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1.
Nat Commun ; 14(1): 34, 2023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36596803

RESUMO

The γ-aminobutyric acid type B (GABAB) receptor is a prototypical family C G protein-coupled receptor (GPCR) that plays a key role in the regulation of synaptic transmission. Although growing evidence suggests that GPCR signaling in neurons might be highly organized in time and space, limited information is available about the mechanisms controlling the nanoscale organization of GABAB receptors and other GPCRs on the neuronal plasma membrane. Using a combination of biochemical assays in vitro, single-particle tracking, and super-resolution microscopy, we provide evidence that the spatial organization and diffusion of GABAB receptors on the plasma membrane are governed by dynamic interactions with filamin A, which tethers the receptors to sub-cortical actin filaments. We further show that GABAB receptors are located together with filamin A in small nanodomains in hippocampal neurons. These interactions are mediated by the first intracellular loop of the GABAB1 subunit and modulate the kinetics of Gαi protein activation in response to GABA stimulation.


Assuntos
Receptores de GABA-B , Receptores de GABA , Receptores de GABA/metabolismo , Filaminas , Receptores de GABA-B/metabolismo , Membrana Celular/metabolismo , Ácido gama-Aminobutírico/metabolismo
2.
J Hypertens ; 41(2): 233-245, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36583351

RESUMO

BACKGROUND: Hypothalamic paraventricular nucleus (PVN) is an important central site for the control of the adipose afferent reflex (AAR) that increases sympathetic outflow and blood pressure in obesity-related hypertension (OH). METHOD: In this study, we investigated the effects of nitric oxide (NO) and cardiovascular bioactive polypeptide adrenomedullin (ADM) in the PVN on AAR and sympathetic nerve activity (SNA) in OH rats induced by a high-fat diet. RESULTS: The results showed that ADM, total neuronal NO synthase (nNOS) and phosphorylated-nNOS protein expression levels in the PVN of the OH rats were down-regulated compared to the control rats. The enhanced AAR in OH rats was attenuated by PVN acute application of NO donor sodium nitroprusside (SNP), but was strengthened by the nNOS inhibitor nNOS-I, guanylyl cyclase inhibitor (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one, ODQ) and gamma-aminobutyric acid A type receptor (GABAA) antagonist Bicuculline. Moreover, PVN ADM microinjection not only decreased basal SNA but also attenuated the enhanced AAR in OH rats, which were effectively inhibited by ADM receptor antagonist ADM22-52, nNOS-I, ODQ or Bicuculline pretreatment. Bilateral PVN acute microinjection of ADM also caused greater increases in NO and cyclic guanosine monophosphate (cGMP) levels, and nNOS phosphorylation. Adeno-associated virus vectors encoding ADM (AAV-ADM) transfection in the PVN of OH rats not only decreased the elevated AAR, basal SNA and blood pressure (BP), but also increased the expression and activation of nNOS. Furthermore, AAV-ADM transfection improved vascular remodeling in OH rats. CONCLUSION: Taken together, our data highlight the roles of ADM in improving sympathetic overactivation, enhanced AAR and hypertension, and its related mechanisms associated with receptors mediated NO-cGMP-GABAA pathway in OH condition.


Assuntos
Hipertensão , Núcleo Hipotalâmico Paraventricular , Ratos , Animais , Núcleo Hipotalâmico Paraventricular/metabolismo , Adrenomedulina , Óxido Nítrico/metabolismo , Ratos Sprague-Dawley , Receptores de GABA/metabolismo , Bicuculina/metabolismo , Bicuculina/farmacologia , Obesidade/complicações , Reflexo/fisiologia , Pressão Sanguínea/fisiologia , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo I/farmacologia , Ácido gama-Aminobutírico/metabolismo , Ácido gama-Aminobutírico/farmacologia , Sistema Nervoso Simpático
3.
ACS Chem Neurosci ; 14(1): 111-118, 2023 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-36535632

