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1.
Int J Mol Sci ; 22(17)2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34502063

RESUMO

α-synuclein is a small protein that is mainly expressed in the synaptic terminals of nervous tissue. Although its implication in neurodegeneration is well established, the physiological role of α-synuclein remains elusive. Given its involvement in the modulation of synaptic transmission and the emerging role of microtubules at the synapse, the current study aimed at investigating whether α-synuclein becomes involved with this cytoskeletal component at the presynapse. We first analyzed the expression of α-synuclein and its colocalization with α-tubulin in murine brain. Differences were found between cortical and striatal/midbrain areas, with substantia nigra pars compacta and corpus striatum showing the lowest levels of colocalization. Using a proximity ligation assay, we revealed the direct interaction of α-synuclein with α-tubulin in murine and in human brain. Finally, the previously unexplored interaction of the two proteins in vivo at the synapse was disclosed in murine striatal presynaptic boutons through multiple approaches, from confocal spinning disk to electron microscopy. Collectively, our data strongly suggest that the association with tubulin/microtubules might actually be an important physiological function for α-synuclein in the synapse, thus suggesting its potential role in a neuropathological context.


Assuntos
Corpo Estriado/metabolismo , Substância Negra/metabolismo , Sinapses/metabolismo , Tubulina (Proteína)/metabolismo , alfa-Sinucleína/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Corpo Estriado/ultraestrutura , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microtúbulos/metabolismo , Pessoa de Meia-Idade , Substância Negra/ultraestrutura , Sinapses/ultraestrutura
2.
Nat Commun ; 12(1): 5305, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489447

RESUMO

Mitochondrial dysfunction is a common hallmark of neurological disorders, and reducing mitochondrial damage is considered a promising neuroprotective therapeutic strategy. Here, we used high-throughput small molecule screening to identify CHIR99021 as a potent enhancer of mitochondrial function. CHIR99021 improved mitochondrial phenotypes and enhanced cell viability in several models of Huntington's disease (HD), a fatal inherited neurodegenerative disorder. Notably, CHIR99201 treatment reduced HD-associated neuropathology and behavioral defects in HD mice and improved mitochondrial function and cell survival in HD patient-derived neurons. Independent of its known inhibitory activity against glycogen synthase kinase 3 (GSK3), CHIR99021 treatment in HD models suppressed the proteasomal degradation of calpastatin (CAST), and subsequently inhibited calpain activation, a well-established effector of neural death, and Drp1, a driver of mitochondrial fragmentation. Our results established CAST-Drp1 as a druggable signaling axis in HD pathogenesis and highlighted CHIR99021 as a mitochondrial function enhancer and a potential lead for developing HD therapies.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Dinaminas/genética , Doença de Huntington/genética , Mitocôndrias/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Calpaína/genética , Calpaína/metabolismo , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Modelos Animais de Doenças , Dinaminas/metabolismo , Regulação da Expressão Gênica , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Doença de Huntington/tratamento farmacológico , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Injeções Intraperitoneais , Masculino , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Cultura Primária de Células , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Transdução de Sinais
3.
Int J Mol Sci ; 22(16)2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34445634

RESUMO

Cannabinoids have been reported as orexigenic, i.e., as promoting food intake that, among others, is controlled by the so-called "hunger" hormone, ghrelin. The aim of this paper was to look for functional and/or molecular interactions between ghrelin GHSR1a and cannabinoid CB2 receptors at the central nervous system (CNS) level. In a heterologous system we identified CB2-GHSR1a receptor complexes with a particular heteromer print consisting of impairment of CB2 receptor/Gi-mediated signaling. The blockade was due to allosteric interactions within the heteromeric complex as it was reverted by antagonists of the GHSR1a receptor. Cannabinoids acting on the CB2 receptor did not affect cytosolic increases of calcium ions induced by ghrelin acting on the GHSR1a receptor. In situ proximity ligation imaging assays confirmed the expression of CB2-GHSR1a receptor complexes in both heterologous cells and primary striatal neurons. We tested heteromer expression in neurons from offspring of high-fat-diet mouse mothers as they have more risk to be obese. Interestingly, there was a marked upregulation of those complexes in striatal neurons from siblings of pregnant female mice under a high-fat diet.


