RESUMO
Bacterial cell wall components provide various unique molecular structures that are detected by pattern recognition receptors (PRRs) of the innate immune system as non-self. Most bacterial species form a cell wall that consists of peptidoglycan (PGN), a polymeric structure comprising alternating amino sugars that form strands cross-linked by short peptides. Muramyl dipeptide (MDP) has been well documented as a minimal immunogenic component of peptidoglycan1-3. MDP is sensed by the cytosolic nucleotide-binding oligomerization domain-containing protein 24 (NOD2). Upon engagement, it triggers pro-inflammatory gene expression, and this functionality is of critical importance in maintaining a healthy intestinal barrier function5. Here, using a forward genetic screen to identify factors required for MDP detection, we identified N-acetylglucosamine kinase (NAGK) as being essential for the immunostimulatory activity of MDP. NAGK is broadly expressed in immune cells and has previously been described to contribute to the hexosamine biosynthetic salvage pathway6. Mechanistically, NAGK functions upstream of NOD2 by directly phosphorylating the N-acetylmuramic acid moiety of MDP at the hydroxyl group of its C6 position, yielding 6-O-phospho-MDP. NAGK-phosphorylated MDP-but not unmodified MDP-constitutes an agonist for NOD2. Macrophages from mice deficient in NAGK are completely deficient in MDP sensing. These results reveal a link between amino sugar metabolism and innate immunity to bacterial cell walls.
Assuntos
Acetilmuramil-Alanil-Isoglutamina , Proteína Adaptadora de Sinalização NOD2 , Fosfotransferases (Aceptor do Grupo Álcool) , Acetilmuramil-Alanil-Isoglutamina/química , Acetilmuramil-Alanil-Isoglutamina/imunologia , Acetilmuramil-Alanil-Isoglutamina/metabolismo , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Animais , Bactérias/química , Bactérias/imunologia , Parede Celular/química , Hexosaminas/biossíntese , Imunidade Inata , Macrófagos/enzimologia , Macrófagos/imunologia , Camundongos , Proteína Adaptadora de Sinalização NOD2/agonistas , Proteína Adaptadora de Sinalização NOD2/metabolismo , Peptidoglicano/química , Peptidoglicano/imunologia , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/deficiência , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismoRESUMO
Cell adhesion is tightly controlled in multicellular organisms, for example, through proteolytic ectodomain shedding of the adhesion-mediating cell surface transmembrane proteins. In the brain, shedding of cell adhesion proteins is required for nervous system development and function, but the shedding of only a few adhesion proteins has been studied in detail in the mammalian brain. One such adhesion protein is the transmembrane protein endoglycan (PODXL2), which belongs to the CD34-family of highly glycosylated sialomucins. Here, we demonstrate that endoglycan is broadly expressed in the developing mouse brains and is proteolytically shed in vitro in mouse neurons and in vivo in mouse brains. Endoglycan shedding in primary neurons was mediated by the transmembrane protease a disintegrin and metalloprotease 10 (ADAM10), but not by its homolog ADAM17. Functionally, endoglycan deficiency reduced the branching of neurites extending from primary neurons in vitro, whereas deletion of ADAM10 had the opposite effect and increased neurite branching. Taken together, our study discovers a function for endoglycan in neurite branching, establishes endoglycan as an ADAM10 substrate and suggests that ADAM10 cleavage of endoglycan may contribute to neurite branching.
