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1.
Front Neuroanat ; 18: 1355940, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38601798

RESUMO

Introduction: The presence of a widespread cortical synucleinopathy is the main neuropathological hallmark underlying clinical entities such as Parkinson's disease with dementia (PDD) and dementia with Lewy bodies (DLB). There currently is a pressing need for the development of non-human primate (NHPs) models of PDD and DLB to further overcome existing limitations in drug discovery. Methods: Here we took advantage of a retrogradely-spreading adeno-associated viral vector serotype 9 coding for the alpha-synuclein A53T mutated gene (AAV9-SynA53T) to induce a widespread synucleinopathy of cortical and subcortical territories innervating the putamen. Four weeks post-AAV deliveries animals were sacrificed and a comprehensive biodistribution study was conducted, comprising the quantification of neurons expressing alpha-synuclein, rostrocaudal distribution and their specific location. Results: Intraputaminal deliveries of AAV9-SynA53T lead to a disseminated synucleinopathy throughout ipsi- and contralateral cerebral cortices, together with transduced neurons located in the ipsilateral caudal intralaminar nuclei and in the substantia nigra pars compacta (leading to thalamostriatal and nigrostriatal projections, respectively). Cortical afferent systems were found to be the main contributors to putaminal afferents (superior frontal and precentral gyri in particular). Discussion: Obtained data extends current models of synucleinopathies in NHPs, providing a reproducible platform enabling the adequate implementation of end-stage preclinical screening of new drugs targeting alpha-synuclein.

2.
Neurobiol Dis ; 188: 106341, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37918757

RESUMO

The antagonistic effect of adenosine on dopaminergic transmission in the basal ganglia indirect motor control pathway is mediated by dopamine D2 (D2R) and adenosine A2A (A2AR) receptors co-expressed on medium spiny striatal neurons. The pathway is unbalanced in Parkinson's disease (PD) and an A2AR blocker has been approved for use with levodopa in the therapy of the disease. However, it is not known whether the therapy is acting on individually expressed receptors or in receptors forming A2A-D2 receptor heteromers, whose functionality is unique. For two proteins prone to interact, a very recently developed technique, MolBoolean, allows to determine the number of proteins that are either non-interacting or interacting. After checking the feasibility of the technique and reliability of data in transfected cells and in striatal primary neurons, the Boolean analysis of receptors in the striatum of rats and monkeys showed a high percentage of D2 receptors interacting with the adenosine receptor, while, on the contrary, a significant proportion of A2A receptors do not interact with dopamine receptors. The number of interacting receptors increased when rats and monkeys were lesioned to become a PD model. The use of a tracer of the indirect pathway in monkeys confirmed that the data was restricted to the population of striatal neurons projecting to the GPe. The results are not only relevant for being the first study quantifying individual versus interacting G protein-coupled receptors, but also for showing that the D2R in these specific neurons, in both control and PD animals, is under the control of the A2AR. The tight adenosine/dopamine receptor coupling suggest benefits of early antiparkinsonian treatment with adenosine receptor blockers.


Assuntos
Doença de Parkinson , Ratos , Animais , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Dopamina/metabolismo , Neurônios Espinhosos Médios , Adenosina/metabolismo , Reprodutibilidade dos Testes , Corpo Estriado/metabolismo , Receptores Dopaminérgicos/metabolismo , Primatas/metabolismo , Receptores Purinérgicos P1/metabolismo , Receptor A2A de Adenosina/metabolismo , Receptores de Dopamina D1/metabolismo
3.
J Mol Med (Berl) ; 101(12): 1587-1601, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37819378