Assuntos
Corpo Estriado/patologia , Dieta Hiperlipídica/efeitos adversos , Grelina/metabolismo , Neurônios/patologia , Obesidade/patologia , Receptor CB2 de Canabinoide/metabolismo , Receptores de Grelina/metabolismo , Animais , Canabinoides/farmacologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Feminino , Grelina/genética , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Obesidade/etiologia , Obesidade/metabolismo , Receptor CB2 de Canabinoide/genética , Receptores de Grelina/genética , Transdução de Sinais , Regulação para Cima
4.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360984

RESUMO

Dopamine D1 receptor (D1R) function is regulated by membrane/lipid raft-resident protein caveolin-1 (Cav1). We examined whether altered expression of Cav1 in the dorsal striatum would affect self-administration of methamphetamine, an indirect agonist at the D1Rs. A lentiviral construct expressing Cav1 (LV-Cav1) or containing a short hairpin RNA against Cav1 (LV-shCav1) was used to overexpress or knock down Cav1 expression respectively, in the dorsal striatum. Under a fixed-ratio schedule, LV-Cav1 enhanced and LV-shCav1 reduced responding for methamphetamine in an extended access paradigm compared to LV-GFP controls. LV-Cav1 and LV-shCav1 also produced an upward and downward shift in a dose-response paradigm, generating a drug vulnerable/resistant phenotype. LV-Cav1 and LV-shCav1 did not alter responding for sucrose. Under a progressive-ratio schedule, LV-shCav1 generally reduced positive-reinforcing effects of methamphetamine and sucrose as seen by reduced breakpoints. Western blotting confirmed enhanced Cav1 expression in LV-Cav1 rats and reduced Cav1 expression in LV-shCav1 rats. Electrophysiological findings in LV-GFP rats demonstrated an absence of high-frequency stimulation (HFS)-induced long-term potentiation (LTP) in the dorsal striatum after extended access methamphetamine self-administration, indicating methamphetamine-induced occlusion of plasticity. LV-Cav1 prevented methamphetamine-induced plasticity via increasing phosphorylation of calcium calmodulin kinase II, suggesting a mechanism for addiction vulnerability. LV-shCav1 produced a marked deficit in the ability of HFS to produce LTP and, therefore, extended access methamphetamine was unable to alter striatal plasticity, indicating a mechanism for resistance to addiction-like behavior. Our results demonstrate that Cav1 expression and knockdown driven striatal plasticity assist with modulating addiction to drug and nondrug rewards, and inspire new strategies to reduce psychostimulant addiction.


Assuntos
Transtornos Relacionados ao Uso de Anfetaminas/metabolismo , Caveolina 1/metabolismo , Corpo Estriado/metabolismo , Potenciação de Longa Duração , Transtornos Relacionados ao Uso de Anfetaminas/genética , Transtornos Relacionados ao Uso de Anfetaminas/fisiopatologia , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Caveolina 1/genética , Corpo Estriado/efeitos dos fármacos , Masculino , Metanfetamina/toxicidade , Ratos , Ratos Long-Evans , Recompensa
5.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360969

RESUMO

Physical exercise reduces the extent, duration, and frequency of drug use in drug addicts during the drug initiation phase, as well as during prolonged addiction, withdrawal, and recurrence. However, information about exercise-induced neurobiological changes is limited. This study aimed to investigate the effects of forced moderate endurance exercise training on methamphetamine (METH)-induced behavior and the associated neurobiological changes. Male Sprague Dawley rats were subjected to the administration of METH (1 mg/kg/day, i.p.) and/or forced moderate endurance exercise (treadmill running, 21 m/min, 60 min/day) for 2 weeks. Over the two weeks, endurance exercise training significantly reduced METH-induced hyperactivity. METH and/or exercise treatment increased striatal dopamine (DA) levels, decreased p(Thr308)-Akt expression, and increased p(Tyr216)-GSK-3ß expression. However, the phosphorylation levels of Ser9-GSK-3ß were significantly increased in the exercise group. METH administration significantly increased the expression of NMDAr1, CaMKK2, MAPKs, and PP1 in the striatum, and exercise treatment significantly decreased the expression of these molecules. Therefore, it is apparent that endurance exercise inhibited the METH-induced hyperactivity due to the decrease in GSK-3ß activation by the regulation of the striatal glutamate signaling pathway.


Assuntos
Ácido Glutâmico/metabolismo , Metanfetamina/toxicidade , Condicionamento Físico Animal/métodos , Agitação Psicomotora/terapia , Corrida , Animais , Corpo Estriado/metabolismo , Dopamina/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Masculino , Proteínas Proto-Oncogênicas c-akt/metabolismo , Agitação Psicomotora/etiologia , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais
6.
Int J Mol Sci ; 22(15)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34360762

RESUMO

Peptide therapeutics offer numerous advantages in the treatment of diseases and disorders of the central nervous system (CNS). However, they are not without limitations, especially in terms of their pharmacokinetics where their metabolic lability and low blood-brain barrier penetration hinder their application. Targeted nanoparticle delivery systems are being tapped for their ability to improve the delivery of therapeutics into the brain non-invasively. We have developed a family of mannosylated glycoliposome delivery systems for targeted drug delivery applications. Herein, we demonstrate via in vivo distribution studies the potential of these glycoliposomes to improve the utility of CNS active therapeutics using dynantin, a potent and selective dynorphin peptide analogue antagonist of the kappa opioid receptor (KOR). Glycoliposomal entrapment protected dynantin against known rapid metabolic degradation and ultimately improved brain levels of the peptide by approximately 3-3.5-fold. Moreover, we linked this improved brain delivery with improved KOR antagonist activity by way of an approximately 30-40% positive modulation of striatal dopamine levels 20 min after intranasal administration. Overall, the results clearly highlight the potential of our glycoliposomes as a targeted delivery system for therapeutic agents of the CNS.