Assuntos
Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Moléculas de Adesão Celular/metabolismo , Desintegrinas/metabolismo , Proteínas de Membrana/metabolismo , Neuritos/metabolismo , Neurônios/metabolismo , Sialoglicoproteínas/metabolismo , Proteína ADAM17/metabolismo , Animais , Encéfalo/metabolismo , Adesão Celular/fisiologia , Linhagem Celular , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurogênese/fisiologia , ProteóliseRESUMO
Single nucleotide polymorphisms (SNPs) in TMEM106B encoding the lysosomal type II transmembrane protein 106B increase the risk for frontotemporal lobar degeneration (FTLD) of GRN (progranulin gene) mutation carriers. Currently, it is unclear if progranulin (PGRN) and TMEM106B are synergistically linked and if a gain or a loss of function of TMEM106B is responsible for the increased disease risk of patients with GRN haploinsufficiency. We therefore compare behavioral abnormalities, gene expression patterns, lysosomal activity, and TDP-43 pathology in single and double knockout animals. Grn-/- /Tmem106b-/- mice show a strongly reduced life span and massive motor deficits. Gene expression analysis reveals an upregulation of molecular signature characteristic for disease-associated microglia and autophagy. Dysregulation of maturation of lysosomal proteins as well as an accumulation of ubiquitinated proteins and widespread p62 deposition suggest that proteostasis is impaired. Moreover, while single Grn-/- knockouts only occasionally show TDP-43 pathology, the double knockout mice exhibit deposition of phosphorylated TDP-43. Thus, a loss of function of TMEM106B may enhance the risk for GRN-associated FTLD by reduced protein turnover in the lysosomal/autophagic system.
Assuntos
Degeneração Lobar Frontotemporal , Peptídeos e Proteínas de Sinalização Intercelular , Animais , Degeneração Lobar Frontotemporal/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Lisossomos , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso , Progranulinas/genéticaRESUMO
Genetic variants in the triggering receptor expressed on myeloid cells 2 (TREM2) increase the risk for several neurodegenerative diseases including Alzheimer's disease and frontotemporal dementia (FTD). Homozygous TREM2 missense mutations, such as p.T66M, lead to the FTD-like syndrome, but how they cause pathology is unknown. Using CRISPR/Cas9 genome editing, we generated a knock-in mouse model for the disease-associated Trem2 p.T66M mutation. Consistent with a loss-of-function mutation, we observe an intracellular accumulation of immature mutant Trem2 and reduced generation of soluble Trem2 similar to patients with the homozygous p.T66M mutation. Trem2 p.T66M knock-in mice show delayed resolution of inflammation upon in vivo lipopolysaccharide stimulation and cultured macrophages display significantly reduced phagocytic activity. Immunohistochemistry together with in vivo TSPO small animal positron emission tomography (µPET) demonstrates an age-dependent reduction in microglial activity. Surprisingly, perfusion magnetic resonance imaging and FDG-µPET imaging reveal a significant reduction in cerebral blood flow and brain glucose metabolism. Thus, we demonstrate that a TREM2 loss-of-function mutation causes brain-wide metabolic alterations pointing toward a possible function of microglia in regulating brain glucose metabolism.
Assuntos
Encéfalo/patologia , Demência Frontotemporal/patologia , Glucose/metabolismo , Glicoproteínas de Membrana/genética , Microglia/fisiologia , Mutação de Sentido Incorreto , Perfusão , Receptores Imunológicos/genética , Animais , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Humanos , Imuno-Histoquímica , Imageamento por Ressonância Magnética , Camundongos , Proteínas Mutantes/genética , Tomografia por Emissão de PósitronsRESUMO
Expansion of a (G4C2)n repeat in C9orf72 causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the link of the five repeat-encoded dipeptide repeat (DPR) proteins to neuroinflammation, TDP-43 pathology, and neurodegeneration is unclear. Poly-PR is most toxic in vitro, but poly-GA is far more abundant in patients. To directly compare these in vivo, we created congenic poly-GA and poly-PR mice. 40% of poly-PR mice were affected with ataxia and seizures, requiring euthanasia by 6 weeks of age. The remaining poly-PR mice were asymptomatic at 14 months of age, likely due to an 80% reduction of the transgene mRNA in this subgroup. In contrast, all poly-GA mice showed selective neuron loss, inflammation, as well as muscle denervation and wasting requiring euthanasia before 7 weeks of age. In-depth analysis of peripheral organs and blood samples suggests that peripheral organ failure does not drive these phenotypes. Although transgene mRNA levels were similar between poly-GA and affected poly-PR mice, poly-GA aggregated far more abundantly than poly-PR in the CNS and was also found in skeletal muscle. In addition, TDP-43 and other disease-linked RNA-binding proteins co-aggregated in rare nuclear inclusions in the hippocampus and frontal cortex only in poly-GA mice. Transcriptome analysis revealed activation of an interferon-responsive pro-inflammatory microglial signature in end-stage poly-GA but not poly-PR mice. This signature was also found in all ALS patients and enriched in C9orf72 cases. In summary, our rigorous comparison of poly-GA and poly-PR toxicity in vivo indicates that poly-GA, but not poly-PR at the same mRNA expression level, promotes interferon responses in C9orf72 disease and contributes to TDP-43 abnormalities and neuron loss selectively in disease-relevant regions.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteína C9orf72/genética , Interferons/biossíntese , Degeneração Neural/patologia , Esclerose Lateral Amiotrófica/imunologia , Esclerose Lateral Amiotrófica/patologia , Animais , Expansão das Repetições de DNA/genética , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Degeneração Neural/genética , Degeneração Neural/imunologia , Neurônios/patologiaRESUMO
The generation of targeted mouse mutants is a key technology for biomedical research. Using the CRISPR/Cas9 system for induction of targeted double-strand breaks, gene editing can be performed in a single step directly in mouse zygotes. This article covers the design of knockout and knockin alleles, preparation of reagents, microinjection or electroporation of zygotes and the genotyping of pups derived from gene editing projects. In addition we include a section for the control of experimental settings by targeting the Rosa26 locus and PCR based genotyping of blastocysts.