RESUMO

Radioligands targeting microglia cells have been developed to identify and determine neuroinflammation in the living brain. One recently discovered ligand is JNJ-64413739 that binds selectively to the purinergic receptor P2X7R. The expression of P2X7R is increased under inflammation; hence, the ligand is considered useful in the detection of neuroinflammation in the brain. [18F]JNJ-64413739 has been evaluated in healthy subjects with positron emission tomography; however, the in vitro binding properties of the ligand in human brain tissue have not been investigated. Therefore, the purpose of this study was to measure Bmax and Kd of [3H]JNJ-64413739 using autoradiography on human cortical tissue sections resected from a total of 48 patients with treatment-resistant epilepsy. Correlations between the specific binding of [3H]JNJ-64413739 with age, sex, and duration of disease were explored. Finally, to examine the relationship between P2X7R and TSPO availability, specific binding of [3H]JNJ-64413739 and [123I]CLINDE was examined in the same tissue. The binding was measured in both cortical gray and subcortical white matter. Saturation revealed a Kd (5 nM) value similar between gray and white matter but a larger Bmax in the white than in the gray matter. The binding was completely displaced by the cold ligand and structurally different P2X7R ligands. The variability in saturable binding among the samples was found to be 38% in gray and white matter but was not correlated to either age, sex, or the duration of the disease. Interestingly, there was no significant correlation between [3H]JNJ-64413739 and [123I]CLINDE binding. These data demonstrate that [3H]JNJ-64413739 is a suitable radioligand for evaluating the distribution and expression of the P2X7R in the human brain.


Assuntos
Doenças Neuroinflamatórias , Receptores Purinérgicos P2X7 , Humanos , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Ligantes , Tomografia por Emissão de Pósitrons/métodos , Receptores de GABA/metabolismo , Peptídeos , Compostos Radiofarmacêuticos , Trítio
4.
Sci Adv ; 8(51): eadc9236, 2022 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-36542715

RESUMO

Aging causes functional decline and degeneration of neurons and is a major risk factor of neurodegenerative diseases. To investigate the molecular mechanisms underlying neuronal aging, we developed a new pipeline for neuronal proteomic profiling in young and aged animals. While the overall translational machinery is down-regulated, certain proteins increase expressions upon aging. Among these aging-up-regulated proteins, the conserved channel protein TMC-1/Tmc has an anti-aging function in all neurons tested, and the neuroprotective function of TMC-1 occurs by regulating GABA signaling. Moreover, our results show that metabotropic GABA receptors and G protein GOA-1/Goα are required for the anti-neuronal aging functions of TMC-1 and GABA, and the activation of GABA receptors prevents neuronal aging by inhibiting the PLCß-PKC pathway. Last, we show that the TMC-1-GABA-PKC signaling axis suppresses neuronal functional decline caused by a pathogenic form of human Tau protein. Together, our findings reveal the neuroprotective function of the TMC-1-GABA-PKC signaling axis in aging and disease conditions.


Assuntos
Caenorhabditis elegans , Proteômica , Animais , Humanos , Idoso , Caenorhabditis elegans/metabolismo , Receptores de GABA/metabolismo , Ácido gama-Aminobutírico , Envelhecimento , Canais Iônicos/metabolismo
5.
Cells ; 11(23)2022 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-36497118

RESUMO

Numerous studies recently showed that the inhibitory neurotransmitter, γ-aminobutyric acid (GABA), can stimulate cerebral angiogenesis and promote neurovascular coupling by activating the ionotropic GABAA receptors on cerebrovascular endothelial cells, whereas the endothelial role of the metabotropic GABAB receptors is still unknown. Preliminary evidence showed that GABAA receptor stimulation can induce an increase in endothelial Ca2+ levels, but the underlying signaling pathway remains to be fully unraveled. In the present investigation, we found that GABA evoked a biphasic elevation in [Ca2+]i that was initiated by inositol-1,4,5-trisphosphate- and nicotinic acid adenine dinucleotide phosphate-dependent Ca2+ release from neutral and acidic Ca2+ stores, respectively, and sustained by store-operated Ca2+ entry. GABAA and GABAB receptors were both required to trigger the endothelial Ca2+ response. Unexpectedly, we found that the GABAA receptors signal in a flux-independent manner via the metabotropic GABAB receptors. Likewise, the full Ca2+ response to GABAB receptors requires functional GABAA receptors. This study, therefore, sheds novel light on the molecular mechanisms by which GABA controls endothelial signaling at the neurovascular unit.


Assuntos
Células Endoteliais , Receptores de GABA , Humanos , Receptores de GABA/metabolismo , Células Endoteliais/metabolismo , Ácido gama-Aminobutírico/farmacologia , Ácido gama-Aminobutírico/metabolismo , Encéfalo/metabolismo
6.
Molecules ; 27(21)2022 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-36363979