RESUMO

The SCN1A gene encodes the alpha subunit of a voltage-gated sodium channel (Nav1.1), which is essential for the function of inhibitory neurons in the brain. Mutations in this gene cause severe encephalopathies such as Dravet syndrome (DS). Upregulation of SCN1A expression by different approaches has demonstrated promising therapeutic effects in preclinical models of DS. Limiting the effect to inhibitory neurons may contribute to the restoration of brain homeostasis, increasing the safety and efficacy of the treatment. In this work, we have evaluated different approaches to obtain preferential expression of the full SCN1A cDNA (6 Kb) in GABAergic neurons, using high-capacity adenoviral vectors (HC-AdV). In order to favour infection of these cells, we considered ErbB4 as a surface target. Incorporation of the EGF-like domain from neuregulin 1 alpha (NRG1α) in the fiber of adenovirus capsid allowed preferential infection in cells lines expressing ErbB4. However, it had no impact on the infectivity of the vector in primary cultures or in vivo. For transcriptional control of transgene expression, we developed a regulatory sequence (DP3V) based on the Distal-less homolog enhancer (Dlx), the vesicular GABA transporter (VGAT) promoter, and a portion of the SCN1A gene. The hybrid DP3V promoter allowed preferential expression of transgenes in GABAergic neurons both in vitro and in vivo. A new HC-AdV expressing SCN1A under the control of this promoter showed improved survival and amelioration of the epileptic phenotype in a DS mouse model. These results increase the repertoire of gene therapy vectors for the treatment of DS and indicate a new avenue for the refinement of gene supplementation in this disease. KEY MESSAGES: Adenoviral vectors can deliver the SCN1A cDNA and are amenable for targeting. An adenoviral vector displaying an ErbB4 ligand in the capsid does not target GABAergic neurons. A hybrid promoter allows preferential expression of transgenes in GABAergic neurons. Preferential expression of SCN1A in GABAergic cells is therapeutic in a Dravet syndrome model.


Assuntos
Epilepsias Mioclônicas , Canal de Sódio Disparado por Voltagem NAV1.1 , Animais , Camundongos , Modelos Animais de Doenças , DNA Complementar , Epilepsias Mioclônicas/terapia , Epilepsias Mioclônicas/tratamento farmacológico , Neurônios GABAérgicos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Fenótipo
4.
Brain ; 146(12): 5000-5014, 2023 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-37769648

RESUMO

Although neuromelanin is a dark pigment characteristic of dopaminergic neurons in the human substantia nigra pars compacta, its potential role in the pathogenesis of Parkinson's disease (PD) has often been neglected since most commonly used laboratory animals lack neuromelanin. Here we took advantage of adeno-associated viral vectors encoding the human tyrosinase gene for triggering a time-dependent neuromelanin accumulation within substantia nigra pars compacta dopaminergic neurons in macaques up to similar levels of pigmentation as observed in elderly humans. Furthermore, neuromelanin accumulation induced an endogenous synucleinopathy mimicking intracellular inclusions typically observed in PD together with a progressive degeneration of neuromelanin-expressing dopaminergic neurons. Moreover, Lewy body-like intracellular inclusions were observed in cortical areas of the frontal lobe receiving dopaminergic innervation, supporting a circuit-specific anterograde spread of endogenous synucleinopathy by permissive trans-synaptic templating. In summary, the conducted strategy resulted in the development and characterization of a new macaque model of PD matching the known neuropathology of this disorder with unprecedented accuracy. Most importantly, evidence is provided showing that intracellular aggregation of endogenous α-synuclein is triggered by neuromelanin accumulation, therefore any therapeutic approach intended to decrease neuromelanin levels may provide appealing choices for the successful implementation of novel PD therapeutics.


Assuntos
Doença de Parkinson , Sinucleinopatias , Animais , Humanos , Idoso , Sinucleinopatias/patologia , Substância Negra/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Doença de Parkinson/patologia , Primatas/metabolismo
6.
Brain Behav Immun ; 108: 255-268, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36535607

