Revistas
Revista:
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
ISSN:
1422-0067
Año:
2021
Vol.:
22
N°:
9
Págs.:
4825
Mutations in the GBA1 gene coding for glucocerebrosidase (GCase) are the main genetic risk factor for Parkinson's disease (PD). Indeed, identifying reduced GCase activity as a common feature underlying the typical neuropathological signatures of PD-even when considering idiopathic forms of PD-has recently paved the way for designing novel strategies focused on enhancing GCase activity to reduce alpha-synuclein burden and preventing dopaminergic cell death. Here we have performed bilateral injections of a viral vector coding for the mutated form of alpha-synuclein (rAAV9-SynA53T) for disease modeling purposes, both in mice as well as in nonhuman primates (NHPs), further inducing a progressive neuronal death in the substantia nigra pars compacta (SNpc). Next, another vector coding for the GBA1 gene (rAAV9-GBA1) was unilaterally delivered in the SNpc of mice and NHPs one month after the initial insult, together with the contralateral delivery of an empty/null rAAV9 for control purposes. Obtained results showed that GCase enhancement reduced alpha-synuclein burden, leading to improved survival of dopaminergic neurons. Data reported here support using GCase gene therapy as a disease-modifying treatment for PD and related synucleinopathies, including idiopathic forms of these disorders.
Autores:
Martínez-Pinilla, E. (Autor de correspondencia); Rico, Alberto José; Rivas-Santisteban, R.; et al.
Revista:
BRAIN STRUCTURE AND FUNCTION
ISSN:
1863-2653
Año:
2020
Vol.:
225
N°:
7
Págs.:
2153 - 2164
Endocannabinoids are neuromodulators acting on specific cannabinoid CB(1)and CB(2)G-protein-coupled receptors (GPCRs), representing potential therapeutic targets for neurodegenerative diseases. Cannabinoids also regulate the activity of GPR55, a recently "deorphanized" GPCR that directly interacts with CB(1)and with CB(2)receptors. Our hypothesis is that these heteromers may be taken as potential targets for Parkinson's disease (PD). This work aims at assessing the expression of heteromers made of GPR55 and CB1/CB(2)receptors in the striatum of control and parkinsonian macaques (with and without levodopa-induced dyskinesia). For this purpose, double blind in situ proximity ligation assays, enabling the detection of GPCR heteromers in tissue samples, were performed in striatal sections of control, MPTP-treated and MPTP-treated animals rendered dyskinetic by chronic treatment with levodopa. Image analysis and statistical assessment were performed using dedicated software. We have previously demonstrated the formation of heteromers between GPR55 and CB(1)receptor (CB1-GPR55_Hets), which is highly expressed in the central nervous system (CNS), but also with the CB(2)receptor (CB2-GPR55_Hets). Compared to the baseline expression of CB1-GPR55_Hets in control animals, our results showed increased expression levels in basal ganglia input nuclei of MPTP-treated animals. These observed increases in CB1-GPR55_Hets returned back to baseline levels upon chronic treatment with levodopa in dyskinetic animals. Obtained data regarding CB2-GPR55_Hets were quite similar, with somehow equivalent amounts in control and dyskinetic animals, and with increased expression levels in MPTP animals. Taken together, the detected increased expression of GPR55-endocannabinoid heteromers appoints these GPCR complexes as potential non-dopaminergic targets for PD therapy.
Revista:
ANNALS OF THE NEW YORK ACADEMY OF SCIENCES
ISSN:
0077-8923
Año:
2020
Vol.:
1475
N°:
1
Págs.:
34 - 42
The cannabinoid CB(1)receptor (CB1R) is the most abundant G protein-coupled receptor in the central nervous system, consistent with the important role of endocannabinoids as neuromodulators. Cannabinoids also modulate the function of G protein-coupled receptor 55 (GPR55), which forms heteroreceptor complexes with the CB1R in the striatum. The aim was to characterize cannabinoid CB1R-GPR55 heteromers (CB1R/GPR55Hets) in the basal ganglia input nuclei of nonhuman primates,Macaca fascicularis, both in projection neurons and interneurons, by thein situproximity ligation assay. Striatal projecting neurons were identified by the retrograde neuroanatomical tracer, biotinylated dextran amine (BDA), injected into external or internal subdivisions of the globus pallidus. Triple immunofluorescent stains were carried out to visualize (1) BDA-labeled neurons, (2) CB1R/GPR55Hets, and (3) striatal interneurons positive for choline acetyltransferase, parvalbumin, calretinin, or nitric oxide synthase. CB1R/GPR55Hets were identified within both types of projection neurons as well as all interneurons except those that are cholinergic. Moreover, CB1R/GPR55Hets were found specifically in the neuronal cell surface, and also in intracellular membranes. Further research efforts will be needed to confirm the intracellular occurrence of heteromers and their potential as therapeutic targets in diseases related to motor control imbalances, particularly within a parkinsonian context (with or without levodopa-induced dyskinesia).