RESUMO

Scientific evidence suggests that quercetin (QUR) has anxiolytic-like effects in experimental animals. However, the mechanism of action responsible for its anxiolytic-like effects is yet to be discovered. The goal of this research is to assess QUR's anxiolytic effects in mouse models to explicate the possible mechanism of action. After acute intraperitoneal (i.p.) treatment with QUR at a dose of 50 mg/kg (i.p.), behavioral models of open-field, hole board, swing box, and light-dark tests were performed. QUR was combined with a GABAergic agonist (diazepam) and/or antagonist (flumazenil) group. Furthermore, in silico analysis was also conducted to observe the interaction of QUR and GABA (α5), GABA (ß1), and GABA (ß2) receptors. In the experimental animal model, QUR had an anxiolytic-like effect. QUR, when combined with diazepam (2 mg/kg, i.p.), drastically potentiated an anxiolytic effect of diazepam. QUR is a more highly competitive ligand for the benzodiazepine recognition site that can displace flumazenil (2.5 mg/kg, i.p.). In all the test models, QUR acted similar to diazepam, with enhanced effects of the standard anxiolytic drug, which were reversed by pre-treatment with flumazenil. QUR showed the best interaction with the GABA (α5) receptor compared to the GABA (ß1) and GABA (ß2) receptors. In conclusion, QUR may exert an anxiolytic-like effect on mice, probably through the GABA-receptor-interacting pathway.


Assuntos
Ansiolíticos , Camundongos , Animais , Ansiolíticos/farmacologia , Flumazenil/farmacologia , Quercetina/farmacologia , Moduladores GABAérgicos/farmacologia , Receptores de GABA/metabolismo , Receptores de GABA-A/metabolismo , Aprendizagem em Labirinto , Diazepam/farmacologia , Ácido gama-Aminobutírico/farmacologia , Ansiedade/tratamento farmacológico , Comportamento Animal
7.
FASEB J ; 36(12): e22637, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36349989

RESUMO

The mitochondrial translocator protein (18 kDa; TSPO) is a high-affinity cholesterol-binding protein that is an integral component of the cholesterol trafficking scaffold responsible for determining the rate of cholesterol import into the mitochondria for steroid biosynthesis. Previous studies have shown that TSPO declines in aging Leydig cells (LCs) and that its decline is associated with depressed circulating testosterone levels in aging rats. However, TSPO's role in the mechanistic decline in LC function is not fully understood. To address the role of TSPO depletion in LC function, we first examined mitochondrial quality in Tspo knockout mouse tumor MA-10 nG1 LCs compared to wild-type MA-10 cells. Tspo deletion caused a disruption in mitochondrial function and membrane dynamics. Increasing mitochondrial fusion via treatment with the mitochondrial fusion promoter M1 or by optic atrophy 1 (OPA1) overexpression resulted in the restoration of mitochondrial function and mitochondrial morphology as well as in steroid formation in TSPO-depleted nG1 LCs. LCs isolated from aged rats form less testosterone than LCs isolated from young rats. Treatment of aging LCs with M1 improved mitochondrial function and increased androgen formation, suggesting that aging LC dysfunction may stem from compromised mitochondrial dynamics caused by the age-dependent LC TSPO decline. These results, taken together, suggest that maintaining or enhancing mitochondrial fusion may provide therapeutic strategies to maintain or restore testosterone levels with aging.


Assuntos
Células Intersticiais do Testículo , Dinâmica Mitocondrial , Camundongos , Masculino , Ratos , Animais , Células Intersticiais do Testículo/metabolismo , Receptores de GABA/genética , Receptores de GABA/metabolismo , Proteínas Mitocondriais/metabolismo , Colesterol/metabolismo , Testosterona/metabolismo
8.
Int J Mol Sci ; 23(20)2022 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-36293329

RESUMO

Diffuse intrinsic pontine gliomas (DIPG), the first cause of cerebral pediatric cancer death, will greatly benefit from specific and non-invasive biomarkers for patient follow-up and monitoring of drug efficacy. Since biopsies are challenging for brain tumors, molecular imaging may be a technique of choice to target and follow tumor evolution. So far, MR remains the imaging technique of reference for DIPG, although it often fails to define the extent of tumors, an essential parameter for therapeutic efficacy assessment. Thanks to its high sensitivity, positron emission tomography (PET) offers a unique way to target specific biomarkers in vivo. We demonstrated in a patient-derived orthotopic xenograft (PDOX) model in the rat that the translocator protein of 18 kDa (TSPO) may be a promising biomarker for monitoring DIPG tumors. We studied the distribution of 18F-DPA-714, a TSPO radioligand, in rats inoculated with HSJD-DIPG-007 cells. The primary DIPG human cell line HSJD-DIPG-007 highly represents this pediatric tumor, displaying the most prevalent DIPG mutations, H3F3A (K27M) and ACVR1 (R206H). Kinetic modeling and parametric imaging using the brain 18F-DPA-714 PET data enabled specific delineation of the DIPG tumor area, which is crucial for radiotherapy dose management.


