RESUMO
Triggering receptor expressed on myeloid cells 2 (TREM2) is an orphan immune receptor expressed on cells of myeloid lineage such as macrophages and microglia. The rare variant R47H TREM2 is associated with an increased risk for Alzheimer's disease, supporting the hypothesis that TREM2 loss of function may exacerbate disease progression. However, a complete knockout of the TREM2 gene in different genetic models of neurodegenerative diseases has been reported to result in both protective and deleterious effects on disease-related end points and myeloid cell function. Here, we describe a Trem2R47H transgenic mouse model and report that even in the absence of additional genetic perturbations, this variant clearly confers a loss of function on myeloid cells. The Trem2R47H variant-containing myeloid cells exhibited subtle defects in survival and migration and displayed an unexpected dysregulation of cytokine responses in a lipopolysaccharide challenge environment. These subtle phenotypic defects with a gradation in severity across genotypes were confirmed in whole-genome RNA-Seq analyses of WT, Trem2-/-, and Trem2R47H myeloid cells under challenge conditions. Of note, TREM2-activating antibodies that boost proximal signaling abrogated survival defects conferred by the variant and also modulated migration and cytokine responses in an antibody-, ligand-, and challenge-dependent manner. In some instances, these antibodies also boosted WT myeloid cell function. Our studies provide a first glimpse into the boost in myeloid cell function that can be achieved by pharmacological modulation of TREM2 activity that can potentially be ameliorative in neurodegenerative diseases such as Alzheimer's disease.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Anticorpos Monoclonais/farmacologia , Apoptose , Movimento Celular , Glicoproteínas de Membrana/fisiologia , Mutação , Células Mieloides/patologia , Receptores Imunológicos/fisiologia , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Animais , Biomarcadores/metabolismo , Encéfalo/imunologia , Encéfalo/metabolismo , Encéfalo/patologia , Células Cultivadas , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/imunologia , Microglia/metabolismo , Microglia/patologia , Células Mieloides/imunologia , Células Mieloides/metabolismoRESUMO
Triggering receptor expressed on myeloid cells 2 (TREM2) is an immune receptor expressed on the surface of microglia, macrophages, dendritic cells, and osteoclasts. The R47H TREM2 variant is a significant risk factor for late-onset Alzheimer's disease (AD), and the molecular basis of R47H TREM2 loss of function is an emerging area of TREM2 biology. Here, we report three high-resolution structures of the extracellular ligand-binding domains (ECDs) of R47H TREM2, apo-WT, and phosphatidylserine (PS)-bound WT TREM2 at 1.8, 2.2, and 2.2 Å, respectively. The structures reveal that Arg47 plays a critical role in maintaining the structural features of the complementarity-determining region 2 (CDR2) loop and the putative positive ligand-interacting surface (PLIS), stabilizing conformations capable of ligand interaction. This is exemplified in the PS-bound structure, in which the CDR2 loop and PLIS drive critical interactions with PS via surfaces that are disrupted in the variant. Together with in vitro and in vivo characterization, our structural findings elucidate the molecular mechanism underlying loss of ligand binding, putative oligomerization, and functional activity of R47H TREM2. They also help unravel how decreased in vitro and in vivo stability of TREM2 contribute to loss of function in disease.
Assuntos
Doença de Alzheimer/genética , Predisposição Genética para Doença , Glicoproteínas de Membrana/química , Proteínas Mutantes/química , Receptores Imunológicos/química , Doença de Alzheimer/patologia , Cristalografia por Raios X , Células Dendríticas/química , Células Dendríticas/patologia , Variação Genética , Humanos , Ligantes , Macrófagos/química , Macrófagos/patologia , Glicoproteínas de Membrana/genética , Microglia/química , Microglia/patologia , Proteínas Mutantes/genética , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Osteoclastos/química , Osteoclastos/patologia , Conformação Proteica , Domínios Proteicos/genética , Receptores Imunológicos/genéticaRESUMO
GPR21 is an orphan G-protein-coupled receptor. We found that mice deficient for the GPR21 gene were resistant to diet-induced obesity. Knockout mice were leaner than their wildtype counterpart, despite that no difference was observed in food intake. No differences were observed in the respiratory exchange rate and thermogenesis. However, knockout mice were more active than wildtype littermates, and this level of activity may be an underlying reason for the difference in energy balance. Mutant mice were more sensitive to insulin than their wildtype control and showed an improved glucose tolerance. Several inflammatory markers MCP-1, CRP and IP-10 were decreased in mutant animals, suggesting that GPR21 may also mediate its effect through anti-inflammatory mechanisms. We found that GPR21 is widely expressed in all tissues, with the highest levels found in the brain and in the spleen. Overall, these findings suggest that GPR21 may play an important role in regulating body weight and glucose metabolism.