Revista:
BRAIN STRUCTURE AND FUNCTION
ISSN:
1863-2653
Año:
2018
Vol.:
223
N°:
1
Págs.:
343 - 355
Glucocerebrosidase (GCase) is a lysosomal enzyme encoded by the GBA1 gene. Mutations in GBA1 gene lead to Gaucher¿s disease, the most prevalent lysosomal storage disorder. GBA1 mutations reduce GCase activity, therefore promoting the aggregation of alpha-synuclein, a common neuropathological finding underlying Parkinson¿s disease (PD) and dementia with Lewy bodies. However, it is also worth noting that a direct link between GBA1 mutations and alpha-synuclein aggregation indicating cause and effect is still lacking, with limited experimental evidence to date. Bearing in mind that a number of strategies increasing GCase expression for the treatment of PD are currently under development, here we sought to analyze the baseline expression of GCase in the brain of Macaca fascicularis, which has often been considered as the gold-standard animal model of PD. Although as with other lysosomal enzymes, GCase is expected to be ubiquitously expressed, here a number of regional variations have been consistently found, together with several specific neurochemical phenotypes expressing very high levels of GCase. In this regard, the most enriched expression of GCase was constantly found in cholinergic neurons from the nucleus basalis of Meynert, dopaminergic cells in the substantia nigra pars compacta, serotoninergic neurons from the raphe nuclei, as well as in noradrenergic neurons located in the locus ceruleus. Moreover, it is also worth noting that moderate levels of expression were also found in a number of areas within the paleocortex and archicortex, such as the entorhinal cortex and the hippocampal formation, respectively.
Autores:
Pignataro, J. D.; Sucunza, D.; Vanrell, L.; et al.
Revista:
FRONTIERS IN NEUROANATOMY
ISSN:
1662-5129
Año:
2017
Vol.:
11
Págs.:
2
Adeno-associated viruses (AAVs) have become highly promising tools for research and clinical applications in the central nervous system (CNS). However, specific delivery of genes to the cell type of interest is essential for the success of gene therapy and therefore a correct selection of the promoter plays a very important role. Here, AAV8 vectors carrying enhanced green fluorescent protein (eGFP) as reporter gene under the transcriptional control of different CNS-specific promoters were used and compared with a strong ubiquitous promoter. Since one of the main limitations of AAV-mediated gene delivery lies in its restricted cloning capacity, we focused our work on small-sized promoters. We tested the transduction efficacy and specificity of each vector after stereotactic injection into the mouse striatum. Three glia-specific AAV vectors were generated using two truncated forms of the human promoter for glial fibrillar acidic protein (GFAP) as well as a truncated form of the murine GFAP promoter. All three vectors resulted in predominantly glial expression; however we also observed eGFP expression in other cell-types such as oligodendrocytes, but never in neurons. In addition, robust and neuron-specific eGFP expression was observed using the minimal promoters for the neural protein BM88 and the neuronal nicotinic receptor ß2 (CHRNB2). In summary, we developed a set of AAV vectors designed for specific expression in cells of the CNS using minimal promoters to drive gene expression when the size of the therapeutic gene matters.