Assuntos
Dinorfinas , Peptídeos , Receptores Opioides kappa/antagonistas & inibidores , Corpo Estriado/metabolismo , Dopamina , Dinorfinas/química , Dinorfinas/farmacocinética , Dinorfinas/farmacologia , Humanos , Lipossomos , Peptídeos/química , Peptídeos/farmacocinética , Peptídeos/farmacologia
7.
Molecules ; 26(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34361780

RESUMO

Parkinson's disease is characterized by the loss of dopaminergic neurons in substantia nigra pars compacta (SNpc) and the resultant loss of dopamine in the striatum. Various studies have shown that oxidative stress and neuroinflammation plays a major role in PD progression. In addition, the autophagy lysosome pathway (ALP) plays an important role in the degradation of aggregated proteins, abnormal cytoplasmic organelles and proteins for intracellular homeostasis. Dysfunction of ALP results in the accumulation of α-synuclein and the loss of dopaminergic neurons in PD. Thus, modulating ALP is becoming an appealing therapeutic intervention. In our current study, we wanted to evaluate the neuroprotective potency of noscapine in a rotenone-induced PD rat model. Rats were administered rotenone injections (2.5 mg/kg, i.p.,) daily followed by noscapine (10 mg/kg, i.p.,) for four weeks. Noscapine, an iso-qinulinin alkaloid found naturally in the Papaveraceae family, has traditionally been used in the treatment of cancer, stroke and fibrosis. However, the neuroprotective potency of noscapine has not been analyzed. Our study showed that administration of noscapine decreased the upregulation of pro-inflammatory factors, oxidative stress, and α-synuclein expression with a significant increase in antioxidant enzymes. In addition, noscapine prevented rotenone-induced activation of microglia and astrocytes. These neuroprotective mechanisms resulted in a decrease in dopaminergic neuron loss in SNpc and neuronal fibers in the striatum. Further, noscapine administration enhanced the mTOR-mediated p70S6K pathway as well as inhibited apoptosis. In addition to these mechanisms, noscapine prevented a rotenone-mediated increase in lysosomal degradation, resulting in a decrease in α-synuclein aggregation. However, further studies are needed to further develop noscapine as a potential therapeutic candidate for PD treatment.


Assuntos
Autofagia/efeitos dos fármacos , Corpo Estriado/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Noscapina/farmacologia , Doença de Parkinson Secundária/tratamento farmacológico , Doença de Parkinson Secundária/genética , Parte Compacta da Substância Negra/efeitos dos fármacos , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Catalase/genética , Catalase/metabolismo , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Dopamina/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Masculino , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Estresse Oxidativo/efeitos dos fármacos , Doença de Parkinson Secundária/induzido quimicamente , Doença de Parkinson Secundária/patologia , Parte Compacta da Substância Negra/metabolismo , Parte Compacta da Substância Negra/patologia , Ratos , Ratos Wistar , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Rotenona/toxicidade , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , alfa-Sinucleína/antagonistas & inibidores , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
8.
Nat Commun ; 12(1): 4855, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381044

RESUMO

The vertebrate brain consists of diverse neuronal types, classified by distinct anatomy and function, along with divergent transcriptomes and proteomes. Defining the cell-type specific neuroproteomes is important for understanding the development and functional organization of neural circuits. This task remains challenging in complex tissue, due to suboptimal protein isolation techniques that often result in loss of cell-type specific information and incomplete capture of subcellular compartments. Here, we develop a genetically targeted proximity labeling approach to identify cell-type specific subcellular proteomes in the mouse brain, confirmed by imaging, electron microscopy, and mass spectrometry. We virally express subcellular-localized APEX2 to map the proteome of direct and indirect pathway spiny projection neurons in the striatum. The workflow provides sufficient depth to uncover changes in the proteome of striatal neurons following chemogenetic activation of Gαq-coupled signaling cascades. This method enables flexible, cell-type specific quantitative profiling of subcellular proteome snapshots in the mouse brain.


