and KCl‐evoked conditions. By contrast, cotransducing the Neurogenin2 and Nurr1 transcription factors produced a significant decrease in the number of TH‐positive neurons. Our results indicate that Nurr1 overexpression in OBSCs induces the formation of two populations of mature dopaminergic neurons with features of the ventral mesencephalon or of the OB, capable of responding to functional dopaminergic stimuli and of releasing dopamine. They also suggest that the accumulation of Fgfr2 by Nurr1 in OBSCs may be involved in the generation of DA neurons.

Human COMT over-expression confers a heightened susceptibility to dyskinesia in mice

Catechol-O-methyltransferase (COMT) degrades dopamine and its precursor l-DOPA and plays a critical role in regulating synaptic dopamine actions. We investigated the effects of heightened levels of COMT on dopamine-regulated motor behaviors and molecular alterations in a mouse model of dyskinesia. Transgenic mice overexpressing human COMT (TG) and their wildtype (WT) littermates received unilateral 6-OHDA lesions in the dorsal striatum and were treated chronically with l-DOPA for two weeks. l-DOPA-induced dyskinesia was exacerbated in TG mice without altering l-DOPA motor efficacy as determined by contralateral rotations or motor coordination. Inductions of FosB and phospho-acetylated histone 3 (molecular correlates of dyskinesia) were potentiated in the lesioned striatum of TG mice compared with their WT littermates. The TG mice had lower basal levels of dopamine in the striatum. In mice with lesions, l-DOPA induces a greater increase in the dopamine metabolite 3-methoxytyramine in the lesioned striatum of dyskinetic TG mice than in WT mice. The levels of serotonin and its metabolite were similar in TG and WT mice. Our results demonstrate that human COMT overexpression confers a heightened susceptibility to l-DOPA-induced dyskinesia and alters molecular and neurochemical responses in the lesioned striatum of mice.

Role of Nurr1 in the Generation and Differentiation of Dopaminergic Neurons from Stem Cells

NURR1 is an essential transcription factor for the differentiation, maturation, and maintenance of midbrain dopaminergic neurons (DA neurons) as it has been demonstrated using knock-out mice. DA neurons of the substantia nigra pars compacta degenerate in Parkinson’s disease (PD) and mutations in the Nurr1 gene have been associated with this human disease. Thus, the study of NURR1 actions in vivo is fundamental to understand the mechanisms of neuron generation and degeneration in the dopaminergic system. Here, we present and discuss findings indicating that NURR1 is a valuable molecular tool for the in vitro generation of DA neurons which could be used for modeling and studying PD in cell culture and in transplantation approaches. Transduction of Nurr1 alone or in combination with other transcription factors such as Foxa2, Ngn2, Ascl1, and Pitx3, induces the generation of DA neurons, which upon transplantation have the capacity to survive and restore motor behavior in animal models of PD. We show that the survival of transplanted neurons is increased when the Nurr1-transduced olfactory bulb stem cells are treated with GDNF. The use of these and other factors with the induced pluripotent stem cell (iPSC)-based technology or the direct reprogramming of astrocytes or fibroblasts into human DA neurons has produced encouraging results for the study of the cellular and molecular mechanisms of neurodegeneration in PD and for the search of new treatments for this disease.

Dopamine receptors: homomeric and heteromeric complexes in l -DOPA-induced dyskinesia

The current standard treatment for Parkinson disease focuses on restoring striatal dopamine levels using l-3,4-dihydroxyphenylalanine (l-DOPA). However, disease progression and chronic treatment are associated with motor side effects such as l-DOPA-induced dyskinesia (LID). Dopamine receptor function is strongly associated with the mechanisms underlying LID. In fact, increased D1R signaling is associated with this motor side effect. Compelling evidence demonstrates that dopamine receptors in the striatum can form heteromeric complexes, and heteromerization can lead to changes in the functional and pharmacological properties of receptors compared to their monomeric subtypes. Currently, the most promising strategy for therapeutic intervention in dyskinesia originates from investigations of the D1R–D3R heteromers. Interestingly, there is a correlation between the expression of D1R–D3R heteromers and the development of LID. Moreover, D3R stimulation can potentiate the D1R signaling pathway. The aim of this review is to summarize current knowledge of the distinct roles of heteromeric dopaminergic receptor complexes in LID.

Genetic enhancement of Ras-ERK pathway does not aggravate L-DOPA-induced dyskinesia in mice but prevents the decrease induced by lovastatin

Increasing evidence supports a close relationship between Ras-ERK1/2 activation in the striatum and L-DOPA-induced dyskinesia (LID). ERK1/2 activation by L-DOPA takes place through the crosstalk between D1R/AC/PKA/DARPP-32 pathway and NMDA/Ras pathway. Compelling genetic and pharmacological evidence indicates that Ras-ERK1/2 inhibition prevents LID onset and may even revert already established dyskinetic symptoms. However, it is currently unclear whether exacerbation of Ras-ERK1/2 activity in the striatum may further aggravate dyskinesia in experimental animal models. Here we took advantage of two genetic models in which Ras-ERK1/2 signaling is hyperactivated, the Nf1+/− mice, in which the Ras inhibitor neurofibromin is reduced, and the Ras-GRF1 overexpressing (Ras-GRF1 OE) transgenic mice in which a specific neuronal activator of Ras is enhanced. Nf1+/− and Ras-GRF1 OE mice were unilaterally lesioned with 6-OHDA and treated with an escalating L-DOPA dosing regimen. In addition, a subset of Nf1+/− hemi-parkinsonian animals was also co-treated with the Ras inhibitor lovastatin. Our results revealed that Nf1+/− and Ras-GRF1 OE mice displayed similar dyskinetic symptoms to their wild-type counterparts. This observation was confirmed by the lack of differences between mutant and wild-type mice in striatal molecular changes associated to LID (i.e., FosB, and pERK1/2 expression). Interestingly, attenuation of Ras activity with lovastatin does not weaken dyskinetic symptoms in Nf1+/− mice. Altogether, these data suggest that ERK1/2-signaling activation in dyskinetic animals is maximal and does not require further genetic enhancement in the upstream Ras pathway. However, our data also demonstrate that such a genetic enhancement may reduce the efficacy of anti-dyskinetic drugs like lovastatin.

Morphological Plasticity in the Striatum Associated With Dopamine Dysfunction

The main entrance for information to the basal ganglia is the striatum. Striatal medium spiny neurons (MSNs) receive glutamate and dopamine inputs that are topographically organized on their dendritic spines. The configuration of the striatal synapses confers on dopamine a central role in regulating spine morphogenesis on MSNs. Dopamine can thus regulate the induction of long-term changes in the strength of corticostriatal synapses, which are implicated in learning and other behavioral plasticity. Indeed, changes in basal ganglia function in Parkinsons disease (PD) or drug addiction are associated with abnormal remodeling of the dendritic arbor of MSNs. In this chapter, we will summarize findings from patients and animal models of PD and drug addiction. We present new results obtained with state-of-the art technology, two-photon microscopy, and Lucifer yellow intracellular injections in identified striatal neurons, which allow the highest currently available resolution of single cell dendritic arbor reconstruction and spine morphology. Images reconstructed with this technique indicate a key role for dopamine in remodeling dendritic and spine morphology in MSNs.