Revista:
BRAIN STRUCTURE AND FUNCTION
ISSN:
1863-2653
Año:
2017
Vol.:
222
N°:
4
Págs.:
1767 - 1784
Although it has long been widely accepted that dopamine receptor types D1 and D2 form GPCR heteromers in the striatum, the presence of D1¿D2 receptor heteromers has been recently challenged. In an attempt to properly characterize D1¿D2 receptor heteromers, here we have used the in situ proximity ligation assay (PLA) in striatal sections comprising the caudate nucleus, the putamen and the core and shell territories of the nucleus accumbens. Experiments were carried out in control macaques as well as in MPTP-treated animals (with and without dyskinesia). Obtained data support the presence of D1¿D2 receptor heteromers within all the striatal subdivisions, with the highest abundance in the accumbens shell. Dopamine depletion by MPTP resulted in an increase of D1¿D2 density in caudate and putamen which was normalized by levodopa treatment. Two different sizes of heteromers were consistently found, thus suggesting that besides individual heteromers, D1¿D2 receptor heteromers are sometimes organized in macromolecular complexes made of a number of D1¿D2 heteromers. Furthermore, the PLA technique was combined with different neuronal markers to properly characterize the identities of striatal neurons expressing D1¿D2 heteromers. We have found that striatal projection neurons giving rise to either the direct or the indirect basal ganglia pathways expressed D1¿D2 heteromers. Interestingly, macromolecular complexes of D1¿D2 heteromers were only found within cholinergic interneurons. In summary, here we provide overwhelming proof that D1 and D2 receptors form heteromeric complexes in the macaque striatum, thus representing a very appealing target for a number of brain diseases involving dopamine dysfunction.
Revista:
BRAIN STRUCTURE AND FUNCTION
ISSN:
1863-2653
Año:
2015
Vol.:
220
N°:
5
Págs.:
2721 - 2738
Although type 1 cannabinoid receptors (CB1Rs) are expressed abundantly throughout the brain, the presence of type 2 cannabinoid receptors (CB2Rs) in neurons is still somewhat controversial. Taking advantage of newly designed CB1R and CB2R mRNA riboprobes, we demonstrate by PCR and in situ hybridization that transcripts for both cannabinoid receptors are present within labeled pallidothalamic-projecting neurons of control and MPTP-treated macaques, whereas the expression is markedly reduced in dyskinetic animals. Moreover, an in situ proximity ligation assay was used to qualitatively assess the presence of CB1Rs and CB2Rs, as well as CB1R¿CB2R heteromers within basal ganglia output neurons in all animal groups (control, parkinsonian and dyskinetic macaques). A marked reduction in the number of CB1Rs, CB2Rs and CB1R¿CB2R heteromers was found in dyskinetic animals, mimicking the observed reduction in CB1R and CB2R mRNA expression levels. The fact that chronic levodopa treatment disrupted CB1R¿CB2R heteromeric complexes should be taken into consideration when designing new drugs acting on cannabinoid receptor heteromers.
Autores:
Farré, D.; Muñoz, A.; Moreno, E.; et al.
Revista:
MOLECULAR NEUROBIOLOGY
ISSN:
0893-7648
Año:
2015
Vol.:
52
N°:
3
Págs.:
1408 - 1420
Radioligand binding assays to rat striatal dopamine D1 receptors showed that brain lateralization of the dopaminergic system were not due to changes in expression but in agonist affinity. D1 receptor-mediated striatal imbalance resulted from a significantly higher agonist affinity in the left striatum. D1 receptors heteromerize with dopamine D3 receptors, which are considered therapeutic targets for dyskinesia in parkinsonian patients. Expression of both D3 and D1¿D3 receptor heteromers were increased in samples from 6-hydroxy-dopamine-hemilesioned rats rendered dyskinetic by treatment with 3, 4-dihydroxyphenyl-l-alanine (l-DOPA). Similar findings were obtained using striatal samples from primates. Radioligand binding studies in the presence of a D3 agonist led in dyskinetic, but not in lesioned or l-DOPA-treated rats, to a higher dopamine sensitivity. Upon D3-receptor activation, the affinity of agonists for binding to the right striatal D1 receptor increased. Excess dopamine coming from l-DOPA medication likely activates D3 receptors thus making right and left striatal D1 receptors equally responsive to dopamine. These results show that dyskinesia occurs concurrently with a right/left striatal balance in D1 receptor-mediated neurotransmission.