Assuntos
Resistência à Insulina/genética , Insulina/farmacologia , Obesidade/genética , Receptores Acoplados a Proteínas G/genética , Animais , Biomarcadores/metabolismo , Peso Corporal/genética , Proteína C-Reativa/metabolismo , Quimiocina CCL2/metabolismo , Quimiocina CXCL10/metabolismo , Dieta/efeitos adversos , Expressão Gênica , Glucose/metabolismo , Teste de Tolerância a Glucose , Camundongos , Camundongos Knockout , Obesidade/etiologia , Distribuição TecidualRESUMO
The present report describes our efforts to convert an existing LXR agonist into an LXR antagonist using a structure-based approach. A series of benzenesulfonamides was synthesized based on structural modification of a known LXR agonist and was determined to be potent dual liver X receptor (LXR α/ß) ligands. Herein we report the identification of compound 54 as the first reported LXR antagonist that is suitable for pharmacological in vivo evaluation in rodents.
Assuntos
Descoberta de Drogas , Receptores Nucleares Órfãos/antagonistas & inibidores , Sulfonamidas/farmacologia , Animais , Relação Dose-Resposta a Droga , Células HEK293 , Células Hep G2 , Humanos , Ligantes , Receptores X do Fígado , Masculino , Camundongos , Estrutura Molecular , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/química , BenzenossulfonamidasRESUMO
Structural modification of a series of dual LXRα/ß agonists led to the identification of a new class of LXRß partial agonists. An X-ray co-crystal structure shows that a representative member of this series, pyrrole 5, binds to LXRß with a reversed orientation compared to 1.
Assuntos
Receptores Nucleares Órfãos/agonistas , Isoformas de Proteínas/agonistas , Pirróis/síntese química , Sítios de Ligação , Células CACO-2 , Cristalografia por Raios X , Genes Reporter , Células HEK293 , Humanos , Receptores X do Fígado , Receptores Nucleares Órfãos/química , Ligação Proteica , Isoformas de Proteínas/química , Pirróis/farmacologia , Relação Estrutura-Atividade , TransfecçãoRESUMO
Axon degeneration is an early pathological event in many neurological diseases. The identification of the nicotinamide adenine dinucleotide (NAD) hydrolase SARM1 as a central metabolic sensor and axon executioner presents an exciting opportunity to develop novel neuroprotective therapies that can prevent or halt the degenerative process, yet limited progress has been made on advancing efficacious inhibitors. We describe a class of NAD-dependent active-site SARM1 inhibitors that function by intercepting NAD hydrolysis and undergoing covalent conjugation with the reaction product adenosine diphosphate ribose (ADPR). The resulting small-molecule ADPR adducts are highly potent and confer compelling neuroprotection in preclinical models of neurological injury and disease, validating this mode of inhibition as a viable therapeutic strategy. Additionally, we show that the most potent inhibitor of CD38, a related NAD hydrolase, also functions by the same mechanism, further underscoring the broader applicability of this mechanism in developing therapies against this class of enzymes.
Assuntos
Proteínas do Domínio Armadillo , NAD , Proteínas do Domínio Armadillo/genética , Proteínas do Domínio Armadillo/metabolismo , NAD/metabolismo , Neuroproteção , Proteínas do Citoesqueleto/metabolismo , Axônios/metabolismo , Hidrolases/metabolismoRESUMO
The NADase SARM1 is a central switch in injury-activated axon degeneration, an early hallmark of many neurological diseases. Here, we present cryo-electron microscopy (cryo-EM) structures of autoinhibited (3.3 Å) and active SARM1 (6.8 Å) and provide mechanistic insight into the tight regulation of SARM1's function by the local metabolic environment. Although both states retain an octameric core, the defining feature of the autoinhibited state is a lock between the autoinhibitory Armadillo/HEAT motif (ARM) and catalytic Toll/interleukin-1 receptor (TIR) domains, which traps SARM1 in an inactive state. Mutations that break this lock activate SARM1, resulting in catastrophic neuronal death. Notably, the mutants cannot be further activated by the endogenous activator nicotinamide mononucleotide (NMN), and active SARM1 is product inhibited by Nicotinamide (NAM), highlighting SARM1's functional dependence on key metabolites in the NAD salvage pathway. Our studies provide a molecular understanding of SARM1's transition from an autoinhibited to an injury-activated state and lay the foundation for future SARM1-based therapies to treat axonopathies.
