RESUMEN
BACKGROUND: Early-onset familial Alzheimer disease (EOFAD) is caused by heterozygous variants in the presenilin 1 (PSEN1), presenilin 2 (PSEN2), and APP genes. Decades after their discovery, the mechanisms by which these genes cause Alzheimer's disease (AD) or promote AD progression are not fully understood. While it is established that presenilin (PS) enzymatic activity produces amyloid-ß (Aß), PSs also regulate numerous other cellular functions, some of which intersect with known pathogenic drivers of neurodegeneration. Accumulating evidence suggests that microglia, resident innate immune cells in the central nervous system, play a key role in AD neurodegeneration. OBJECTIVE: Previous work has identified a regulatory role for PS2 in microglia. We hypothesized that PSEN2 variants lead to dysregulated microglia, which could further contribute to disease acceleration. To mimic the genotype of EOFAD patients, we created a transgenic mouse expressing PSEN2 N141I on a mouse background expressing one wildtype PS2 and two PS1 alleles. RESULTS: Microglial expression of PSEN2 N141I resulted in impaired γ-secretase activity as well as exaggerated inflammatory cytokine release, NFκB activity, and Aß internalization. In vivo, PS2 N141I mice showed enhanced IL-6 and TREM2 expression in brain as well as reduced branch number and length, an indication of "activated" morphology, in the absence of inflammatory stimuli. LPS intraperitoneal injection resulted in higher inflammatory gene expression in PS2 N141I mouse brain relative to controls. CONCLUSION: Our findings demonstrate that PSEN2 N141I heterozygosity is associated with disrupted innate immune homeostasis, suggesting EOFAD variants may promote disease progression through non-neuronal cells beyond canonical dysregulated Aß production.
Asunto(s)
Enfermedad de Alzheimer/genética , Variación Genética/genética , Heterocigoto , Microglía/fisiología , Fenotipo , Presenilina-2/genética , Enfermedad de Alzheimer/patología , Animales , Línea Celular Tumoral , Células Cultivadas , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/patologíaRESUMEN
Microglia are the primary innate immune cell type in the brain, and their dysfunction has been linked to a variety of central nervous system disorders. Human microglia are extraordinarily difficult to obtain for experimental investigation, limiting our ability to study the impact of human genetic variants on microglia functions. Previous studies have reported that microglia-like cells can be derived from human monocytes or pluripotent stem cells. Here, we describe a reproducible relatively simple method for generating microglia-like cells by first deriving embryoid body mesoderm followed by exposure to microglia relevant cytokines. Our approach is based on recent studies demonstrating that microglia originate from primitive yolk sac mesoderm distinct from peripheral macrophages that arise during definitive hematopoiesis. We hypothesized that functional microglia could be derived from human stem cells by employing BMP-4 mesodermal specification followed by exposure to microglia-relevant cytokines, M-CSF, GM-CSF, IL-34, and TGF-ß. Using immunofluorescence microscopy, flow cytometry, and reverse transcription polymerase chain reaction, we observed cells with microglia morphology expressing a repertoire of markers associated with microglia: Iba1, CX3CR1, CD11b, TREM2, HexB, and P2RY12. These microglia-like cells maintain myeloid functional phenotypes including Aß peptide phagocytosis and induction of pro-inflammatory gene expression in response to lipopolysaccharide stimulation. Addition of small molecules BIO and SB431542, previously demonstrated to drive definitive hematopoiesis, resulted in decreased surface expression of TREM2. Together, these data suggest that mesodermal lineage specification followed by cytokine exposure produces microglia-like cells in vitro from human pluripotent stem cells and that this phenotype can be modulated by factors influencing hematopoietic lineage in vitro.