Revista:
EXPERIMENTAL NEUROLOGY
ISSN:
0014-4886
Año:
2014
Vol.:
261
Págs.:
44 - 52
Heteromerization of G-protein-coupled receptors is an important event as they integrate the actions of extracellular signals to give heteromer-selective ligand binding and signaling, opening new avenues in the development of potential drug targets in pharmacotherapy. The aim of the present paper was to check for cannabinoid CB1¿GPR55 receptor heteromers in the central nervous system (CNS), specifically in striatum. First, a direct interaction was demonstrated in cells transfected with the cDNA for the human version of the receptors, using bioluminescence resonance energy transfer and in situ proximity ligation assays (PLA). In the heterologous system, a biochemical fingerprint consisting on cross-antagonism in ERK1/2 phosphorylation was detected. The cross-antagonism was also observed on GPR55-mediated NFAT activation. Direct identification of GPR55 receptors in striatum is here demonstrated in rat brain slices using a specific agonist. Moreover, the heteromer fingerprint was identified in these rat slices by ERK1/2 phosphorylation assays whereas PLA assays showed results consistent with receptor¿receptor interactions in both caudate and putamen nuclei of a non-human primate. The results indicate not only that GPR55 is expressed in striatum but also that CB1 and GPR55 receptors form heteromers in this specific CNS region.
Revista:
FRONTIERS IN NEUROANATOMY
ISSN:
1662-5129
Calbindin (CB) is a calcium binding protein reported to protect dopaminergic neurons from degeneration. Although a direct link between CB content and differential vulnerability of dopaminergic neurons has long been accepted, factors other than CB have also been suggested, particularly those related to the dopamine transporter. Indeed, several studies have reported that CB levels are not causally related to the differential vulnerability of dopaminergic neurons against neurotoxins. Here we have used dual stains for tyrosine hydroxylase (TH) and CB in 3 control and 3 MPTP-treated monkeys to visualize dopaminergic neurons in the ventral tegmental area (VTA) and in the dorsal and ventral tiers of the substantia nigra pars compacta (SNcd and SNcv) co-expressing TH and CB. In control animals, the highest percentages of co-localization were found in VTA (58.2%), followed by neurons located in the SNcd (34.7%). As expected, SNcv neurons lacked CB expression. In MPTP-treated animals, the percentage of CB-ir/TH-ir neurons in the VTA was similar to control monkeys (62.1%), whereas most of the few surviving neurons in the SNcd were CB-ir/TH-ir (88.6%). Next, we have elucidated the presence of CB within identified nigrostriatal and nigroextrastriatal midbrain dopaminergic projection neurons. For this purpose, two control monkeys received one injection of Fluoro-Gold into the caudate nucleus and one injection of cholera toxin (CTB) into the postcommissural putamen, whereas two more monkeys were injected with CTB into the internal division of the globus pallidus (GPi). As expected, all the nigrocaudate- and nigroputamen-projecting neurons were TH-ir, although surprisingly, all of these nigrostriatal-projecting neurons were negative for CB. Furthermore, all the nigropallidal-projecting neurons co-expressed both TH and CB. In summary, although CB-ir dopaminergic neurons seem to be less prone to MPTP-induced degeneration, our data clearly demonstrated that these neurons are not giving rise to nigrostriatal projections and indeed CB-ir/TH-ir neurons only originate nigroextrastriatal projections.
Revista:
NEUROPHARMACOLOGY
ISSN:
0028-3908
Año:
2014
Vol.:
79
Págs.:
90 - 100
The molecular basis of priming for l-DOPA-induced dyskinesias in Parkinson's disease (PD), which depends on the indirect pathway of motor control, is not known. In rodents, the indirect pathway contains striatopallidal GABAergic neurons that express heterotrimers composed of A2A adenosine, CB1 cannabinoid and D2 dopamine receptors that regulate dopaminergic neurotransmission. The present study was designed to investigate the expression of these heteromers in the striatum of a primate model of Parkinson's disease and to determine whether their expression and pharmacological properties are altered upon l-DOPA treatment. By using the recently developed in situ proximity ligation assay and by identification of a biochemical fingerprint, we discovered a regional distribution of A2A/CB1/D2 receptor heteromers that predicts differential D2-mediated neurotransmission in the caudate¿putamen of Macaca fascicularis. Whereas heteromers were abundant in the caudate nucleus of both naïve and MPTP-treated monkeys, l-DOPA treatment blunted the biochemical fingerprint and led to weak heteromer expression. These findings constitute the first evidence of altered receptor heteromer expression in pathological conditions and suggest that drugs targeting A2A¿CB1¿D2 receptor heteromers may be successful to either normalize basal ganglia output or prevent L-DOPA-induced side effects.