Assuntos
Proteínas de Bactérias/genética , Sistemas CRISPR-Cas , Endonucleases/genética , Edição de Genes/métodos , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Técnicas de Transferência de Genes , RNA Guia de Cinetoplastídeos/genética , Animais , Animais Recém-Nascidos , Proteínas de Bactérias/metabolismo , Proteína 9 Associada à CRISPR , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA/genética , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Eletroporação/métodos , Endonucleases/metabolismo , Marcação de Genes/métodos , Genoma , Camundongos , Camundongos Transgênicos , Microinjeções , RNA Guia de Cinetoplastídeos/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Reparo de DNA por Recombinação , Zigoto/citologia , Zigoto/metabolismoRESUMO
DNA double-strand breaks (DSBs) are produced intentionally by RNA-guided nucleases to achieve genome editing through DSB repair. These breaks are repaired by one of two main repair pathways, classic non-homologous end joining (c-NHEJ) and homology-directed repair (HDR), the latter being restricted to the S/G2 phases of the cell cycle and notably less frequent. Precise genome editing applications rely on HDR, with the abundant c-NHEJ formed mutations presenting a barrier to achieving high rates of precise sequence modifications. Here, we give an overview of HDR- and c-NHEJ-mediated DSB repair in gene editing and summarize the current efforts to promote HDR over c-NHEJ.
Assuntos
Reparo do DNA , Edição de Genes , Animais , Biomarcadores , Sistemas CRISPR-Cas , Reparo do DNA por Junção de Extremidades , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Testes Genéticos , Recombinação Homóloga , Humanos , Reparo de DNA por Recombinação , Transdução de SinaisRESUMO
The study of genetic disease mechanisms relies mostly on targeted mouse mutants that are derived from engineered embryonic stem (ES) cells. Nevertheless, the establishment of mutant ES cells is laborious and time-consuming, restricting the study of the increasing number of human disease mutations discovered by high-throughput genomic analysis. Here, we present an advanced approach for the production of mouse disease models by microinjection of transcription activator-like effector nucleases (TALENs) and synthetic oligodeoxynucleotides into one-cell embryos. Within 2 d of embryo injection, we created and corrected chocolate missense mutations in the small GTPase RAB38; a regulator of intracellular vesicle trafficking and phenotypic model of Hermansky-Pudlak syndrome. Because ES cell cultures and targeting vectors are not required, this technology enables instant germline modifications, making heterozygous mutants available within 18 wk. The key features of direct mutagenesis by TALENs and oligodeoxynucleotides, minimal effort and high speed, catalyze the generation of future in vivo models for the study of human disease mechanisms and interventions.
