RESUMO
Dysregulated microglia are intimately involved in neurodegeneration, including Alzheimer's disease (AD) pathogenesis, but the mechanisms controlling pathogenic microglial gene expression remain poorly understood. The transcription factor CCAAT/enhancer binding protein beta (c/EBPß) regulates pro-inflammatory genes in microglia and is upregulated in AD. We show expression of c/EBPß in microglia is regulated post-translationally by the ubiquitin ligase COP1 (also called RFWD2). In the absence of COP1, c/EBPß accumulates rapidly and drives a potent pro-inflammatory and neurodegeneration-related gene program, evidenced by increased neurotoxicity in microglia-neuronal co-cultures. Antibody blocking studies reveal that neurotoxicity is almost entirely attributable to complement. Remarkably, loss of a single allele of Cebpb prevented the pro-inflammatory phenotype. COP1-deficient microglia markedly accelerated tau-mediated neurodegeneration in a mouse model where activated microglia play a deleterious role. Thus, COP1 is an important suppressor of pathogenic c/EBPß-dependent gene expression programs in microglia.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Ligases/metabolismo , Microglia/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/genética , Doença de Alzheimer/metabolismo , Animais , Linhagem Celular , Técnicas de Cocultura/métodos , Feminino , Expressão Gênica/fisiologia , Regulação da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismoRESUMO
Human genetics and preclinical studies have identified key contributions of TREM2 to several neurodegenerative conditions, inspiring efforts to modulate TREM2 therapeutically. Here, we characterize the activities of three TREM2 agonist antibodies in multiple mixed-sex mouse models of Alzheimer's disease (AD) pathology and remyelination. Receptor activation and downstream signaling are explored in vitro, and active dose ranges are determined in vivo based on pharmacodynamic responses from microglia. For mice bearing amyloid-ß (Aß) pathology (PS2APP) or combined Aß and tau pathology (TauPS2APP), chronic TREM2 agonist antibody treatment had limited impact on microglia engagement with pathology, overall pathology burden, or downstream neuronal damage. For mice with demyelinating injuries triggered acutely with lysolecithin, TREM2 agonist antibodies unexpectedly disrupted injury resolution. Likewise, TREM2 agonist antibodies limited myelin recovery for mice experiencing chronic demyelination from cuprizone. We highlight the contributions of dose timing and frequency across models. These results introduce important considerations for future TREM2-targeting approaches.
Assuntos
Doença de Alzheimer , Glicoproteínas de Membrana , Microglia , Esclerose Múltipla , Receptores Imunológicos , Animais , Receptores Imunológicos/agonistas , Receptores Imunológicos/metabolismo , Receptores Imunológicos/genética , Glicoproteínas de Membrana/agonistas , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Camundongos , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/imunologia , Feminino , Masculino , Microglia/efeitos dos fármacos , Microglia/metabolismo , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Anticorpos/farmacologia , Humanos , Peptídeos beta-Amiloides/metabolismo , Proteínas tau/metabolismoRESUMO
INTRODUCTION: Triggering receptor expressed on myeloid cells 2 (TREM2) agonists are being clinically evaluated as disease-modifying therapeutics for Alzheimer's disease. Clinically translatable pharmacodynamic (PD) biomarkers are needed to confirm drug activity and select the appropriate therapeutic dose in clinical trials. METHODS: We conducted multi-omic analyses on paired non-human primate brain and cerebrospinal fluid (CSF), and stimulation of human induced pluripotent stem cell-derived microglia cultures after TREM2 agonist treatment, followed by validation of candidate fluid PD biomarkers using immunoassays. We immunostained microglia to characterize proliferation and clustering. RESULTS: We report CSF soluble TREM2 (sTREM2) and CSF chitinase-3-like protein 1 (CHI3L1/YKL-40) as PD biomarkers for the TREM2 agonist hPara.09. The respective reduction of sTREM2 and elevation of CHI3L1 in brain and CSF after TREM2 agonist treatment correlated with transient microglia proliferation and clustering. DISCUSSION: CSF CHI3L1 and sTREM2 reflect microglial TREM2 agonism and can be used as clinical PD biomarkers to monitor TREM2 activity in the brain. HIGHLIGHTS: CSF soluble triggering receptor expressed on myeloid cells 2 (sTREM2) reflects brain target engagement for a novel TREM2 agonist, hPara.09. CSF chitinase-3-like protein 1 reflects microglial TREM2 agonism. Both can be used as clinical fluid biomarkers to monitor TREM2 activity in brain.