Revista:
JOURNAL OF ALZHEIMERS DISEASE
ISSN:
1387-2877
Año:
2014
Vol.:
42
N°:
Suppl. 4
Págs.:
S561 - S573
Understanding the cellular and molecular processes involved in learning and memory will help in the development of safe and effective cognitive enhancers. The cAMP response element-binding (CREB) may be a universal modulator of processes required for memory formation, and increasing the levels of second messengers like cAMP and cGMP could ultimately lead to CREB activation. Phosphodiesterase (PDE) inhibitors regulate signaling pathways by elevating cAMP and/or cGMP levels, and they have been demonstrated to improve learning and memory in a number of rodent models of impaired cognition. The aim of this review is to summarize the outstanding progress that has been made in the application of PDE inhibitors for memory dysfunction. In addition, we have introduced some recent data we generated demonstrating that tadalafil could be considered as an optimal candidate for drug re-positioning and as a good candidate to enhance cognition.
Revista:
NEUROPHARMACOLOGY
ISSN:
0028-3908
Año:
2013
Vol.:
64
Págs.:
114 - 123
Previous studies have demonstrated that cognitive function can be restored in mouse models of Alzheimer's disease (AD) following administration of sildenafil, a specific PDE5 inhibitor (Puzzo et al., 2009; Cuadrado-Tejedor et al.). Another very potent PDE5 inhibitor with a longer half-life and safe in chronic treatments, tadalafil, may represent a better alternative candidate for AD therapy. However, tadalafil was proven unable to achieve similar benefits than those of sildenafil in AD animal models (Puzzo et al., 2009). The lack of efficacy was attributed to inability to cross the blood-brain barrier (BBB). In this paper we first measured the blood and brain levels of tadalafil to prove that the compound crosses BBB and that chronic treatment leads to accumulation in the brain of the J20 transgenic mouse model of AD. We demonstrated the presence of PDE5 mRNA in the brain of the mice and also in the human brain. After a 10 week treatment with either of these PDE5 inhibitors, the performance of the J20 mice in the Morris water maze test improved when compared with the transgenic mice that received vehicle. Biochemical analysis revealed that neither sildenafil nor tadalafil altered the amyloid burden, although both compounds reduced Tau phosphorylation in the mouse hippocampus. This study provides evidence of the potential benefits of a chronic tadalafil treatment in AD therapy. This article is part of a Special Issue entitled 'Cognitive Enhancers'.
Revista:
J PSYCHOPHARMACOL
ISSN:
0269-8811
Año:
2011
Vol.:
25
N°:
1
Págs.:
97 - 104
Revista:
BRAIN STRUCTURE AND FUNCTION
ISSN:
1863-2653
Año:
2011
Vol.:
216
N°:
4
Págs.:
319 - 330
The tegmental pedunculopontine nucleus (PPN) is a basal ganglia-related structure that has recently gained renewed interest as a potential surgical target for the treatment of several aspects of Parkinson's disease. However, the underlying anatomical substrates sustaining the choice of the PPN nucleus as a surgical candidate remain poorly understood. Here, we characterized the chemical phenotypes of different subtypes of PPN efferent neurons innervating the rat parafascicular (PF) nucleus. Emphasis was placed on elucidating the impact of unilateral nigrostriatal denervation on the expression patterns of the mRNA coding the vesicular glutamate transporter type 2 (vGlut2 mRNA). We found a bilateral projection from the PPN nucleus to the PF nucleus arising from cholinergic and glutamatergic efferent neurons, with a small fraction of projection neurons co-expressing both cholinergic and glutamatergic markers. Furthermore, the unilateral nigrostriatal depletion induced a bilateral twofold increase in the expression levels of vGlut2 mRNA within the PPN nucleus. Our results support the view that heterogeneous chemical profiles account for PPN efferent neurons innervating thalamic targets. Moreover, a bilateral enhancement of glutamatergic transmission arising from the PPN nucleus occurs following unilateral dopaminergic denervation, therefore sustaining the well-known hyperactivity of the PF nucleus in parkinsonian-like conditions. In conclusion, our data suggest that the ascending projections from the PPN that reach basal ganglia-related targets could play an important role in the pathophysiology of Parkinson's disease.