Assuntos
Modelos Animais de Doenças , Doenças Genéticas Inatas/genética , Mutação em Linhagem Germinativa , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Fase de Clivagem do Zigoto , Desoxirribonucleases/administração & dosagem , Desoxirribonucleases/genética , Feminino , Técnicas Genéticas , Vetores Genéticos , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Microinjeções , Dados de Sequência Molecular , Mutagênese , Mutação de Sentido Incorreto , Gravidez , Homologia de Sequência do Ácido Nucleico , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Gene engineering for generating targeted mouse mutants is a key technology for biomedical research. Using TALENs as nucleases to induce targeted double-strand breaks, the mouse genome can be directly modified in zygotes in a single step, without the need for embryonic stem cells. Thereby, knockout and knockin alleles can be generated fast and efficiently by embryo microinjection of TALEN mRNAs and targeting vectors. In this article we present an introduction into the TALEN technology and provide protocols for the application of TALENs in mouse zygotes.
Assuntos
Mutagênese Sítio-Dirigida/métodos , Animais , Desoxirribonucleases/química , Desoxirribonucleases/genética , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Camundongos , MicroinjeçõesRESUMO
Innate immune responses are linked to key metabolic pathways, yet the proximal signaling events that connect these systems remain poorly understood. Here we show that phosphofructokinase 1, liver type (PFKL), a rate-limiting enzyme of glycolysis, is phosphorylated at Ser775 in macrophages following several innate stimuli. This phosphorylation increases the catalytic activity of PFKL, as shown by biochemical assays and glycolysis monitoring in cells expressing phosphorylation-defective PFKL variants. Using a genetic mouse model in which PFKL Ser775 phosphorylation cannot take place, we observe that upon activation, glycolysis in macrophages is lower than in the same cell population of wild-type animals. Consistent with their higher glycolytic activity, wild-type cells have higher levels of HIF1α and IL-1ß than PfklS775A/S775A after LPS treatment. In an in vivo inflammation model, PfklS775A/S775A mice show reduced levels of MCP-1 and IL-1ß. Our study thus identifies a molecular link between innate immune activation and early induction of glycolysis.
Assuntos
Glicólise , Subunidade alfa do Fator 1 Induzível por Hipóxia , Imunidade Inata , Interleucina-1beta , Macrófagos , Animais , Macrófagos/metabolismo , Macrófagos/imunologia , Camundongos , Fosforilação , Interleucina-1beta/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Receptores de Reconhecimento de Padrão/metabolismo , Receptores de Reconhecimento de Padrão/genética , Fosfofrutoquinase-1/metabolismo , Fosfofrutoquinase-1/genética , Lipopolissacarídeos/farmacologia , Camundongos Endogâmicos C57BL , Humanos , Quimiocina CCL2/metabolismo , Quimiocina CCL2/genética , Inflamação/metabolismo , Masculino , Reprogramação MetabólicaRESUMO
Restless legs syndrome (RLS) is a neurological disorder characterized by uncomfortable or unpleasant sensations in the legs during rest periods. To relieve these sensations, patients move their legs, causing sleep disruption. While the pathogenesis of RLS has yet to be resolved, there is a strong genetic association with the MEIS1 gene. A missense variant in MEIS1 is enriched sevenfold in people with RLS compared to non-affected individuals. We generated a mouse line carrying this mutation (p.Arg272His/c.815G>A), referred to herein as Meis1R272H/R272H (Meis1 point mutation), to determine whether it would phenotypically resemble RLS. As women are more prone to RLS, driven partly by an increased risk of developing RLS during pregnancy, we focused on female homozygous mice. We evaluated RLS-related outcomes, particularly sensorimotor behavior and sleep, in young and aged mice. Compared to noncarrier littermates, homozygous mice displayed very few differences. Significant hyperactivity occurred before the lights-on (rest) period in aged female mice, reflecting the age-dependent incidence of RLS. Sensory experiments involving tactile feedback (rotarod, wheel running, and hotplate) were only marginally different. Overall, RLS-like phenomena were not recapitulated except for the increased wake activity prior to rest. This is likely due to the focus on young mice. Nevertheless, the Meis1R272H mouse line is a potentially useful RLS model, carrying a clinically relevant variant and showing an age-dependent phenotype.