Assuntos
Ascorbato Peroxidases/metabolismo , Núcleo Celular/metabolismo , Corpo Estriado/metabolismo , Proteoma/metabolismo , Animais , Ascorbato Peroxidases/genética , Corpo Estriado/citologia , Citosol/metabolismo , Espectrometria de Massas , Camundongos , Vias Neurais , Neurônios/citologia , Neurônios/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Coloração e Rotulagem , Fluxo de Trabalho
9.
Nat Commun ; 12(1): 4775, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34362914

RESUMO

Dopamine controls diverse behaviors and their dysregulation contributes to many disorders. Our ability to understand and manipulate the function of dopamine is limited by the heterogenous nature of dopaminergic projections, the diversity of neurons that are regulated by dopamine, the varying distribution of the five dopamine receptors (DARs), and the complex dynamics of dopamine release. In order to improve our ability to specifically modulate distinct DARs, here we develop a photo-pharmacological strategy using a Membrane anchored Photoswitchable orthogonal remotely tethered agonist for the Dopamine receptor (MP-D). Our design selectively targets D1R/D5R receptor subtypes, most potently D1R (MP-D1ago), as shown in HEK293T cells. In vivo, we targeted dorsal striatal medium spiny neurons where the photo-activation of MP-D1ago increased movement initiation, although further work is required to assess the effects of MP-D1ago on neuronal function. Our method combines ligand and cell type-specificity with temporally precise and reversible activation of D1R to control specific aspects of movement. Our results provide a template for analyzing dopamine receptors.


Assuntos
Dopamina/metabolismo , Neurônios/metabolismo , Receptores Dopaminérgicos/química , Receptores Dopaminérgicos/metabolismo , Animais , Encéfalo/metabolismo , Corpo Estriado/metabolismo , Dopamina/química , Agonistas de Dopamina/farmacologia , Feminino , Células HEK293 , Humanos , Ligantes , Masculino , Camundongos , Receptores de Dopamina D1/química , Receptores de Dopamina D1/metabolismo , Transmissão Sináptica/fisiologia
10.
Eur J Neurosci ; 54(4): 5327-5340, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34273137

RESUMO

Dopamine D1 receptors play an important role in the effects of cocaine. Here, we investigated the role of neurons which express these receptors (D1-neurons) in the acute locomotor effects of cocaine and the locomotor sensitization observed after a second injection of this drug, using the previously established two-injection protocol of sensitization. We inhibited D1-neurons using double transgenic mice conditionally expressing the inhibitory Gi-coupled designer receptor exclusively activated by designer drugs (Gi-DREADD) in D1-neurons. Chemogenetic inhibition of D1-neurons by a low dose of clozapine (0.1 mg/kg) decreased the cocaine-induced expression of Fos in striatal neurons. It diminished the basal locomotor activity and acute hyper-locomotion induced by cocaine (20 mg/kg). Clozapine 0.1 mg/kg had no effect by itself and did not alter cocaine effects in wild-type mice. Inhibition of D1-neurons during the first cocaine administration prevented the sensitization of the locomotor response in response to a second cocaine administration 10 days later. On Day 11, inhibition of D1-neurons by clozapine stimulation of Gi-DREADD blocked cocaine-induced locomotion including in sensitized mice, whereas on Day 12, in the absence of clozapine and D1-neurons inhibition, all mice displayed a sensitized response to cocaine. These results show that chemogenetic inhibition of D1-neurons decreases spontaneous and cocaine-induced locomotor activity. It prevents sensitization induction and blocks sensitized locomotion in a two-injection protocol of sensitization but does not reverse established sensitization. Our study further supports the central role of D1-neurons in mediating the acute locomotor effects of cocaine and its sensitization.


Assuntos
Cocaína , Animais , Cocaína/farmacologia , Corpo Estriado/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Camundongos , Atividade Motora , Neurônios/metabolismo , Receptores de Dopamina D1/metabolismo
11.
Behav Neurosci ; 135(3): 369-379, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34264690

RESUMO

Ventral striatal dopamine is thought to be important for associative learning. Dopamine exerts its role via activation of dopamine D1 and D2 receptors in the ventral striatum. Upregulation of dopamine D2R in ventral striatopallidal neurons impairs incentive motivation via inhibiting synaptic transmission to the ventral pallidum. Here, we determined whether upregulation of D2Rs and the resulting impairment in ventral striatopallidal pathway function modulates associative learning in an auditory Pavlovian reward learning task as well as Go/No-Go learning in an operant based reward driven Go/No-Go task. We found that upregulation of D2Rs did not affect Pavlovian learning or the extinction of Pavlovian responses, and neither did it alter No-Go learning. We however observed a delay in the Go component of the task which may indicate a deficit in learning though it could also be attributed to the established locomotor hyperactivity of the mice. In combination with previously published findings, our data suggest that D2Rs in ventral striatopallidal neurons play a specific role in regulating motivation by balancing cost/benefit computations but do not necessarily affect associative learning. (PsycInfo Database Record (c) 2021 APA, all rights reserved).