Asunto(s)
Linaje de la Célula/fisiología , Hematopoyesis/fisiología , Glicoproteínas de Membrana/metabolismo , Microglía/metabolismo , Células Madre Pluripotentes/metabolismo , Receptores Inmunológicos/metabolismo , Péptidos beta-Amiloides/metabolismo , Técnicas de Cultivo de Célula , Fibroblastos/citología , Fibroblastos/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Inflamación/metabolismo , Inflamación/patología , Lipopolisacáridos , Microglía/citología , Fagocitosis/fisiología , Células Madre Pluripotentes/citología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Indole-based compounds, such as the alkyl-indole (AI) compound WIN55212-2, activate the cannabinoid receptors, CB1 and CB2, two well-characterized G protein-coupled receptors (GPCR). Reports indicate that several indole-based cannabinoid agonists, including WIN55212-2, lack selectivity and interact with at least two additional targets: AI-sensitive GPCRs and microtubules. Studying how indole-based compounds modulate the activity of these 4 targets has been difficult as selective chemical tools were not available. Here we report the pharmacological characterization of six newly-developed indole-based compounds (ST-11, ST-23, ST-25, ST-29, ST-47 and ST-48) that exhibit distinct binding affinities at AI-sensitive receptors, cannabinoid CB1 and CB2 receptors and the colchicine site of tubulin. Several compounds exhibit some level of selectivity for AI-sensitive receptors, including ST-11 that binds AI-sensitive receptors with a Kd of 52nM and appears to have a weaker affinity for the colchicine site of tubulin (Kd=3.2µM) and does not bind CB1/CB2 receptors. Leveraging these characteristics, we show that activation of AI-sensitive receptors with ST-11 inhibits both the basal and stimulated migration of the Delayed Brain Tumor (DBT) mouse glioma cell line. Our study describes a new series of indole-based compounds that enable the pharmacological and functional differentiation of alkylindole-sensitive receptors from cannabinoid receptors and microtubules.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Glioma/tratamiento farmacológico , Indoles/farmacología , Microtúbulos/efectos de los fármacos , Receptores de Cannabinoides/metabolismo , Animales , Benzoxazinas/farmacología , Unión Competitiva/fisiología , Agonistas de Receptores de Cannabinoides/farmacología , Línea Celular , Colchicina/metabolismo , Glioma/metabolismo , Células HEK293 , Humanos , Ratones , Morfolinas/farmacología , Naftalenos/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
Glioblastoma multiforme is a devastating and intractable type of cancer. Current antineoplastic drugs do not improve the median survival of patients diagnosed with glioblastoma multiforme beyond 14 to 15 months, in part because the blood-brain barrier is generally impermeable to many therapeutic agents. Drugs that target microtubules (MT) have shown remarkable efficacy in a variety of cancers, yet their use as glioblastoma multiforme treatments has also been hindered by the scarcity of brain-penetrant MT-targeting compounds. We have discovered a new alkylindole compound, ST-11, that acts directly on MTs and rapidly attenuates their rate of assembly. Accordingly, ST-11 arrests glioblastoma multiforme cells in prometaphase and triggers apoptosis. In vivo analyses reveal that unlike current antitubulin agents, ST-11 readily crosses the blood-brain barrier. Further investigation in a syngeneic orthotopic mouse model of glioblastoma multiforme shows that ST-11 activates caspase-3 in tumors to reduce tumor volume without overt toxicity. Thus, ST-11 represents the first member of a new class of brain-penetrant antitubulin therapeutic agents. Mol Cancer Ther; 15(9); 2018-29. ©2016 AACR.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Microtúbulos/metabolismo , Moduladores de Tubulina/farmacología , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Caspasa 3/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Humanos , Ratones , Nanopartículas , Proyectos Piloto , Solubilidad , Moduladores de Tubulina/administración & dosificación , Moduladores de Tubulina/farmacocinética , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Ligands targeting G protein-coupled receptors (GPCR) expressed by microglia have been shown to regulate distinct components of their activation process, including cell proliferation, migration and differentiation into M1 or M2 phenotypes. Cannabinoids, including the active component of the Cannabis plant, tetrahydrocannabinol (THC), and the synthetic alkylindole (AI) compound, WIN55212-2 (WIN-2), activate two molecularly identified GPCRs: CB1 and CB2 . Previous studies reported that WIN-2 activates an additional unknown GPCR that is not activated by plant-derived cannabinoids, and evidence indicates that microglia express these receptors. Detailed studies on the role of AI-sensitive receptors in microglial cell activation were difficult as no selective pharmacological tools were available. Here, three newly-developed AI analogues allowed us to determine if microglia express AI-sensitive receptors and if so, study how they regulate the microglial cell activation process. We found that mouse microglia in primary culture express functional AI-sensitive receptors as measured by radioligand binding and changes in intracellular cAMP levels, and that these receptors control both basal and ATP-stimulated migration. AI analogues inhibit cell proliferation stimulated by macrophage-colony stimulating factor (M-CSF) without affecting basal cell proliferation. Remarkably, AI analogues do not control the expression of effector proteins characteristic of M1 or M2 phenotypes; yet activating microglia with M1 and M2 cytokines reduces the microglial response to AI analogues. Our results suggest that microglia express functional AI-sensitive receptors that control select components of their activation process. Agonists of these novel targets might represent a novel class of therapeutics to influence the microglial cell activation process.
Asunto(s)
Movimiento Celular/fisiología , Proliferación Celular/fisiología , Microglía/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Adenosina Trifosfato/farmacología , Animales , Animales Recién Nacidos , Benzoxazinas/farmacología , Encéfalo/citología , Bloqueadores de los Canales de Calcio/farmacología , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Quimiocina CXCL10/metabolismo , AMP Cíclico/metabolismo , Citocinas/farmacología , Dronabinol/farmacología , Ratones , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Morfolinas/farmacología , Naftalenos/farmacología , Óxido Nítrico/metabolismoRESUMEN
The serine hydrolase α/ß-hydrolase domain 6 (ABHD6) hydrolyzes the most abundant endocannabinoid (eCB) in the brain, 2-arachidonoylglycerol (2-AG), and controls its availability at cannabinoid receptors. We show that ABHD6 inhibition decreases pentylenetetrazole (PTZ)-induced generalized tonic-clonic and myoclonic seizure incidence and severity. This effect is retained in Cnr1(-/-) or Cnr2(-/-) mice, but blocked by addition of a subconvulsive dose of picrotoxin, suggesting the involvement of GABAA receptors. ABHD6 inhibition also blocked spontaneous seizures in R6/2 mice, a genetic model of juvenile Huntington's disease known to exhibit dysregulated eCB signaling. ABHD6 blockade retained its antiepileptic activity over chronic dosing and was not associated with psychomotor or cognitive effects. While the etiology of seizures in R6/2 mice remains unsolved, involvement of the hippocampus is suggested by interictal epileptic discharges, increased expression of vGLUT1 but not vGAT, and reduced Neuropeptide Y (NPY) expression. We conclude that ABHD6 inhibition may represent a novel antiepileptic strategy.