Assuntos
Proteínas de Homeodomínio , Proteína Meis1 , Síndrome das Pernas Inquietas , Animais , Feminino , Humanos , Camundongos , Fatores Etários , Modelos Animais de Doenças , Proteínas de Homeodomínio/genética , Camundongos Endogâmicos C57BL , Mutação de Sentido Incorreto/genética , Proteína Meis1/genética , Proteínas de Neoplasias/genética , Fenótipo , Mutação Puntual/genética , Síndrome das Pernas Inquietas/genética , Síndrome das Pernas Inquietas/fisiopatologia , Sono/genética , Sono/fisiologiaRESUMO
NMDA receptors (NMDARs) are ionotropic receptors crucial for brain information processing. Yet, evidence also supports an ion-flux-independent signaling mode mediating synaptic long-term depression (LTD) and spine shrinkage. Here, we identify AETA (Aη), an amyloid-ß precursor protein (APP) cleavage product, as an NMDAR modulator with the unique dual regulatory capacity to impact both signaling modes. AETA inhibits ionotropic NMDAR activity by competing with the co-agonist and induces an intracellular conformational modification of GluN1 subunits. This favors non-ionotropic NMDAR signaling leading to enhanced LTD and favors spine shrinkage. Endogenously, AETA production is increased by in vivo chemogenetically induced neuronal activity. Genetic deletion of AETA production alters NMDAR transmission and prevents LTD, phenotypes rescued by acute exogenous AETA application. This genetic deletion also impairs contextual fear memory. Our findings demonstrate AETA-dependent NMDAR activation (ADNA), characterizing AETA as a unique type of endogenous NMDAR modulator that exerts bidirectional control over NMDAR signaling and associated information processing.
Assuntos
Precursor de Proteína beta-Amiloide , Receptores de N-Metil-D-Aspartato , Transdução de Sinais , Animais , Humanos , Camundongos , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Espinhas Dendríticas/metabolismo , Medo/fisiologia , Hipocampo/metabolismo , Depressão Sináptica de Longo Prazo/fisiologia , Depressão Sináptica de Longo Prazo/efeitos dos fármacos , Memória/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais/fisiologia , RatosRESUMO
Loss-of-function mutations in the homotrimeric serine protease HTRA1 cause cerebral vasculopathy. Here, we establish independent approaches to achieve the functional correction of trimer assembly defects. Focusing on the prototypical R274Q mutation, we identify an HTRA1 variant that promotes trimer formation thus restoring enzymatic activity in vitro. Genetic experiments in Htra1R274Q mice further demonstrate that expression of this protein-based corrector in trans is sufficient to stabilize HtrA1-R274Q and restore the proteomic signature of the brain vasculature. An alternative approach employs supramolecular chemical ligands that shift the monomer-trimer equilibrium towards proteolytically active trimers. Moreover, we identify a peptidic ligand that activates HTRA1 monomers. Our findings open perspectives for tailored protein repair strategies.
Assuntos
Serina Peptidase 1 de Requerimento de Alta Temperatura A , Serina Peptidase 1 de Requerimento de Alta Temperatura A/metabolismo , Serina Peptidase 1 de Requerimento de Alta Temperatura A/genética , Animais , Humanos , Camundongos , Conformação Proteica , Multimerização Proteica , Células HEK293 , Encéfalo/metabolismo , Encéfalo/patologia , Mutação , Mutação com Perda de FunçãoRESUMO
Engineering of the mouse germline is a key technology in biomedical research for studying the function of genes in health and disease. Since the first knockout mouse was described in 1989, gene targeting was based on recombination of vector encoded sequences in mouse embryonic stem cell lines and their introduction into preimplantation embryos to obtain germline chimeric mice. This approach has been replaced in 2013 by the application of the RNA-guided CRISPR/Cas9 nuclease system, which is introduced into zygotes and directly creates targeted modifications in the mouse genome. Upon the introduction of Cas9 nuclease and guide RNAs into one-cell embryos, sequence-specific double-strand breaks are created that are highly recombinogenic and processed by DNA repair enzymes. Gene editing commonly refers to the diversity of DSB repair products that include imprecise deletions or precise sequence modifications copied from repair template molecules. Since gene editing can now be easily applied directly in mouse zygotes, it has rapidly become the standard procedure for generating genetically engineered mice. This article covers the design of guide RNAs, knockout and knockin alleles, options for donor delivery, preparation of reagents, microinjection or electroporation of zygotes, and the genotyping of pups derived from gene editing projects.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Camundongos , Edição de Genes/métodos , Sistemas CRISPR-Cas/genética , Zigoto/metabolismo , Marcação de Genes/métodos , Camundongos Knockout , RNA Guia de Sistemas CRISPR-CasRESUMO