Assuntos
Dopamina , Receptores de Dopamina D2 , Animais , Corpo Estriado/metabolismo , Aprendizagem , Camundongos , Neurônios/metabolismo , Receptores de Dopamina D1 , Receptores de Dopamina D2/metabolismo , Regulação para Cima
12.
Int J Mol Sci ; 22(14)2021 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-34299000

RESUMO

Parkinson's disease (PD) is the most common movement disorder, characterized by progressive degeneration of the nigrostriatal pathway, which consists of dopaminergic cell bodies in substantia nigra and their neuronal projections to the striatum. Moreover, PD is associated with an array of non-motor symptoms such as olfactory dysfunction, gastrointestinal dysfunction, impaired regulation of the sleep-wake cycle, anxiety, depression, and cognitive impairment. Inflammation and concomitant oxidative stress are crucial in the pathogenesis of PD. Thus, this study aimed to model PD via intrastriatal injection of the inflammagen lipopolysaccharide (LPS)to investigate if the lesion causes olfactory and motor impairments, inflammation, oxidative stress, and alteration in synaptic proteins in the olfactory bulb, striatum, and colon. Ten µg of LPS was injected unilaterally into the striatum of 27 male C57BL/6 mice, and behavioural assessment was conducted at 4 and 8 weeks post-treatment, followed by tissue collection. Intrastriatal LPS induced motor impairment in C57BL/6 mice at 8 weeks post-treatment evidenced by reduced latency time in the rotarod test. LPS also induced inflammation in the striatum characterized by increased expression of microglial marker Iba-1 and astrocytic marker GFAP, with degeneration of dopaminergic neuronal fibres (reduced tyrosine hydroxylase immunoreactivity), and reduction of synaptic proteins and DJ-1 protein. Additionally, intrastriatal LPS induced inflammation, oxidative stress and alterations in synaptic proteins within the olfactory bulb, although this did not induce a significant impairment in olfactory function. Intrastriatal LPS induced mild inflammatory changes in the distal colon, accompanied by increased protein expression of 3-nitrotyrosine-modified proteins. This model recapitulated the major features of PD such as motor impairment and degeneration of dopaminergic neuronal fibres in the striatum, as well as some pathological changes in the olfactory bulb and colon; thus, this model could be suitable for understanding clinical PD and testing neuroprotective strategies.


Assuntos
Astrócitos/metabolismo , Colo/metabolismo , Corpo Estriado/metabolismo , Neurônios Dopaminérgicos/metabolismo , Lipopolissacarídeos/metabolismo , Bulbo Olfatório/metabolismo , Doença de Parkinson/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Escala de Avaliação Comportamental , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Colo/patologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/patologia , Modelos Animais de Doenças , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/patologia , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Inflamação/metabolismo , Inflamação/patologia , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Microglia/metabolismo , Microglia/patologia , Bulbo Olfatório/efeitos dos fármacos , Bulbo Olfatório/patologia , Estresse Oxidativo/efeitos dos fármacos , Doença de Parkinson/patologia , Doença de Parkinson/psicologia , Proteína Desglicase DJ-1/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Substância Negra/metabolismo , Substância Negra/patologia , Tirosina 3-Mono-Oxigenase/metabolismo , alfa-Sinucleína/metabolismo
13.
Life Sci ; 282: 119822, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34271058

RESUMO

AIMS: The harmful cellular environment leads to brain damage, and each brain subregion exhibits a differential vulnerability to its effects. This study investigated the causes of selectively striatal cell loss in systemic 3-nitropropionic acid (3-NP) infused mice. MAIN METHODS: This study was performed in the neuronal cell line, primary neuron, cultured mouse brain, and mice brain tissues. The 3-NP solution was delivered using an osmotic mini-pump system for 7 days. ROS in brain tissue were detected and evaluated with the signals of CM-H2DCFDA for total cellular ROS and MitoSOX Red for mitochondrial ROS. Cellular ROS and the functional status of mitochondria were assessed with a detection kit and analyzed using flow cytometry. To quantify oxidative damaged DNA, apurinic/apyrimidinic (AP) site numbers in DNA were measured. The protein expression level was assessed using Western blotting, and immunohistochemistry was performed. Cleaved caspase-3 activities were measured by using an enzyme-linked immunosorbent assay (ELISA) kit. KEY FINDINGS: By 3-NP, mitochondrial dysfunction was higher in the striatum than in the cortex, and mitochondria-derived ROS levels were higher in the striatum than in the cortex. However, autophagy that may restore the energy depletion resulting from mitochondrial dysfunction occurred comparably less in the striatum than in the cortex. Inhibition of ASK1 by NQDI1 regulates MAPK signaling, apoptosis, and autophagy. Regulated autophagy of the cortex improved non-cell autonomously striatal damaged condition. SIGNIFICANCE: This study illustrated that the different vulnerabilities of the brain subregions, striatum or cortex, against 3-NP are rooted in different mitochondria-derived ROS amounts and autophagic capacity.