Revista:
BRAIN STRUCTURE AND FUNCTION
ISSN:
1863-2653
Año:
2011
Vol.:
216
N°:
4
Págs.:
371 - 386
GABAergic neurons within the internal division of the globus pallidus (GPi) are the main source of basal ganglia output reaching the thalamic ventral nuclei in monkeys. Following dopaminergic denervation, pallidothalamic-projecting neurons are known to be hyperactive, whereas a reduction in GPi activity is typically observed in lesioned animals showing levodopa-induced dyskinesia. Besides the mRNAs coding for GABAergic markers (GAD65 and GAD67), we show that all GPi neurons innervating thalamic targets also express transcripts for the isoforms 1 and 2 of the vesicular glutamate transporter (vGlut1 and vGlut2 mRNA). Indeed, dual immunofluorescent detection of GAD67 and vGlut1/2 confirmed the data gathered from in situ hybridization experiments, therefore demonstrating that the detected mRNAs are translated into the related proteins. Furthermore, the dopaminergic lesion resulted in an up-regulation of expression levels for both GAD65 and GAD67 mRNA within identified pallidothalamic-projecting neurons. This was coupled with a down-regulation of GAD65/67 mRNA expression levels in GPi neurons innervating thalamic targets in monkeys showing levodopa-induced dyskinesia. By contrast, the patterns of gene expression for both vGlut1 and vGlut2 mRNAs remained unchanged across GPi projection neurons in control, MPTP-treated and dyskinetic monkeys. In summary, both GABAergic and glutamatergic markers were co-expressed by GPi efferent neurons in primates. Although the status of the dopaminergic system directly modulates the expression levels of GAD65/67 mRNA, the observed expression of vGlut1/2 mRNA is not regulated by either dopaminergic removal or by continuous stimulation with dopaminergic agonists.
Revista:
Neurobiology of Disease
ISSN:
0969-9961
Año:
2010
Vol.:
39
N°:
3
Págs.:
381 - 392
Revista:
Journal of Neuroscience Methods
ISSN:
0165-0270
Año:
2010
Vol.:
194
N°:
1
Págs.:
28 - 33
Most of our current understanding of brain circuits is based on hodological studies carried out using neuroanatomical tract-tracing. Our aim is to advance one step further by visualizing the functional correlate in a given circuit. In this regard, we believe it is feasible to combine retrograde tracing with fluorescence, non-radioactive in situ hybridization (ISH) protocols. The subsequent detection at the single-cell level of the expression of a given mRNA within retrograde-labeled neurons provides information regarding cellular function. This may be of particular interest when trying to elucidate the performance of brain circuits of interest in animal models of brain diseases. Several combinations of retrograde tracing with either single- and double-ISH are presented here, together with some criteria that influence the selection of the tracer to be used in conjunction with the strong demands of the ISH.
Revista:
Journal of Neuroscience Methods
ISSN:
0165-0270
Año:
2010
Vol.:
194
N°:
1
Págs.:
21 - 27
Rabies virus (RV) has widely been used as a trans-synaptic retrograde tracer to analyze chains of connected neurons. The use of antibodies directed against the viral nucleoprotein enables viral nucleocapsids to be visualized within the cell soma, as well as within the thickest main dendrites. However, through this approach it is often difficult to accurately define post-synaptic elements (thin dendrites and/or dendritic spines). This limitation can now easily been circumvented by taking advantage of antibodies directed against a soluble viral phosphoprotein that spreads throughout the cytoplasm of the infected neuron, thereby producing Golgi-like immunofluorescent labeling of first-order projection neurons that are infected with RV. Furthermore, when combined with anterograde tracers such as Phaseolus vulgaris-leucoagglutinin (PHA-L) and biotinylated dextran amine (BDA), this procedure to detect RV facilitates the accurate visualization of both the pre- and post-synaptic elements. Finally, this method of viral detection is sufficiently sensitive to detect weakly labeled second-order neurons, which can then be further characterized neurochemically. Several examples are provided to illustrate why retrograde trans-synaptic tracing using RV can be regarded as an important breakthrough in the analysis of brain circuits, providing an unprecedented level of resolution.