RESUMO
Disability in multiple sclerosis (MS) is driven in part by the failure of remyelination and progressive neurodegeneration. Microglia, and specifically triggering receptor expressed on myeloid cells 2 (TREM2), a factor highly expressed in microglia, have been shown to play an important role in remyelination. Here, using a focal demyelination model in the brain, we demonstrate that demyelination is persistent in TREM2 knockout mice, lasting more than 6 weeks after lysolecithin injection and resulting in substantial neurodegeneration. We also find that TREM2 knockout mice exhibit an altered glial response following demyelination. TREM2 knockout microglia demonstrate defects in migration and phagocytosis of myelin debris. In addition, human monocyte-derived macrophages from subjects with a TREM2 mutation prevalent in human disease also show a defect in myelin debris phagocytosis. Together, we highlight the central role of TREM2 signaling in remyelination and neuroprotection. These findings provide insights into how chronic demyelination might lead to axonal damage and could help identify novel neuroprotective therapeutic targets for MS.
Assuntos
Esclerose Múltipla , Remielinização , Animais , Camundongos , Humanos , Microglia/fisiologia , Neuroproteção , Esclerose Múltipla/tratamento farmacológico , Bainha de Mielina , Camundongos Knockout , Camundongos Endogâmicos C57BL , Glicoproteínas de Membrana/genética , Receptores Imunológicos/genéticaRESUMO
Parkinson's disease (PD), a neurodegenerative disease affecting dopaminergic (DA) neurons, is characterized by decline of motor function and cognition. Dopaminergic cell loss is associated with accumulation of toxic alpha synuclein aggregates. As DA neuron death occurs late in the disease, therapeutics that block the spread of alpha synuclein may offer functional benefit and delay disease progression. To test this hypothesis, we generated antibodies to the C terminal region of synuclein with high nanomolar affinity and characterized them in in vitro and in vivo models of spread. Interestingly, we found that only antibodies with high affinity to the distal most portion of the C-terminus robustly reduced uptake of alpha synuclein preformed fibrils (PFF) and accumulation of phospho (S129) alpha synuclein in cell culture. Additionally, the antibody treatment blocked the spread of phospho (S129) alpha synuclein associated-pathology in a mouse model of synucleinopathy. Blockade of neuronal PFF uptake by different antibodies was more predictive of in vivo activity than their binding potency to monomeric or oligomeric forms of alpha synuclein. These data demonstrate that antibodies directed to the C-terminus of the alpha synuclein have differential effects on target engagement and efficacy. Furthermore, our data provides additional support for the development of alpha synuclein antibodies as a therapeutic strategy for PD patients.
Assuntos
Doenças Neurodegenerativas , Doença de Parkinson , Sinucleinopatias , Camundongos , Animais , alfa-Sinucleína/metabolismo , Doença de Parkinson/metabolismo , Doenças Neurodegenerativas/metabolismo , Sinucleinopatias/patologia , Neurônios Dopaminérgicos/metabolismoRESUMO
Familial neurodegenerative diseases commonly involve mutations that result in either aberrant proteins or dysfunctional components of the proteolytic machinery that act on aberrant proteins. UBQLN2 is a ubiquitin receptor of the UBL/UBA family that binds the proteasome through its ubiquitin-like domain and is thought to deliver ubiquitinated proteins to proteasomes for degradation. UBQLN2 mutations result in familial amyotrophic lateral sclerosis (ALS)/frontotemporal dementia in humans through an unknown mechanism. Quantitative multiplexed proteomics was used to provide for the first time an unbiased and global analysis of the role of Ubqln2 in controlling the composition of the proteome. We studied several murine models of Ubqln2-linked ALS and also generated Ubqln2 null mutant mice. We identified impacts of Ubqln2 on diverse physiological pathways, most notably serotonergic signaling. Interestingly, we observed an upregulation of proteasome subunits, suggesting a compensatory response to diminished proteasome output. Among the specific proteins whose abundance is linked to UBQLN2 function, the strongest hits were the ubiquitin ligase TRIM32 and two retroelement-derived proteins, PEG10 and CXX1B. Cycloheximide chase studies using induced human neurons and HEK293 cells suggested that PEG10 and TRIM32 are direct clients. Although UBQLN2 directs the degradation of multiple proteins via the proteasome, it surprisingly conferred strong protection from degradation on the Gag-like protein CXX1B, which is expressed from the same family of retroelement genes as PEG10. In summary, this study charts the proteomic landscape of ALS-related Ubqln2 mutants and identifies candidate client proteins that are altered in vivo in disease models and whose degradation is promoted by UBQLN2.