Assuntos
Autofagia/efeitos dos fármacos , Córtex Cerebral/metabolismo , Corpo Estriado/metabolismo , Nitrocompostos/toxicidade , Propionatos/toxicidade , Animais , Linhagem Celular , Córtex Cerebral/patologia , Corpo Estriado/patologia , Camundongos
14.
RNA ; 27(10): 1220-1240, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34244459

RESUMO

Metabotropic glutamate receptor 4 (mGlu4) is one of eight mGlu receptors within the Class C G protein-coupled receptor superfamily. mGlu4 is primarily localized to the presynaptic membrane of neurons where it functions as an auto and heteroreceptor controlling synaptic release of neurotransmitter. mGlu4 is implicated in numerous disorders and is a promising drug target; however, more remains to be understood about its regulation and pharmacology. Using high-throughput sequencing, we have validated and quantified an adenosine-to-inosine (A-to-I) RNA editing event that converts glutamine 124 to arginine in mGlu4; additionally, we have identified a rare but novel K129R site. Using an in vitro editing assay, we then validated the pre-mRNA duplex that allows for editing by ADAR enzymes and predicted its conservation across the mammalian species. Structural modeling of the mGlu4 protein predicts the Q124R substitution to occur in the B helix of the receptor that is critical for receptor dimerization and activation. Interestingly, editing of a receptor homodimer does not disrupt G protein activation in response to the endogenous agonist, glutamate. Using an assay designed to specifically measure heterodimer populations at the surface, however, we found that Q124R substitution decreased the propensity of mGlu4 to heterodimerize with mGlu2 and mGlu7 Our study is the first to extensively describe the extent and regulatory factors of RNA editing of mGlu4 mRNA transcripts. In addition, we have proposed a novel functional consequence of this editing event that provides insights regarding its effects in vivo and expands the regulatory capacity for mGlu receptors.


Assuntos
Edição de RNA , RNA Mensageiro/genética , Receptores de Glutamato Metabotrópico/genética , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Sequência de Aminoácidos , Animais , Pareamento de Bases , Sequência de Bases , Aves , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Corpo Estriado/citologia , Corpo Estriado/metabolismo , Células HEK293 , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Modelos Moleculares , Neurônios/citologia , Neurônios/metabolismo , Conformação de Ácido Nucleico , Mutação Puntual , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/metabolismo , Répteis , Homologia de Sequência de Aminoácidos
15.
Genes (Basel) ; 12(5)2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068799

RESUMO

White matter atrophy has been shown to precede the massive loss of striatal GABAergic neurons in Huntington's disease (HD). This study investigated the effects of in vivo expression of reprogramming factor octamer-binding transcription factor 4 (OCT4) on neural stem cell (NSC) niche activation in the subventricular zone (SVZ) and induction of cell fate specific to the microenvironment of HD. R6/2 mice randomly received adeno-associated virus 9 (AAV9)-OCT4, AAV9-Null, or phosphate-buffered saline into both lateral ventricles at 4 weeks of age. The AAV9-OCT4 group displayed significantly improved behavioral performance compared to the control groups. Following AAV9-OCT4 treatment, the number of newly generated NSCs and oligodendrocyte progenitor cells (OPCs) significantly increased in the SVZ, and the expression of OPC-related genes and glial cell-derived neurotrophic factor (GDNF) significantly increased. Further, amelioration of myelination deficits in the corpus callosum was observed through electron microscopy and magnetic resonance imaging, and striatal DARPP32+ GABAergic neurons significantly increased in the AAV9-OCT4 group. These results suggest that in situ expression of the reprogramming factor OCT4 in the SVZ induces OPC proliferation, thereby attenuating myelination deficits. Particularly, GDNF released by OPCs seems to induce striatal neuroprotection in HD, which explains the behavioral improvement in R6/2 mice overexpressing OCT4.


Assuntos
Doença de Huntington/genética , Bainha de Mielina/genética , Fator 3 de Transcrição de Octâmero/genética , Animais , Proliferação de Células/genética , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Feminino , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Humanos , Masculino , Camundongos , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/metabolismo , Neuroproteção/genética , Nicho de Células-Tronco/genética
16.
FASEB J ; 35(7): e21748, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34152016