RESUMO

The metabolic syndrome has been associated to chronic peripheral inflammation and related with neuroinflammation and neurodegeneration, including Parkinson's disease. However, the responsible mechanisms are unclear. Previous studies have involved the brain renin-angiotensin system in progression of Parkinson's disease and the angiotensin receptor type 1 (AT1) has been recently revealed as a major marker of dopaminergic vulnerability in humans. Dysregulation of tissue renin-angiotensin system is a key common mechanism for all major components of metabolic syndrome. Circulating AT1 agonistic autoantibodies have been observed in several inflammation-related peripheral processes, and activation of AT1 receptors of endothelial cells, dopaminergic neurons and glial cells have been observed to disrupt endothelial blood -brain barrier and induce neurodegeneration, respectively. Using a rat model, we observed that metabolic syndrome induces overactivity of nigral pro-inflammatory renin-angiotensin system axis, leading to increase in oxidative stress and neuroinflammation and enhancing dopaminergic neurodegeneration, which was inhibited by treatment with AT1 receptor blockers (ARBs). In rats, metabolic syndrome induced the increase in circulating levels of LIGHT and other major pro-inflammatory cytokines, and 27-hydroxycholesterol. Furthermore, the rats showed a significant increase in serum levels of proinflammatory AT1 and angiotensin converting enzyme 2 (ACE2) autoantibodies, which correlated with levels of several metabolic syndrome parameters. We also found AT1 and ACE2 autoantibodies in the CSF of these rats. Effects of circulating autoantibodies were confirmed by chronic infusion of AT1 autoantibodies, which induced blood-brain barrier disruption, an increase in the pro-inflammatory renin-angiotensin system activity in the substantia nigra and a significant enhancement in dopaminergic neuron death in two different rat models of Parkinson's disease. Observations in the rat models, were analyzed in a cohort of parkinsonian and non-parkinsonian patients with or without metabolic syndrome. Non-parkinsonian patients with metabolic syndrome showed significantly higher levels of AT1 autoantibodies than non-parkinsonian patients without metabolic syndrome. However, there was no significant difference between parkinsonian patients with metabolic syndrome or without metabolic syndrome, which showed higher levels of AT1 autoantibodies than non-parkinsonian controls. This is consistent with our recent studies, showing significant increase of AT1 and ACE2 autoantibodies in parkinsonian patients, which was related to dopaminergic degeneration and neuroinflammation. Altogether may lead to a vicious circle enhancing the progression of the disease that may be inhibited by strategies against production of these autoantibodies or AT1 receptor blockers (ARBs).


Assuntos
Síndrome Metabólica , Doença de Parkinson , Animais , Humanos , Ratos , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Antagonistas de Receptores de Angiotensina/metabolismo , Antagonistas de Receptores de Angiotensina/farmacologia , Enzima de Conversão de Angiotensina 2/metabolismo , Inibidores da Enzima Conversora de Angiotensina/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Autoanticorpos/metabolismo , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Células Endoteliais/metabolismo , Inflamação/metabolismo , Síndrome Metabólica/metabolismo , Doenças Neuroinflamatórias , Doença de Parkinson/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo
7.
Curr Res Neurobiol ; 4: 100064, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36582401

RESUMO

Future neuroscience and biomedical projects involving non-human primates (NHPs) remain essential in our endeavors to understand the complexities and functioning of the mammalian central nervous system. In so doing, the NHP neuroscience researcher must be allowed to incorporate state-of-the-art technologies, including the use of novel viral vectors, gene therapy and transgenic approaches to answer continuing and emerging research questions that can only be addressed in NHP research models. This perspective piece captures these emerging technologies and some specific research questions they can address. At the same time, we highlight some current caveats to global NHP research and collaborations including the lack of common ethical and regulatory frameworks for NHP research, the limitations involving animal transportation and exports, and the ongoing influence of activist groups opposed to NHP research.

8.
Life (Basel) ; 12(11)2022 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-36430993

RESUMO

Rare diseases, especially monogenic diseases, which usually affect a single target protein, have attracted growing interest in drug research by encouraging pharmaceutical companies to design and develop therapeutic products to be tested in the clinical arena. Acute intermittent porphyria (AIP) is one of these rare diseases. AIP is characterized by haploinsufficiency in the third enzyme of the heme biosynthesis pathway. Identification of the liver as the target organ and a detailed molecular characterization have enabled the development and approval of several therapies to manage this disease, such as glucose infusions, heme replenishment, and, more recently, an siRNA strategy that aims to down-regulate the key limiting enzyme of heme synthesis. Given the involvement of hepatic hemoproteins in essential metabolic functions, important questions regarding energy supply, antioxidant and detoxifying responses, and glucose homeostasis remain to be elucidated. This review reports recent insights into the pathogenesis of acute attacks and provides an update on emerging treatments aimed at increasing the activity of the deficient enzyme in the liver and restoring the physiological regulation of the pathway. While further studies are needed to optimize gene therapy vectors or large-scale production of liver-targeted PBGD proteins, effective protection of PBGD mRNA against the acute attacks has already been successfully confirmed in mice and large animals, and mRNA transfer technology is being tested in several clinical trials for metabolic diseases.