Assuntos
Astrocitoma , Neoplasias do Tronco Encefálico , Glioma Pontino Intrínseco Difuso , Glioma , Criança , Animais , Humanos , Ratos , Glioma/diagnóstico por imagem , Glioma/genética , Glioma/metabolismo , Linhagem Celular Tumoral , Neoplasias do Tronco Encefálico/diagnóstico por imagem , Neoplasias do Tronco Encefálico/genética , Tomografia por Emissão de Pósitrons/métodos , Proteínas de Transporte , Modelos Animais de Doenças , Biomarcadores , Receptores de GABA/genética , Receptores de GABA/metabolismo , Receptores de GABA-A
9.
Signal Transduct Target Ther ; 7(1): 340, 2022 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-36184627

RESUMO

Maintaining a proper balance between the glutamate receptor-mediated neuronal excitation and the A type of GABA receptor (GABAAR) mediated inhibition is essential for brain functioning; and its imbalance contributes to the pathogenesis of many brain disorders including neurodegenerative diseases and mental illnesses. Here we identify a novel glutamate-GABAAR interaction mediated by a direct glutamate binding of the GABAAR. In HEK293 cells overexpressing recombinant GABAARs, glutamate and its analog ligands, while producing no current on their own, potentiate GABA-evoked currents. This potentiation is mediated by a direct binding at a novel glutamate binding pocket located at the α+/ß- subunit interface of the GABAAR. Moreover, the potentiation does not require the presence of a γ subunit, and in fact, the presence of γ subunit significantly reduces the potency of the glutamate potentiation. In addition, the glutamate-mediated allosteric potentiation occurs on native GABAARs in rat neurons maintained in culture, as evidenced by the potentiation of GABAAR-mediated inhibitory postsynaptic currents and tonic currents. Most importantly, we found that genetic impairment of this glutamate potentiation in knock-in mice resulted in phenotypes of increased neuronal excitability, including decreased thresholds to noxious stimuli and increased seizure susceptibility. These results demonstrate a novel cross-talk between excitatory transmitter glutamate and inhibitory GABAAR. Such a rapid and short feedback loop between the two principal excitatory and inhibitory neurotransmission systems may play a critical homeostatic role in fine-tuning the excitation-inhibition balance (E/I balance), thereby maintaining neuronal excitability in the mammalian brain under both physiological and pathological conditions.


Assuntos
Ácido Glutâmico , Receptores de GABA-A , Animais , Encéfalo/metabolismo , Ácido Glutâmico/farmacologia , Células HEK293 , Humanos , Mamíferos/metabolismo , Camundongos , Ratos , Receptores de GABA/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Ácido gama-Aminobutírico/farmacologia
10.
Int J Mol Sci ; 23(19)2022 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-36233185

RESUMO

Urothelium is a transitional, stratified epithelium that lines the lower urinary tract, providing a tight barrier to urine whilst retaining the capacity to stretch and rapidly resolve damage. The role of glycerophospholipids in urothelial barrier function is largely unknown, despite their importance in membrane structural integrity, protein complex assembly, and the master regulatory role of PPARγ in urothelial differentiation. We performed lipidomic and transcriptomic characterisation of urothelial differentiation, revealing a metabolic switch signature from fatty acid synthesis to lipid remodelling, including 5-fold upregulation of LPCAT4. LPCAT4 knockdown urothelial cultures exhibited an impaired proliferation rate but developed elevated trans-epithelial electrical resistances upon differentiation, associated with a reduced and delayed capacity to restitute barrier function after wounding. Specific reduction in 18:1 PC fatty acyl chains upon knockdown was consistent with LPCAT4 specificity, but was unlikely to elicit broad barrier function changes. However, transcriptomic analysis of LPCAT4 knockdown supported an LPC-induced reduction in DAG availability, predicted to limit PKC activity, and TSPO abundance, predicted to limit endogenous ATP. These phenotypes were confirmed by PKC and TSPO inhibition. Together, these data suggest an integral role for lipid mediators in urothelial barrier function and highlight the strength of combined lipidomic and transcriptomic analyses for characterising tissue homeostasis.


Assuntos
1-Acilglicerofosfocolina O-Aciltransferase , PPAR gama , Urotélio , 1-Acilglicerofosfocolina O-Aciltransferase/genética , Trifosfato de Adenosina/metabolismo , Diferenciação Celular/genética , Metabolismo Energético , Ácidos Graxos/metabolismo , Glicerofosfolipídeos/metabolismo , Humanos , Lipídeos , PPAR gama/genética , PPAR gama/metabolismo , Receptores de GABA/metabolismo , Urotélio/metabolismo
11.
Artigo em Inglês | MEDLINE | ID: mdl-36229188