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Esclerose Lateral Amiotrófica/genética , Proteínas Relacionadas à Autofagia/genética , Demência Frontotemporal/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteômica/métodos , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Relacionadas à Autofagia/deficiência , Proteínas Relacionadas à Autofagia/metabolismo , Linhagem Celular , Cicloeximida/farmacologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Regulação da Expressão Gênica , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Estabilidade Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Serotonina/metabolismo , Transdução de Sinais , Transativadores/genética , Transativadores/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Receptor-interacting protein kinase 1 (RIPK1) promotes cell survival-mice lacking RIPK1 die perinatally, exhibiting aberrant caspase-8-dependent apoptosis and mixed lineage kinase-like (MLKL)-dependent necroptosis. However, mice expressing catalytically inactive RIPK1 are viable, and an ill-defined pro-survival function for the RIPK1 scaffold has therefore been proposed. Here we show that the RIP homotypic interaction motif (RHIM) in RIPK1 prevents the RHIM-containing adaptor protein ZBP1 (Z-DNA binding protein 1; also known as DAI or DLM1) from activating RIPK3 upstream of MLKL. Ripk1RHIM/RHIM mice that expressed mutant RIPK1 with critical RHIM residues IQIG mutated to AAAA died around birth and exhibited RIPK3 autophosphorylation on Thr231 and Ser232, which is a hallmark of necroptosis, in the skin and thymus. Blocking necroptosis with catalytically inactive RIPK3(D161N), RHIM mutant RIPK3, RIPK3 deficiency, or MLKL deficiency prevented lethality in Ripk1RHIM/RHIM mice. Loss of ZBP1, which engages RIPK3 in response to certain viruses but previously had no defined role in development, also prevented perinatal lethality in Ripk1RHIM/RHIM mice. Consistent with the RHIM of RIPK1 functioning as a brake that prevents ZBP1 from engaging the RIPK3 RHIM, ZBP1 interacted with RIPK3 in Ripk1RHIM/RHIMMlkl-/- macrophages, but not in wild-type, Mlkl-/- or Ripk1RHIM/RHIMRipk3RHIM/RHIM macrophages. Collectively, these findings indicate that the RHIM of RIPK1 is critical for preventing ZBP1/RIPK3/MLKL-dependent necroptosis during development.
Assuntos
Apoptose , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/metabolismo , Necrose , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Motivos de Aminoácidos , Animais , Animais Recém-Nascidos , Caspase 8/genética , Caspase 8/metabolismo , Embrião de Mamíferos/citologia , Feminino , Glicoproteínas/química , Glicoproteínas/deficiência , Macrófagos/metabolismo , Masculino , Camundongos , Mutação , Fosforilação , Ligação Proteica , Proteínas Quinases/deficiência , Proteínas Quinases/metabolismo , Proteínas de Ligação a RNA , Proteína Serina-Treonina Quinases de Interação com Receptores/química , Proteína Serina-Treonina Quinases de Interação com Receptores/deficiência , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
TREM2 is an Alzheimer's disease (AD) risk gene expressed in microglia. To study the role of Trem2 in a mouse model of ß-amyloidosis, we compared PS2APP transgenic mice versus PS2APP mice lacking Trem2 (PS2APP;Trem2ko) at ages ranging from 4 to 22 months. Microgliosis was impaired in PS2APP;Trem2ko mice, with Trem2-deficient microglia showing compromised expression of proliferation/Wnt-related genes and marked accumulation of ApoE. Plaque abundance was elevated in PS2APP;Trem2ko females at 6-7 months; but by 12 or 19-22 months of age, it was notably diminished in female and male PS2APP;Trem2ko mice, respectively. Across all ages, plaque morphology was more diffuse in PS2APP;Trem2ko brains, and the Aß42:Aß40 ratio was elevated. The amount of soluble, fibrillar Aß oligomers also increased in PS2APP;Trem2ko hippocampi. Associated with these changes, axonal dystrophy was exacerbated from 6 to 7 months onward in PS2APP;Trem2ko mice, notwithstanding the reduced plaque load at later ages. PS2APP;Trem2ko mice also exhibited more dendritic spine loss around plaque and more neurofilament light chain in CSF. Thus, aggravated neuritic dystrophy is a more consistent outcome of Trem2 deficiency than amyloid plaque load, suggesting that the microglial packing of Aß into dense plaque is an important neuroprotective activity.SIGNIFICANCE STATEMENT Genetic studies indicate that TREM2 gene mutations confer increased Alzheimer's disease (AD) risk. We studied the effects of Trem2 deletion in the PS2APP mouse AD model, in which overproduction of Aß peptide leads to amyloid plaque formation and associated neuritic dystrophy. Interestingly, neuritic dystrophies were intensified in the brains of Trem2-deficient mice, despite these mice displaying reduced plaque accumulation at later ages (12-22 months). Microglial clustering around plaques was impaired, plaques were more diffuse, and the Aß42:Aß40 ratio and amount of soluble, fibrillar Aß oligomers were elevated in Trem2-deficient brains. These results suggest that the Trem2-dependent compaction of Aß into dense plaques is a protective microglial activity, limiting the exposure of neurons to toxic Aß species.