The recent CRISPR revolution has provided researchers with powerful tools to perform genome editing in a variety of organisms. However, recent reports indicate widespread occurrence of unintended CRISPR-induced on-target effects (OnTEs) at the edited site in mice and human induced pluripotent stem cells (iPSCs) that escape standard quality controls. By altering gene expression of targeted or neighbouring genes, OnTEs can severely affect phenotypes of CRISPR-edited cells and organisms and thus lead to data misinterpretation, which can undermine the reliability of CRISPR-based studies. Here we describe a broadly applicable framework for detecting OnTEs in genome-edited cells and organisms after non-homologous end joining-mediated and homology-directed repair-mediated editing. Our protocol enables identification of OnTEs such as large deletions, large insertions, rearrangements or loss of heterozygosity (LOH). This is achieved by subjecting genomic DNA first to quantitative genotyping PCR (qgPCR), which determines the number of intact alleles at the target site using the same PCR amplicon that has been optimized for genotyping. This combination of genotyping and quantitation makes it possible to exclude clones with monoallelic OnTEs and hemizygous editing, which are often mischaracterized as correctly edited in standard Sanger sequencing. Second, occurrence of LOH around the edited locus is detected by genotyping neighbouring single-nucleotide polymorphisms (SNPs), using either a Sanger sequencing-based method or SNP microarrays. All steps are optimized to maximize simplicity and minimize cost to promote wide dissemination and applicability across the field. The entire protocol from genomic DNA extraction to OnTE exclusion can be performed in 6-9 d.
Assuntos
Edição de Genes/métodos , Engenharia Genética/métodos , Polimorfismo de Nucleotídeo Único/genética , Animais , Sequência de Bases/genética , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Reparo do DNA por Junção de Extremidades/genética , Genótipo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , RNA Guia de Cinetoplastídeos/genética , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Microglia-specific genetic variants are enriched in several neurodegenerative diseases, including Alzheimer's disease (AD), implicating a central role for alterations of the innate immune system in the disease etiology. A rare coding variant in the PLCG2 gene (rs72824905, p.P522R) expressed in myeloid lineage cells was recently identified and shown to reduce the risk for AD. METHODS: To assess the role of the protective variant in the context of immune cell functions, we generated a Plcγ2-P522R knock-in (KI) mouse model using CRISPR/Cas9 gene editing. RESULTS: Functional analyses of macrophages derived from homozygous KI mice and wild type (WT) littermates revealed that the P522R variant potentiates the primary function of Plcγ2 as a Pip2-metabolizing enzyme. This was associated with improved survival and increased acute inflammatory response of the KI macrophages. Enhanced phagocytosis was observed in mouse BV2 microglia-like cells overexpressing human PLCγ2-P522R, but not in PLCγ2-WT expressing cells. Immunohistochemical analyses did not reveal changes in the number or morphology of microglia in the cortex of Plcγ2-P522R KI mice. However, the brain mRNA signature together with microglia-related PET imaging suggested enhanced microglial functions in Plcγ2-P522R KI mice. CONCLUSION: The AD-associated protective Plcγ2-P522R variant promotes protective functions associated with TREM2 signaling. Our findings provide further support for the idea that pharmacological modulation of microglia via TREM2-PLCγ2 pathway-dependent stimulation may be a novel therapeutic option for the treatment of AD.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/imunologia , Fosfolipase C gama/genética , Animais , Técnicas de Introdução de Genes , Variação Genética , Humanos , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Microglia/imunologia , Fosfolipase C gama/imunologiaRESUMO
Genetic variations in TMEM106B, coding for a lysosomal membrane protein, affect frontotemporal lobar degeneration (FTLD) in GRN- (coding for progranulin) and C9orf72-expansion carriers and might play a role in aging. To determine the physiological function of TMEM106B, we generated TMEM106B-deficient mice. These mice develop proximal axonal swellings caused by drastically enlarged LAMP1-positive vacuoles, increased retrograde axonal transport of lysosomes, and accumulation of lipofuscin and autophagosomes. Giant vacuoles specifically accumulate at the distal end and within the axon initial segment, but not in peripheral nerves or at axon terminals, resulting in an impaired facial-nerve-dependent motor performance. These data implicate TMEM106B in mediating the axonal transport of LAMP1-positive organelles in motoneurons and axonal sorting at the initial segment. Our data provide mechanistic insight into how TMEM106B affects lysosomal proteolysis and degradative capacity in neurons.