RESUMO

BACKGROUND AND OBJECTIVES: Recent imaging studies have suggested a possible involvement of the choroid plexus (CP) in multiple sclerosis (MS). Here, we investigated whether CP changes are already detectable at the earliest stage of MS, preceding symptom onset. METHODS: This study is a retrospective analysis of 27 patients with presymptomatic MS, 97 patients with clinically definite MS (CDMS), and 53 healthy controls (HCs) who underwent a cross-sectional 3T-MRI acquisition; of which, 22 MS, 19 HCs, and 1 presymptomatic MS (evaluated 8 months before conversion to CDMS) also underwent translocator protein (TSPO) 18F-DPA-714 PET and were included in the analysis. CPs were manually segmented on 3D T1-weighted images for volumetric analysis. CP 18F-DPA-714 uptake, reflecting inflammation, was calculated as the average standardized uptake value (SUV). Multivariable regressions adjusted for age, sex, and ventricular and brain volume were fitted to test CP volume differences between presymptomatic patients and MS or HCs. For the presymptomatic case who also had 18F-DPA-714 PET, CP SUV differences with MS and HCs were assessed through Crawford-Howell tests. To provide further insight into the interpretation of 18F-DPA-714-PET uptake at the CP level, a postmortem analysis of CPs in MS vs HCs was performed to characterize the cellular localization of TSPO expression. RESULTS: Compared with HCs, patients with presymptomatic MS had 32% larger CPs (ß = 0.38, p = 0.001), which were not dissimilar to MS CPs (p = 0.69). Moreover, in the baseline scan of the presymptomatic case who later on developed MS, TSPO PET showed 33% greater CP inflammation vs HCs (p = 0.04), although no differences in 18F-DPA-714 uptake were found in parenchymal regions vs controls. CP postmortem analysis identified a population of CD163+ mononuclear phagocytes expressing TSPO in MS, possibly contributing to the increased 18F-DPA-714 uptake. DISCUSSION: We identified an imaging signature in CPs at the presymptomatic MS stage using MRI; in addition, we found an increased CP inflammation with PET in a single presymptomatic patient. These findings suggest a role of CP imaging as an early biomarker and argue for the involvement of the blood-CSF barrier dysfunction in disease development. TRIAL REGISTRATION INFORMATION: APHP-20210727144630, EudraCT-Number: 2008-004174-40; ClinicalTrials.gov: NCT02305264, NCT01651520, and NCT02319382.


Assuntos
Esclerose Múltipla , Biomarcadores , Proteínas de Transporte , Corioide/metabolismo , Plexo Corióideo/diagnóstico por imagem , Ensaios Clínicos como Assunto , Estudos Transversais , Feminino , Humanos , Inflamação/metabolismo , Masculino , Esclerose Múltipla/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodos , Receptores de GABA/metabolismo , Estudos Retrospectivos
12.
Mol Med Rep ; 26(6)2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36205135

RESUMO

The present study aimed to investigate the level of translocator protein (TSPO) and its correlation with different inflammatory cytokines in diabetic retinopathy (DR) patients. Peripheral blood samples were obtained from 54 DR patients and 22 age-related cataract (ARC) patients. The mRNA expression of TSPO, voltage-dependent anion channel (VDAC), apoptosis-associated speck like protein with a caspase recruitment domain (ASC), NOD-like receptors pyrin domain-containing 3 (NLRP3) and caspase-1 were examined by reverse transcription-quantitative PCR. Interleukin-1ß and interleukin-18 levels were detected by enzyme-linked immunosorbent assay. The mRNA levels of TSPO, VDAC, ASC, NLRP3 and capase-1, the protein levels of IL-ß and IL-18 were all significantly higher in the DR group compared with those in the ARC group. The expression levels of those aforementioned cytokines/proteins were more significantly higher in the subgroup of active proliferative DR (PDR) compared with those in the inactive PDR group (P<0.05). Significant positive correlations between TSPO/VDAC complex and ASC, NLRP3, capase-1, IL-ß and IL-18 were found in DR patients. These outcomes suggested that TSPO/VDAC complex and NLRP3 inflammasomes may play an important role in the development and progression of DR.