9.
Biomedicines ; 10(4)2022 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-35453499

RESUMO

It is without doubt that the gene therapy field is currently in the spotlight for the development of new therapeutics targeting unmet medical needs. Thus, considering the gene therapy scenario, neurological diseases in general and neurodegenerative disorders in particular are emerging as the most appealing choices for new therapeutic arrivals intended to slow down, stop, or even revert the natural progressive course that characterizes most of these devastating neurodegenerative processes. Since an extensive coverage of all available literature is not feasible in practical terms, here emphasis was made in providing some advice to beginners in the field with a narrow focus on elucidating the best delivery route available for fulfilling any given AAV-based therapeutic approach. Furthermore, it is worth nothing that the number of ongoing clinical trials is increasing at a breath-taking speed. Accordingly, a landscape view of preclinical and clinical initiatives is also provided here in an attempt to best illustrate what is ongoing in this quickly expanding field.

10.
Sci Transl Med ; 14(627): eabc0700, 2022 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-35020410

RESUMO

Correction of enzymatic deficits in hepatocytes by systemic administration of a recombinant protein is a desired therapeutic goal for hepatic enzymopenic disorders such as acute intermittent porphyria (AIP), an inherited porphobilinogen deaminase (PBGD) deficiency. Apolipoprotein A-I (ApoAI) is internalized into hepatocytes during the centripetal transport of cholesterol. Here, we generated a recombinant protein formed by linking ApoAI to the amino terminus of human PBGD (rhApoAI-PBGD) in an attempt to transfer PBGD into liver cells. In vivo experiments showed that, after intravenous injection, rhApoAI-PBGD circulates in blood incorporated into high-density lipoprotein (HDL), penetrates into hepatocytes, and crosses the blood-brain barrier, increasing PBGD activity in both the liver and brain. Consistently, the intravenous administration of rhApoAI-PBGD or the hyperfunctional rApoAI-PBGD-I129M/N340S (rApoAI-PBGDms) variant efficiently prevented and abrogated phenobarbital-induced acute attacks in a mouse model of AIP. One month after a single intravenous dose of rApoAI-PBGDms, the protein was still detectable in the liver, and hepatic PBGD activity remained increased above control values. A long-lasting therapeutic effect of rApoAI-PBGDms was observed after either intravenous or subcutaneous administration. These data describe a method to deliver PBGD to hepatocytes with resulting enhanced hepatic enzymatic activity and protection against AIP attacks in rodent models, suggesting that the approach might be an effective therapy for AIP.


Assuntos
Hidroximetilbilano Sintase , Porfiria Aguda Intermitente , Animais , Modelos Animais de Doenças , Terapia Genética/métodos , Hidroximetilbilano Sintase/metabolismo , Hidroximetilbilano Sintase/uso terapêutico , Camundongos , Porfiria Aguda Intermitente/tratamento farmacológico , Porfiria Aguda Intermitente/metabolismo
11.
Redox Biol ; 46: 102078, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34333284

RESUMO

ACE2 plays a pivotal role in the balance between the pro-oxidative pro-inflammatory and the anti-oxidative anti-inflammatory arms of the renin-angiotensin system. Furthermore, ACE2 is the entry receptor for SARS-CoV-2. Clarification of ACE2-related mechanisms is crucial for the understanding of COVID-19 and other oxidative stress and inflammation-related processes. In rat and monkey brain, we discovered that the intracellular ACE2 and its products Ang 1-7 and alamandine are highly concentrated in the mitochondria and bind to a new mitochondrial Mas-related receptor MrgE (MrgE) to produce nitric oxide. We found MrgE expressed in neurons and glia of rodents and primates in the substantia nigra and different brain regions. In the mitochondria, ACE2 and MrgE expressions decreased and NOX4 increased with aging. This new ACE2/MrgE/NO axis may play a major role in mitochondrial regulation of oxidative stress in neurons, and possibly other cells. Therefore, dysregulation of the mitochondrial ACE2/MrgE/NO axis may play a major role in neurodegenerative processes of dopaminergic neurons, where mitochondrial dysfunction and oxidative stress play a crucial role. Since ACE2 binds SARS-CoV-2 spike protein, the mitochondrial ACE2/MrgE/NO axis may also play a role in SARS-CoV-2 cellular effects.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Neurônios Dopaminérgicos/metabolismo , Mitocôndrias/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , COVID-19 , Humanos , Primatas , Ratos , Roedores , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus
12.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203739