Asunto(s)
Anticonvulsivantes/uso terapéutico , Encéfalo/efectos de los fármacos , Carbamatos/uso terapéutico , Monoacilglicerol Lipasas/antagonistas & inhibidores , Convulsiones/tratamiento farmacológico , Animales , Anticonvulsivantes/farmacología , Conducta Animal/efectos de los fármacos , Encéfalo/fisiopatología , Carbamatos/farmacología , Masculino , Ratones , Ratones Noqueados , Pentilenotetrazol , Receptores de Cannabinoides/genética , Convulsiones/inducido químicamente , Convulsiones/fisiopatologíaRESUMEN
Cannabinoid receptor 1 (CB(1) receptor) controls several neuronal functions, including neurotransmitter release, synaptic plasticity, gene expression and neuronal viability. Downregulation of CB(1) expression in the basal ganglia of patients with Huntington's disease (HD) and animal models represents one of the earliest molecular events induced by mutant huntingtin (mHtt). This early disruption of neuronal CB(1) signaling is thought to contribute to HD symptoms and neurodegeneration. Here we determined whether CB(1) downregulation measured in patients with HD and mouse models was ubiquitous or restricted to specific striatal neuronal subpopulations. Using unbiased semi-quantitative immunohistochemistry, we confirmed previous studies showing that CB(1) expression is downregulated in medium spiny neurons of the indirect pathway, and found that CB(1) is also downregulated in neuropeptide Y (NPY)/neuronal nitric oxide synthase (nNOS)-expressing interneurons while remaining unchanged in parvalbumin- and calretinin-expressing interneurons. CB(1) downregulation in striatal NPY/nNOS-expressing interneurons occurs in R6/2 mice, Hdh(Q150/Q150) mice and the caudate nucleus of patients with HD. In R6/2 mice, CB(1) downregulation in NPY/nNOS-expressing interneurons correlates with diffuse expression of mHtt in the soma. This downregulation also occludes the ability of cannabinoid agonists to activate the pro-survival signaling molecule cAMP response element-binding protein in NPY/nNOS-expressing interneurons. Loss of CB(1) signaling in NPY/nNOS-expressing interneurons could contribute to the impairment of basal ganglia functions linked to HD.
Asunto(s)
Ganglios Basales/metabolismo , Regulación hacia Abajo , Enfermedad de Huntington/metabolismo , Interneuronas/metabolismo , Neuropéptido Y/metabolismo , Receptor Cannabinoide CB1/metabolismo , Adulto , Anciano , Animales , Ganglios Basales/citología , Calbindina 2 , Agonistas de Receptores de Cannabinoides/farmacología , Estudios de Casos y Controles , AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Humanos , Proteína Huntingtina , Interneuronas/clasificación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Proteínas del Tejido Nervioso/genética , Neuropéptido Y/genética , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Proteínas Nucleares/genética , Parvalbúminas/genética , Parvalbúminas/metabolismo , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/genética , Proteína G de Unión al Calcio S100/genética , Proteína G de Unión al Calcio S100/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismoRESUMEN
The endocannabinoid 2-arachidonoylglycerol (2-AG) regulates neurotransmission and neuroinflammation by activating CB1 cannabinoid receptors on neurons and CB2 cannabinoid receptors on microglia. Enzymes that hydrolyze 2-AG, such as monoacylglycerol lipase, regulate the accumulation and efficacy of 2-AG at cannabinoid receptors. We found that the recently described serine hydrolase alpha-beta-hydrolase domain 6 (ABHD6) also controls the accumulation and efficacy of 2-AG at cannabinoid receptors. In cells from the BV-2 microglia cell line, ABHD6 knockdown reduced hydrolysis of 2-AG and increased the efficacy with which 2-AG can stimulate CB2-mediated cell migration. ABHD6 was expressed by neurons in primary culture and its inhibition led to activity-dependent accumulation of 2-AG. In adult mouse cortex, ABHD6 was located postsynaptically and its selective inhibition allowed the induction of CB1-dependent long-term depression by otherwise subthreshold stimulation. Our results indicate that ABHD6 is a rate-limiting step of 2-AG signaling and is therefore a bona fide member of the endocannabinoid signaling system.