Assuntos
Proteínas do Domínio Armadillo/química , Proteínas do Domínio Armadillo/metabolismo , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , NAD/metabolismo , Animais , Morte Celular , Linhagem Celular Tumoral , Microscopia Crioeletrônica , Feminino , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Neurônios/citologia , Mononucleotídeo de Nicotinamida/metabolismo , Domínios ProteicosRESUMO
GPR103 is a G-protein-coupled receptor with reported expression in brain, heart, kidney, adrenal gland, retina, and testis. It encodes a 455-amino-acid protein homologous to neuropeptide FF2, neuropeptide Y2, and galanin GalR1 receptors. Its natural ligand was recently identified as 26RFa, a novel human RF-amide-related peptide with orexigenic activity. To identify the function of GPR103, we generated GPR103-deficient mice. Homozygous mutant mice were viable and fertile. Their body weight was undistinguishable from that of their wild-type littermates. Histological analysis revealed that GPR103-/- mice exhibited a thinned osteochondral growth plate, a thickening of trabecular branches, and a reduction in osteoclast number, suggestive of an early arrest of osteochondral bone formation. Microcomputed tomography confirmed the reduction in trabecular bone and connective tissue densities in GPR103 knockout animals. Whole-body radiography followed by morphometric analysis revealed a kyphosis in mutant animals. Reverse transcription-PCR analysis showed that GPR103 was expressed in human skull, mouse spine, and several osteoblast cell lines. Dexamethasone, a known inhibitor of osteoblast growth and inducer of osteoblast differentiation, inhibited GPR103 expression in human osteoblast primary cultures. Altogether, these results suggest that osteopenia in GPR103-/- mice may be mediated directly by the loss of GPR103 expression in bone.
Assuntos
Osteogênese/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Animais , Densidade Óssea , Osso e Ossos/metabolismo , Encéfalo/metabolismo , Diferenciação Celular , Linhagem Celular , Feminino , Lâmina de Crescimento/fisiologia , Humanos , Rim/metabolismo , Cifose/genética , Cifose/patologia , Masculino , Camundongos , Camundongos Knockout , Especificidade de Órgãos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Ovariectomia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Crânio/metabolismo , Coluna Vertebral/diagnóstico por imagem , Coluna Vertebral/metabolismo , Tomografia Computadorizada por Raios XRESUMO
The biological effects of the melanin-concentrating hormone (MCH) are mediated by the melanin concentrating hormone receptor 1 (MCHR1) in mice. This receptor is enriched in brain areas that are involved in the modulation of mood and affect, suggesting that MCH-dependent signaling may influence neurobiological mechanisms underlying fear and anxiety processes. To test this, we have generated mice lacking functional MCHR1 and characterized phenotypic traits using a number of behavioral tests. Mice carrying a null mutation of the MCHR1 gene display anxiolytic-like behavior across a battery different behavioral paradigms commonly used to assess fear and anxiety responses in rodents: open field, elevated plus maze, social interaction, and stress-induced hyperthermia. The brain serotonin (5-HT) system is central to the control of mood- and anxiety-related processes. To examine the impact of MCHR1 receptor deletion on 5-HT neurotransmission, we used in vivo microdialysis in freely moving knockout and wild-type mice. Baseline dialysate 5-HT levels were significantly lower in MCHR1 knockout mice as compared with wild-type controls (9.53+/-0.24 fmol for wild types vs 6.91+/-0.36 fmol for knockouts) in the prefrontal cortex (PFC), one of the main target structures of the serotonergic system and one that is highly associated with the control of emotional processes. Moreover, forced swim increased 5-HT efflux in the PFC of wild-type but not MCHR1 knockout mice. In summary, we show that MCHR1 can modulate stress- and anxiety-like behaviors and suggest that this may be due to changes in serotonergic transmission in forebrain regions.
Assuntos
Ansiedade/genética , Receptores do Hormônio Hipofisário/genética , Animais , Comportamento Animal/fisiologia , Química Encefálica/genética , Química Encefálica/fisiologia , Cromatografia Líquida de Alta Pressão , Meio Ambiente , Espaço Extracelular/metabolismo , Febre/genética , Febre/fisiopatologia , Genótipo , Relações Interpessoais , Camundongos , Camundongos Knockout , Microdiálise , Atividade Motora/fisiologia , Fenótipo , Córtex Pré-Frontal/metabolismo , Serotonina/metabolismo , Estresse Psicológico/genética , Estresse Psicológico/fisiopatologiaRESUMO
We report the identification of a PDE10A clinical candidate by optimizing potency and in vivo efficacy of promising keto-benzimidazole leads 1 and 2. Significant increase in biochemical potency was observed when the saturated rings on morpholine 1 and N-acetyl piperazine 2 were changed by a single atom to tetrahydropyran 3 and N-acetyl piperidine 5. A second single atom modification from pyrazines 3 and 5 to pyridines 4 and 6 improved the inhibitory activity of 4 but not 6. In the in vivo LC-MS/MS target occupancy (TO) study at 10 mg/kg, 3, 5, and 6 achieved 86-91% occupancy of PDE10A in the brain. Furthermore, both CNS TO and efficacy in PCP-LMA behavioral model were observed in a dose dependent manner. With superior in vivo TO, in vivo efficacy and in vivo PK profiles in multiple preclinical species, compound 5 (AMG 579) was advanced as our PDE10A clinical candidate.