RESUMO

It is without any doubt that precision medicine therapeutic strategies targeting neurodegenerative disorders are currently witnessing the spectacular rise of newly designed approaches based on the use of viral vectors as Trojan horses for the controlled release of a given genetic payload. Among the different types of viral vectors, adeno-associated viruses (AAVs) rank as the ones most commonly used for the purposes of either disease modeling or for therapeutic strategies. Here, we reviewed the current literature dealing with the use of AAVs within the field of Parkinson's disease with the aim to provide neuroscientists with the advice and background required when facing a choice on which AAV might be best suited for addressing a given experimental challenge. Accordingly, here we will be summarizing some insights on different AAV serotypes, and which would be the most appropriate AAV delivery route. Next, the use of AAVs for modeling synucleinopathies is highlighted, providing potential readers with a landscape view of ongoing pre-clinical and clinical initiatives pushing forward AAV-based therapeutic approaches for Parkinson's disease and related synucleinopathies.


Assuntos
Pesquisa Biomédica , Dependovirus/genética , Vetores Genéticos/uso terapêutico , Doença de Parkinson/genética , Animais , Modelos Animais de Doenças , Técnicas de Transferência de Genes , Humanos
13.
Biology (Basel) ; 10(6)2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-34073238

RESUMO

Glioblastoma multiforme, the most common type of malignant brain tumor as well as the most aggressive one, lacks an effective therapy. Glioblastoma presents overexpression of mesenchymal markers Snail, Slug, and N-Cadherin and of the autophagic marker p62. Glioblastoma cell lines also present increased autophagy, overexpression of mesenchymal markers, Shh pathway activation, and lack of primary cilia. In this study, we aimed to evaluate the role of HDAC6 in the pathogenesis of glioblastoma, as HDAC6 is the most overexpressed of all HDACs isoforms in this tumor. We treated glioblastoma cell lines with siHDAC6. HDAC6 silencing inhibited proliferation, migration, and clonogenicity of glioblastoma cell lines. They also reversed the mesenchymal phenotype, decreased autophagy, inhibited Shh pathway, and recovered the expression of primary cilia in glioblastoma cell lines. These results demonstrate that HDAC6 might be a good target for glioblastoma treatment.

14.
Int J Mol Sci ; 22(9)2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-34062940

RESUMO

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.


Assuntos
Neurônios Dopaminérgicos/metabolismo , Terapia Genética , Glucosilceramidase/genética , Doença de Parkinson/terapia , alfa-Sinucleína/genética , Animais , Dopamina/genética , Neurônios Dopaminérgicos/patologia , Vetores Genéticos/uso terapêutico , Humanos , Macaca/genética , Mesencéfalo/metabolismo , Mesencéfalo/patologia , Camundongos , Mutação/genética , Neuroproteção/genética , Doença de Parkinson/genética , Doença de Parkinson/patologia , Substância Negra/metabolismo , Substância Negra/patologia
15.
Hum Mol Genet ; 29(19): 3211-3223, 2020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-32916704