RESUMO

Although adipose-derived human mesenchymal stem cell (hADSC) transplantation has recently emerged as a promising therapeutic modality for Parkinson's disease (PD), its underlying mechanism of action has not been fully elucidated. This study evaluated the therapeutic effects of stereotaxic injection of hADSCs in the striatum of the 6-OHDA-induced mouse model. Furthermore, an in vitro PD model was constructed using tissue-organized brain slices. The therapeutic effect was also evaluated using a co-culture of the hADSCs and 6-OHDA-treated brain slice. The analysis of hADSC exocrine proteins using RNA-sequencing, human protein cytokine arrays, and label-free quantitative proteomics identified key extracellular factors in the hADSC secretion environment. The degeneration and apoptosis of the dopaminergic neurons were measured in the PD samples in vivo and in vitro, and the beneficial effects were evaluated using quantitative reverse transcription-polymerase chain reaction, western blotting, Fluoro-Jade C, TUNEL assay, and immunofluorescence analysis. This study found that hADSCs protected the dopaminergic neurons in the in vivo and vitro models. We identified Pentraxin 3 (PTX3) as a key extracellular factor in the hADSC secretion environment. Moreover, we found that human recombinant PTX3 (rhPTX3) treatment could rescue the pathophysiological behavior of the PD mice in vivo, prevent dopaminergic neuronal death, and increase neuronal terminals in the ventral tegmental area + substantia nigra pars compacta and striatum in the PD brain slices in vitro. Furthermore, testing of the pro-apoptotic markers in the PD mouse brain following rhPTX3 treatment revealed that rhPTX3 can prevent apoptosis and degeneration of the dopaminergic neurons. This study discovered that PTX3, a hADSC-secreted protein, potentially protected the dopaminergic neurons against apoptosis and degeneration during PD progression and improved motor performance in PD mice, indicating the possible mechanism of action of hADSC replacement therapy for PD. Thus, our study discovered potential translational implications for the development of PTX3-based therapeutics for PD.


Assuntos
Tecido Adiposo/metabolismo , Apoptose/fisiologia , Proteína C-Reativa/metabolismo , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Doença de Parkinson/metabolismo , Componente Amiloide P Sérico/metabolismo , Animais , Morte Celular/fisiologia , Células Cultivadas , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL
17.
Medicine (Baltimore) ; 100(26): e26534, 2021 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-34190190

RESUMO

ABSTRACT: Many previous studies have estimated the rate of dopaminergic denervation in Parkinson disease (PD) via imaging studies. However, they lack the considerations of onset age, disease duration at onset, gender, and dopaminergic denervation due to normal aging. Herein, using a large prospective cohort, we estimated the rate of dopaminergic denervation in PD patients, compared with an age- and gender-matched normal control group.One hundred forty-one normal controls and 301 PD patients were enrolled. Striatal specific binding ratios (SBRs) of I-123 FP-CIT single positron emission tomography images were analyzed according to the age of onset, gender, and the duration of motor symptoms.In the PD group, symptom duration was significantly correlated with caudate SBRs, but with putamen SBRs (P  < .05, R2 = 0.02). Moreover, was significantly inversely related to caudate SBRs, but not with putamen SBRs (P  < .05, R2 = 0.02). Patients of different age onsets did not show any significant correlation between symptom durations and striatal SBRs. In the age-matched group, no significant relationship was observed between symptom duration and percent decrease of caudate SBRs, but there was a significant relationship between symptom duration and percent decrease of the putamen SBRs (P  < .01, R2 = 0.06). There was no significant relationship between the symptom duration and the percent decrease of striatal SBRs in the age- and gender-matched group.The significance and R2 values from the regression analysis between symptom duration, age, and dopaminergic denervation are low. This suggests that, contrary to previous knowledge, there is a relatively weak association between dopaminergic denervation and age or symptom duration.


Assuntos
Corpo Estriado , Diagnóstico por Imagem , Dopamina/metabolismo , Neurônios Dopaminérgicos , Degeneração Neural , Doença de Parkinson , Idade de Início , Biomarcadores/análise , Corpo Estriado/diagnóstico por imagem , Corpo Estriado/metabolismo , Diagnóstico por Imagem/classificação , Diagnóstico por Imagem/métodos , Diagnóstico por Imagem/estatística & dados numéricos , Progressão da Doença , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Degeneração Neural/diagnóstico , Degeneração Neural/fisiopatologia , Doença de Parkinson/diagnóstico , Doença de Parkinson/epidemiologia , Doença de Parkinson/metabolismo , Doença de Parkinson/fisiopatologia , Gravidade do Paciente , República da Coreia/epidemiologia , Avaliação de Sintomas/métodos , Tomografia Computadorizada de Emissão de Fóton Único/métodos
18.
Nutrients ; 13(5)2021 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068460

RESUMO

Parkinson's disease (PD) is a debilitating neurodegenerative disease, which progresses over time, causing pathological depigmentation of the substantia nigra (SN) in the midbrain due to loss of dopaminergic neurons. Emerging studies revealed the promising effects of some nutrient compounds in reducing the risk of PD. One such nutrient compound that possess neuroprotective effects and prevents neurodegeneration is tocotrienol (T3), a vitamin E family member. In the present study, a single dose intracisternal injection of 250 µg 6-hydroxydopamine (6-OHDA) was used to induce parkinsonism in male Sprague Dawley (SD) rats. Forty-eight hours post injection, the SD rats were orally supplemented with alpha (α)- and gamma (γ)-T3 for 28 days. The neuroprotective effects of α- and γ-T3 were evaluated using behavioural studies and immunohistochemistry (IHC). The findings from this study revealed that supplementation of α- and γ-T3 was able to ameliorate the motor deficits induced by 6-OHDA and improve the neuronal functions by reducing inflammation, reversing the neuronal degradation, and preventing further reduction of dopaminergic neurons in the SN and striatum (STR) fibre density.