Assuntos
Segmento Inicial do Axônio/metabolismo , Degeneração Lobar Frontotemporal/genética , Predisposição Genética para Doença , Lisossomos/metabolismo , Proteínas de Membrana/genética , Neurônios Motores/metabolismo , Proteínas do Tecido Nervoso/genética , Animais , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Segmento Inicial do Axônio/ultraestrutura , Transporte Axonal , Tronco Encefálico/patologia , Núcleo Celular/metabolismo , Nervo Facial/patologia , Lisossomos/ultraestrutura , Proteínas de Membrana/deficiência , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios Motores/ultraestrutura , Músculos/inervação , Proteínas do Tecido Nervoso/deficiência , Fatores de RiscoRESUMO
In recent years RNA interference (RNAi) has become a useful genetic tool to downregulate candidate disease genes for which pharmaceutical inhibitors are not available. In combination with viral vectors to trigger RNAi in the mammalian body, it allows the localized and specific manipulation of the expression of single or multiple genes in vivo. The MAP kinases ERK1 and ERK2 are involved in the transduction of extracellular signals to nuclear effectors. A role for ERKs has been proposed in the adult brain in mediating neuronal functions, as for fear learning in the lateral amygdala. To study the role of ERK in anxiety disorders characterized by disturbed fear learning processes we developed Erk-specific RNAi tools and tested the efficacy of a viral Erk2 vector in the adult mouse brain. We found shRNAs that showed silencing of either both ERK1/2 or only ERK2. In particular, our analysis showed that an Erk2-specific shRNA reduced the activity of this gene at comparable efficiency both in vitro and in vivo. This reagent provides a useful tool to study the role of ERK2, for which small molecule inhibitors are not available, in the development of anxiety and other psychiatric disorders.
Assuntos
Tonsila do Cerebelo/enzimologia , Dependovirus/genética , Técnicas de Silenciamento de Genes/métodos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Interferência de RNA , Tonsila do Cerebelo/citologia , Análise de Variância , Animais , Transtornos de Ansiedade/enzimologia , Dependovirus/metabolismo , Regulação para Baixo , Vetores Genéticos , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: The R47H variant of the Triggering Receptor Expressed on Myeloid cells 2 (TREM2) significantly increases the risk for late onset Alzheimer's disease. Mouse models accurately reproducing phenotypes observed in Alzheimer' disease patients carrying the R47H coding variant are required to understand the TREM2 related dysfunctions responsible for the enhanced risk for late onset Alzheimer's disease. METHODS: A CRISPR/Cas9-assisted gene targeting strategy was used to generate Trem2 R47H knock-in mice. Trem2 mRNA and protein levels as well as Trem2 splicing patterns were assessed in these mice, in iPSC-derived human microglia-like cells, and in human brains from Alzheimer's patients carrying the TREM2 R47H risk factor. RESULTS: Two independent Trem2 R47H knock-in mouse models show reduced Trem2 mRNA and protein production. In both mouse models Trem2 haploinsufficiency was due to atypical splicing of mouse Trem2 R47H, which introduced a premature stop codon. Cellular splicing assays using minigene constructs demonstrate that the R47H variant induced abnormal splicing only occurs in mice but not in humans. TREM2 mRNA levels and splicing patterns were both normal in iPSC-derived human microglia-like cells and patient brains with the TREM2 R47H variant. CONCLUSIONS: The Trem2 R47H variant activates a cryptic splice site that generates miss-spliced transcripts leading to Trem2 haploinsufficiency only in mice but not in humans. Since Trem2 R47H related phenotypes are mouse specific and do not occur in humans, humanized TREM2 R47H knock-in mice should be generated to study the cellular consequences caused by the human TREM2 R47H coding variant. Currently described phenotypes of Trem2 R47H knock-in mice can therefore not be translated to humans.