Assuntos
Diabetes Mellitus , Retinopatia Diabética , Proteínas de Transporte/genética , Caspase 1/genética , Caspase 1/metabolismo , Citocinas/metabolismo , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Humanos , Inflamassomos/metabolismo , Interleucina-18/genética , Interleucina-18/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas NLR/metabolismo , RNA Mensageiro/genética , Receptores de GABA/genética , Receptores de GABA/metabolismo , Canais de Ânion Dependentes de Voltagem
13.
Biomolecules ; 12(10)2022 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-36291606

RESUMO

The mitochondrial translocator protein (TSPO) is a modulator of the apoptotic pathway involving reactive oxygen species (ROS) generation, mitochondrial membrane potential (Δψm) collapse, activation of caspases, and eventually initiation of the apoptotic process. In this in vitro study, H1299 lung cells and BV-2 microglial cells were exposed to the hypoxia-like effect of CoCl2 with or without PK 11195. Exposing the H1299 cells to 0.5 mM CoCl2 for 24 h resulted in decreases in cell viability (63%, p &lt; 0.05), elevation of cardiolipin peroxidation levels (38%, p &lt; 0.05), mitochondrial membrane potential depolarization (13%, p &lt; 0.001), and apoptotic cell death (117%, p &lt; 0.05). Pretreatment with PK 11195 (25 µM) exhibited significant protective capacity on CoCl2-induced alterations in the mentioned processes. Exposure of BV-2 cells to increasing concentrations of CoCl2 (0.3, 0.5, 0.7 mM) for 4 h resulted in alterations in the same cellular processes. These alterations were obtained in a dose-dependent manner, except the changes in caspases 3 and 9. The novel ligands as well as PK 1195 attenuated the in vitro hypoxia-like effects of CoCl2. It appears that the TSPO ligand PK 11195 can prevent CoCl2-induced cellular damage in both non-neuronal and brain cell lines, and they may offer a novel approach to the treatment of hypoxia-related lung and brain diseases in some cases that fail to respond to conventional therapies.


Assuntos
Apoptose , Cardiolipinas , Humanos , Espécies Reativas de Oxigênio/metabolismo , Cardiolipinas/metabolismo , Ligantes , Caspases/metabolismo , Sobrevivência Celular , Linhagem Celular , Hipóxia , Hipóxia Celular , Encéfalo/metabolismo , Pulmão/metabolismo , Receptores de GABA/metabolismo
14.
ACS Chem Neurosci ; 13(22): 3188-3197, 2022 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-36300862

RESUMO

The 18 kDa translocator protein (TSPO) is predominantly located in the mitochondrial outer membrane, playing an important role in steroidogenesis, inflammation, survival, and cell proliferation. Its expression in the CNS, and mainly in glial cells, is upregulated in neuropathologies and brain injury. In this study, the potential of targeting TSPO for the therapeutic treatment of inflammatory-based retinal neurodegeneration was evaluated by means of an in vitro model of lipopolysaccharide (LPS)-induced degeneration in 661 W cells, a photoreceptor-like cell line. After the assessment of the expression of TSPO in 661W cells, which, to the best of our knowledge, was never investigated so far, the anti-inflammatory and cytoprotective effects of a number of known TSPO ligands, belonging to the class of N,N-dialkyl-2-arylindol-3-ylglyoxylamides (PIGAs), were evaluated, using the classic TSPO ligand PK11195 as the reference standard. All tested PIGAs showed the ability to modulate the inflammatory and apoptotic processes in 661 W photoreceptor-like cells and to reduce LPS-driven cellular cytotoxicity. The protective effect of PIGAs was, in all cases, reduced by cotreatment with the pregnenolone synthesis inhibitor SU-10603, suggesting the involvement of neurosteroids in the protective mechanism. As inflammatory processes play a crucial role in the retinal neurodegenerative disease progression toward photoreceptors' death and complete blindness, targeting TSPO might represent a successful strategy to slow down this degenerative process that may lead to the inexorable loss of vision.


Assuntos
Doenças Neurodegenerativas , Degeneração Retiniana , Humanos , Degeneração Retiniana/tratamento farmacológico , Degeneração Retiniana/patologia , Lipopolissacarídeos/farmacologia , Receptores de GABA/metabolismo , Inflamação/metabolismo , Apoptose , Proteínas de Transporte , Ligantes
15.
ACS Chem Neurosci ; 13(20): 3008-3022, 2022 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-36183275

RESUMO

Dopaminergic pathways control highly consequential aspects of physiology and behavior. One of the most therapeutically important and best-studied receptors in these pathways is dopamine receptor D2 (DRD2). Unfortunately, DRD2 is challenging to study with traditional molecular biological techniques, and most drugs designed to target DRD2 are ligands for many other receptors. Here, we developed probes able to both covalently bind to DRD2 using photoaffinity labeling and provide a chemical handle for detection or affinity purification. These probes behaved like good DRD2 agonists in traditional biochemical assays and were able to perform in chemical-biological assays of cell and receptor labeling. Rat whole brain labeling and affinity enrichment using the probes permitted proteomic analysis of the probes' interacting proteins. Bioinformatic study of the hits revealed that the probes bound noncanonically targeted proteins in Parkinson's disease network as well as the retrograde endocannabinoid signaling, neuronal nitric oxide synthase, muscarinic acetylcholine receptor M1, GABA receptor, and dopamine receptor D1 (DRD1) signaling networks. Follow-up analysis may yield insights into how this pathway relates specifically to Parkinson's disease symptoms or provide new targets for treatments. This work reinforces the notion that the combination of chemical biology and omics-based approaches provides a broad picture of a molecule's "interactome" and may also give insight into the pleiotropy of effects observed for a drug or perhaps indicate new applications.