Assuntos
Oxidopamina/toxicidade , Doença de Parkinson/tratamento farmacológico , Tocotrienóis/farmacologia , Animais , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Imuno-Histoquímica , Masculino , Fármacos Neuroprotetores/farmacologia , Doença de Parkinson/etiologia , Transtornos Parkinsonianos/tratamento farmacológico , Transtornos Parkinsonianos/etiologia , Ratos , Ratos Sprague-Dawley , Substância Negra/efeitos dos fármacos , Substância Negra/metabolismo
19.
Int J Mol Sci ; 22(10)2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-34065515

RESUMO

α-Synuclein (α-syn) is a hallmark amyloidogenic protein component of Lewy bodies in dopaminergic neurons affected by Parkinson's disease (PD). Despite the multi-faceted gene regulation of α-syn in the nucleus, the mechanism underlying α-syn crosstalk in chromatin remodeling in PD pathogenesis remains elusive. Here, we identified transcriptional adapter 2-alpha (TADA2a) as a novel binding partner of α-syn using the BioID system. TADA2a is a component of the p300/CBP-associated factor and is related to histone H3/H4 acetylation. We found that α-syn A53T was more preferentially localized in the nucleus than the α-syn wild-type (WT), leading to a stronger disturbance of TADA2a. Indeed, α-syn A53T significantly reduced the level of histone H3 acetylation in SH-SY5Y cells; its reduction was also evident in the striatum (STR) and substantia nigra (SN) of mice that were stereotaxically injected with α-syn preformed fibrils (PFFs). Interestingly, α-syn PFF injection resulted in a decrease in TADA2a in the STR and SN of α-syn PFF-injected mice. Furthermore, the levels of TADA2a and acetylated histone H3 were significantly decreased in the SN of patients with PD. Therefore, histone modification through α-syn A53T-TADA2a interaction may be associated with α-syn-mediated neurotoxicity in PD pathology.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Fatores de Transcrição/metabolismo , alfa-Sinucleína/metabolismo , Acetilação , Animais , Linhagem Celular Tumoral , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Humanos , Corpos de Lewy/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Degeneração Neural/metabolismo , Doença de Parkinson/metabolismo , Substância Negra/metabolismo
20.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34069782

RESUMO

Parkinson's disease (PD) is the second most common neurodegenerative disorder, mainly characterized by motor deficits correlated with progressive dopaminergic neuronal loss in the substantia nigra pars compacta (SN). Necroptosis is a caspase-independent form of regulated cell death mediated by the concerted action of receptor-interacting protein 3 (RIP3) and the pseudokinase mixed lineage domain-like protein (MLKL). It is also usually dependent on RIP1 kinase activity, influenced by further cellular clues. Importantly, necroptosis appears to be strongly linked to several neurodegenerative diseases, including PD. Here, we aimed at identifying novel chemical inhibitors of necroptosis in a PD-mimicking model, by conducting a two-step screening. Firstly, we phenotypically screened a library of 31 small molecules using a cellular model of necroptosis and, thereafter, the hit compound effect was validated in vivo in a sub-acute 1-methyl-1-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP) PD-related mouse model. From the initial compounds, we identified one hit-Oxa12-that strongly inhibited necroptosis induced by the pan-caspase inhibitor zVAD-fmk in the BV2 murine microglia cell line. More importantly, mice exposed to MPTP and further treated with Oxa12 showed protection against MPTP-induced dopaminergic neuronal loss in the SN and striatum. In conclusion, we identified Oxa12 as a hit compound that represents a new chemotype to tackle necroptosis. Oxa12 displays in vivo effects, making this compound a drug candidate for further optimization to attenuate PD pathogenesis.


Assuntos
Neurônios Dopaminérgicos/metabolismo , Necroptose/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Encéfalo/metabolismo , Morte Celular/efeitos dos fármacos , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Dopamina/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Proteínas Ativadoras de GTPase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Necroptose/fisiologia , Doenças Neurodegenerativas/metabolismo , Doença de Parkinson/metabolismo , Parte Compacta da Substância Negra/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Substância Negra/metabolismo
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