RESUMO

The morphological changes that occur in the central nervous system of patients with severe acute intermittent porphyria (AIP) have not yet been clearly established. The aim of this work was to analyze brain involvement in patients with severe AIP without epileptic seizures or clinical posterior reversible encephalopathy syndrome, as well as in a mouse model receiving or not liver-directed gene therapy aimed at correcting the metabolic disorder. We conducted neuroradiologic studies in 8 severely affected patients (6 women) and 16 gender- and age-matched controls. Seven patients showed significant enlargement of the cerebral ventricles and decreased brain perfusion was observed during the acute attack in two patients in whom perfusion imaging data were acquired. AIP mice exhibited reduced cerebral blood flow and developed chronic dilatation of the cerebral ventricles even in the presence of slightly increased porphyrin precursors. While repeated phenobarbital-induced attacks exacerbated ventricular dilation in AIP mice, correction of the metabolic defect using liver-directed gene therapy restored brain perfusion and afforded protection against ventricular enlargement. Histological studies revealed no signs of neuronal loss but a denser neurofilament pattern in the periventricular areas, suggesting compression probably caused by imbalance in cerebrospinal fluid dynamics. In conclusion, severely affected AIP patients exhibit cerebral ventricular enlargement. Liver-directed gene therapy protected against the morphological consequences of the disease seen in the brain of AIP mice. The observational study was registered at Clinicaltrial.gov as NCT02076763.


Assuntos
Encéfalo/patologia , Ventrículos Cerebrais/patologia , Modelos Animais de Doenças , Hidroximetilbilano Sintase/genética , Porfiria Aguda Intermitente/fisiopatologia , Adulto , Animais , Encéfalo/metabolismo , Estudos de Casos e Controles , Ventrículos Cerebrais/metabolismo , Ensaios Clínicos Fase I como Assunto , Feminino , Terapia Genética , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Porfiria Aguda Intermitente/genética , Porfiria Aguda Intermitente/metabolismo , Estudos Prospectivos
16.
Brain Struct Funct ; 225(7): 2153-2164, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32691218

RESUMO

Endocannabinoids are neuromodulators acting on specific cannabinoid CB1 and CB2 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 CB1 and with CB2 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/CB2 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 CB1 receptor (CB1-GPR55_Hets), which is highly expressed in the central nervous system (CNS), but also with the CB2 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.


Assuntos
Núcleo Caudado/metabolismo , Discinesias/metabolismo , Núcleo Accumbens/metabolismo , Transtornos Parkinsonianos/metabolismo , Putamen/metabolismo , Receptores de Canabinoides/metabolismo , Animais , Modelos Animais de Doenças , Macaca fascicularis , Masculino , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo
17.
Ann N Y Acad Sci ; 1475(1): 34-42, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32594556

RESUMO

The cannabinoid CB1 receptor (CB1 R) 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 CB1 R in the striatum. The aim was to characterize cannabinoid CB1 R-GPR55 heteromers (CB1 R/GPR55Hets) in the basal ganglia input nuclei of nonhuman primates, Macaca fascicularis, both in projection neurons and interneurons, by the in situ proximity 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) CB1 R/GPR55Hets, and (3) striatal interneurons positive for choline acetyltransferase, parvalbumin, calretinin, or nitric oxide synthase. CB1 R/GPR55Hets were identified within both types of projection neurons as well as all interneurons except those that are cholinergic. Moreover, CB1 R/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).


Assuntos
Corpo Estriado/metabolismo , Neurônios/metabolismo , Multimerização Proteica , Receptor CB1 de Canabinoide/metabolismo , Receptores de Canabinoides/metabolismo , Animais , Anticorpos/metabolismo , Biomarcadores/metabolismo , Interneurônios/metabolismo , Macaca fascicularis , Masculino
18.
Brain Struct Funct ; 225(4): 1193-1224, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32062721

RESUMO

Neuroanatomical tracing methods remain fundamental for elucidating the complexity of brain circuits. During the past decades, the technical arsenal at our disposal has been greatly enriched, with a steady supply of fresh arrivals. This paper provides a landscape view of classical and modern tools for tract-tracing purposes. Focus is placed on methods that have gone viral, i.e., became most widespread used and fully reliable. To keep an historical perspective, we start by reviewing one-dimensional, standalone transport-tracing tools; these including today's two most favorite anterograde neuroanatomical tracers such as Phaseolus vulgaris-leucoagglutinin and biotinylated dextran amine. Next, emphasis is placed on several classical tools widely used for retrograde neuroanatomical tracing purposes, where Fluoro-Gold in our opinion represents the best example. Furthermore, it is worth noting that multi-dimensional paradigms can be designed by combining different tracers or by applying a given tracer together with detecting one or more neurochemical substances, as illustrated here with several examples. Finally, it is without any doubt that we are currently witnessing the unstoppable and spectacular rise of modern molecular-genetic techniques based on the use of modified viruses as delivery vehicles for genetic material, therefore, pushing the tract-tracing field forward into a new era. In summary, here, we aim to provide neuroscientists with the advice and background required when facing a choice on which neuroanatomical tracer-or combination thereof-might be best suited for addressing a given experimental design.