Assuntos
Doença de Parkinson , Receptores de Dopamina D2 , Animais , Ratos , Receptores de Dopamina D2/metabolismo , Doença de Parkinson/tratamento farmacológico , Óxido Nítrico Sintase Tipo I/metabolismo , Ligantes , Proteômica , Endocanabinoides , Receptores de Dopamina D1 , Proteínas de Transporte , Receptores de GABA/metabolismo , Agonistas de Dopamina/farmacologia
16.
Mol Biochem Parasitol ; 252: 111521, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36100173

RESUMO

The UNC-49 receptor is a Cys-loop GABA receptor that is unique to the nematode phylum. The receptor differs from mammalian GABA receptors both in amino acid sequence and pharmacology which highlights its potential as a novel anthelmintic target. Sequence differences within and near the various ligand-binding loops of the nematode receptor suggest that there could be structural differences compared to mammalian receptors that result in different pharmacological and functional features. Here we investigated three residues in the UNC-49 receptor from the parasitic nematode Haemonchus contortus: K181, E183, and T230. Analysis of these residues was conducted via site-directed mutagenesis, electrophysiology, MD simulations, and mutant cycling analysis. In the UNC-49 receptor, E183 lies in close proximity to K181 where together they appear to play a role in GABA sensitivity and pharmacology, possibly interacting via an ionic bond. While the introduction of single alanine residues at each position separately had a negative impact on GABA EC50, the double alanine mutant (K181A/E183A) exhibited wildtype-level GABA EC50 and some differences in pharmacology. Overall, this study has revealed a potentially novel role for these two residues in nematode UNC-49 GABA receptors that could aid in understanding their function.


Assuntos
Nematoides , Receptores de GABA , Animais , Receptores de GABA/genética , Receptores de GABA/química , Receptores de GABA/metabolismo , Sítios de Ligação , Nematoides/metabolismo , Ácido gama-Aminobutírico/metabolismo , Alanina , Mamíferos
17.
Int J Mol Sci ; 23(18)2022 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-36142469

RESUMO

Glyght is a new photochromic compound described as an effective modulator of glycine receptors at heterologous expression, in brain slices and in zebrafish larvae. Glyght also caused weak inhibition of GABAA-mediated currents in a cell line expressing α1/ß2/γ2 GABAA receptors. However, the effects of Glyght on GABAergic transmission in the brain have not been analysed, which does not allow a sufficiently comprehensive assessment of the effects of the compound on the nervous system. Therefore, in this study using whole-cell patch-clamp recording, we analysed the Glyght (100 µM) action on evoked GABAergic inhibitory postsynaptic currents (eIPSCs) in mice hippocampal slices. Two populations of cells were found: the first responded by reducing the GABAergic eIPSCs' amplitude, whereas the second showed no sensitivity to the compound. Glyght did not affect the ionic currents' amplitude induced by GABA application, suggesting the absence of action on postsynaptic GABA receptors. Additionally, Glyght had no impact on the paired-pulse modulation of GABAergic eIPSCs, indicating that Glyght does not modulate the neurotransmitter release mechanisms. In the presence of strychnine, an antagonist of glycine receptors, the Glyght effect on GABAergic synaptic transmission was absent. Our results suggest that Glyght can modulate GABAergic synaptic transmission via action on extrasynaptic glycine receptors.


Assuntos
Receptores de Glicina , Estricnina , Animais , Encéfalo/metabolismo , Camundongos , Neurônios/metabolismo , Neurotransmissores/metabolismo , Receptores de GABA/metabolismo , Receptores de GABA-A/metabolismo , Receptores de Glicina/metabolismo , Estricnina/farmacologia , Transmissão Sináptica , Peixe-Zebra/metabolismo , Ácido gama-Aminobutírico/metabolismo
18.
Neural Plast ; 2022: 8057854, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36071748

RESUMO

Chronic pain is an enormous modern public health problem, with significant numbers of people debilitated by chronic pain from a variety of etiologies. Translocator protein 18 kDa (TSPO) was discovered in 1977 as a peripheral benzodiazepine receptor. It is a five transmembrane domain protein, mainly localized in the outer mitochondrial membrane. Recent and increasing studies have found changes in TSPO and its ligands in various chronic pain models. Reversing their expressions has been shown to alleviate chronic pain in these models, illustrating the effects of TSPO and its ligands. Herein, we review recent evidence and the mechanisms of TSPO in the development of chronic pain associated with peripheral nerve injury, spinal cord injury, cancer, and inflammatory responses. The cumulative evidence indicates that TSPO-based therapy may become an alternative strategy for treating chronic pain.