Assuntos
Encéfalo/citologia , Técnicas de Rastreamento Neuroanatômico , Neurônios/citologia , Animais , Transporte Axonal , História do Século XX , História do Século XXI , Humanos , Processamento de Imagem Assistida por Computador , Vias Neurais/citologia , Técnicas de Rastreamento Neuroanatômico/história
19.
Mol Neurobiol ; 56(8): 5900-5910, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30687889

RESUMO

Cannabinoid CB1 receptors (CB1R) and the GPR55 receptor are expressed in striatum and are potential targets in the therapy of Parkinson's disease (PD), one of the most prevalent neurodegenerative diseases in developed countries. The aim of this paper was to address the potential of ligands acting on those receptors to prevent the action of a neurotoxic agent, MPP+, that specifically affects neurons of the substantia nigra due to uptake via the dopamine DAT transporter. The SH-SY5Y cell line model was used as it expresses DAT and, therefore, is able to uptake MPP+ that inhibits complex I of the respiratory mitochondrial chain and leads to cell death. Cells were transfected with cDNAs coding for either or both receptors. Receptors in cotransfected cells formed heteromers as indicated by the in situ proximity ligation assays. Cell viability was assayed by oxygen rate consumption and by the bromide-based MTT method. Assays of neuroprotection using two concentrations of MPP+ showed that cells expressing receptor heteromers were more resistant to the toxic effect. After correction by effects on cell proliferation, the CB1R antagonist, SR141716, afforded an almost full neuroprotection in CB1R-expressing cells even when a selective agonist, ACEA, was present. In contrast, SR141716 was not effective in cells expressing CB1/GPR55 heteromeric complexes. In addition, an agonist of GPR55, CID1792197, did not enhance neuroprotection in GPR55-expressing cells. These results show that neurons expressing heteromers are more resistant to cell death but question the real usefulness of CB1R, GPR55, and their heteromers as targets to afford PD-related neuroprotection.


Assuntos
Terapia de Alvo Molecular , Fármacos Neuroprotetores/uso terapêutico , Doença de Parkinson/tratamento farmacológico , Receptor CB1 de Canabinoide/antagonistas & inibidores , Linhagem Celular Tumoral , Humanos , Ligantes , Modelos Biológicos , Neuroproteção/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Consumo de Oxigênio/efeitos dos fármacos , Receptor CB1 de Canabinoide/metabolismo , Receptores de Canabinoides/metabolismo
20.
Mov Disord ; 34(1): 9-21, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30589955

RESUMO

Glucocerebrosidase is a lysosomal enzyme. The characterization of a direct link between mutations in the gene coding for glucocerebrosidase (GBA1) with the development of Parkinson's disease and dementia with Lewy bodies has heightened interest in this enzyme. Although the mechanisms through which glucocerebrosidase regulates the homeostasis of α-synuclein remains poorly understood, the identification of reduced glucocerebrosidase activity in the brains of patients with PD and dementia with Lewy bodies has paved the way for the development of novel therapeutic strategies directed at enhancing glucocerebrosidase activity and reducing α-synuclein burden, thereby slowing down or even preventing neuronal death. Here we reviewed the current literature relating to the mechanisms underlying the cross talk between glucocerebrosidase and α-synuclein, the GBA1 mutation-associated clinical phenotypes, and ongoing therapeutic approaches targeting glucocerebrosidase. © 2018 International Parkinson and Movement Disorder Society.


Assuntos
Glucosilceramidase/genética , Lisossomos/genética , Medicina de Precisão , Sinucleinopatias/genética , Doença de Gaucher/genética , Humanos , Lisossomos/metabolismo , Medicina de Precisão/métodos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
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