Assuntos
Dor Crônica , Receptores de GABA , Proteínas de Transporte/metabolismo , Dor Crônica/tratamento farmacológico , Dor Crônica/metabolismo , Humanos , Ligantes , Membranas Mitocondriais/metabolismo , Receptores de GABA/metabolismo
19.
Ann Neurol ; 92(5): 768-781, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36053756

RESUMO

OBJECTIVE: Alzheimer disease (AD) is characterized by amyloid ß (Aß) plaques and neurofibrillary tau tangles, but increasing evidence suggests that neuroinflammation also plays a key role, driven by the activation of microglia. Aß and tau pathology appear to spread along pathways of highly connected brain regions, but it remains elusive whether microglial activation follows a similar distribution pattern. Here, we assess whether connectivity is associated with microglia activation patterns. METHODS: We included 32 Aß-positive early AD subjects (18 women, 14 men) and 18 Aß-negative age-matched healthy controls (10 women, 8 men) from the prospective ActiGliA (Activity of Cerebral Networks, Amyloid and Microglia in Aging and Alzheimer's Disease) study. All participants underwent microglial activation positron emission tomography (PET) with the third-generation mitochondrial 18 kDa translocator protein (TSPO) ligand [18 F]GE-180 and magnetic resonance imaging (MRI) to measure resting-state functional and structural connectivity. RESULTS: We found that inter-regional covariance in TSPO-PET and standardized uptake value ratio was preferentially distributed along functionally highly connected brain regions, with MRI structural connectivity showing a weaker association with microglial activation. AD patients showed increased TSPO-PET tracer uptake bilaterally in the anterior medial temporal lobe compared to controls, and higher TSPO-PET uptake was associated with cognitive impairment and dementia severity in a disease stage-dependent manner. INTERPRETATION: Microglial activation distributes preferentially along highly connected brain regions, similar to tau pathology. These findings support the important role of microglia in neurodegeneration, and we speculate that pathology spreads throughout the brain along vulnerable connectivity pathways. ANN NEUROL 2022;92:768-781.


Assuntos
Doença de Alzheimer , Masculino , Humanos , Feminino , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Microglia/metabolismo , Proteínas tau/metabolismo , Ligantes , Estudos Prospectivos , Tomografia por Emissão de Pósitrons/métodos , Placa Amiloide/metabolismo , Encéfalo/patologia , Receptores de GABA/metabolismo
20.
Drug Dev Res ; 83(7): 1519-1533, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36074736

RESUMO

Inflammation has been linked to the onset and progression of a wide range of neuropathological disorders. The well-conserved outer mitochondrial membrane 18 kDa translocator protein (TSPO) is perceived as an in vivo neuroinflammation marker. A dearth of a reference region, genetic disparity influencing the ligand's affinity for TSPO, and a substantial signal in the endothelium of the brain veins contributes toward complications in quantifying TSPO positron emission tomography (PET) image. Up to the present time several radiotracers based on different pharmacophore such as (R)[11 C]PK11195, [18 F]DPA714, [11 C]PBR28, [11 C]ER176, and many more have been recognized for envisaging the prominent TSPO level observed in neurological conditions. Recently acetamidobenzoxazolone (ABO) scaffold, a bicyclic ring system composed of a phenyl ring fused to a carbamate and its substituted radiolabelled analogues especially at C-5 position has evidenced encouraging outcomes as next generation of TSPO PET ligands. Diverse ABO framework-based TSPO ligands have been designed embracing imperative aspects such as lipophilicity, metabolic profile, and capability to penetrate the blood-brain barrier apart from least effect of polymorphism (rs6971). Over the years numerous systematic literature reviews compiling different structural class of TSPO ligands characterized on the grounds of their binding affinity and metabolite profile have been reported but none is especially focused toward a fascinating benzoxazolone scaffold. This review exclusively bestows an overview of the recent advancements on ABO derivatives with neuroinflammation imaging potential and emphases on the structural features accountable for visualizing TSPO in-vivo with collation of published reports during last 10 years.


Assuntos
Doenças Neuroinflamatórias , Receptores de GABA , Humanos , Receptores de GABA/metabolismo , Ligantes , Tomografia por Emissão de Pósitrons/métodos , Proteínas de Transporte , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo
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