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Axônios/patologia , Espinhas Dendríticas/patologia , Glicoproteínas de Membrana/genética , Fragmentos de Peptídeos/metabolismo , Placa Amiloide/genética , Receptores Imunológicos/genética , Fator Trefoil-1/metabolismo , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/patologia , Neuritos/patologia , Proteínas de Neurofilamentos/líquido cefalorraquidiano , Placa Amiloide/patologiaRESUMO
The gene GPNMB is known to play roles in phagocytosis and tissue repair, and is upregulated in microglia in many mouse models of neurodegenerative disease as well as in human patients. Nearby genomic variants are associated with both elevated Parkinson's disease (PD) risk and higher expression of this gene, suggesting that inhibiting GPNMB activity might be protective in Parkinson's disease. We tested this hypothesis in three different mouse models of neurological diseases: a remyelination model and two models of alpha-synuclein pathology. We found that Gpnmb deletion had no effect on histological, cellular, behavioral, neurochemical or gene expression phenotypes in any of these models. These data suggest that Gpnmb does not play a major role in the development of pathology or functional defects in these models and that further work is necessary to study its role in the development or progression of Parkinson's disease.
Assuntos
Proteínas do Olho/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Doença de Parkinson/metabolismo , Remielinização/genética , Substância Negra/metabolismo , Sinucleinopatias/genética , Idoso , Idoso de 80 Anos ou mais , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Doença de Parkinson/patologia , Substância Negra/patologia , Sinucleinopatias/metabolismo , Sinucleinopatias/patologiaRESUMO
The E3 ubiquitin ligase CRL4COP1/DET1 is active in the absence of ERK signaling, modifying the transcription factors ETV1, ETV4, ETV5, and c-JUN with polyubiquitin that targets them for proteasomal degradation. Here we show that this posttranslational regulatory mechanism is active in neurons, with ETV5 and c-JUN accumulating within minutes of ERK activation. Mice with constitutive photomorphogenesis 1 (Cop1) deleted in neural stem cells showed abnormally elevated expression of ETV1, ETV4, ETV5, and c-JUN in the developing brain and spinal cord. Expression of c-JUN target genes Vimentin and Gfap was increased, whereas ETV5 and c-JUN both contributed to an expanded number of cells expressing genes associated with gliogenesis, including Olig1, Olig2, and Sox10. The mice had subtle morphological abnormalities in the cerebral cortex, hippocampus, and cerebellum by embryonic day 18 and died soon after birth. Elevated c-JUN, ETV5, and ETV1 contributed to the perinatal lethality, as several Cop1-deficient mice also lacking c-Jun and Etv5, or lacking Etv5 and heterozygous for Etv1, were viable.
Assuntos
Encéfalo/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS), a neurodegenerative disease affecting motor neurons, is characterized by rapid decline of motor function and ultimately respiratory failure. As motor neuron death occurs late in the disease, therapeutics that prevent the initial disassembly of the neuromuscular junction may offer optimal functional benefit and delay disease progression. To test this hypothesis, we treated the SOD1G93A mouse model of ALS with an agonist antibody to muscle specific kinase (MuSK), a receptor tyrosine kinase required for the formation and maintenance of the neuromuscular junction. Chronic MuSK antibody treatment fully preserved innervation of the neuromuscular junction when compared with control-treated mice; however, no preservation of diaphragm function, motor neurons, or survival benefit was detected. These data show that anatomical preservation of neuromuscular junctions in the diaphragm via MuSK activation does not correlate with functional benefit in SOD1G93A mice, suggesting caution in employing MuSK activation as a therapeutic strategy for ALS patients.
Assuntos
Esclerose Lateral Amiotrófica/enzimologia , Esclerose Lateral Amiotrófica/fisiopatologia , Diafragma/fisiopatologia , Junção Neuromuscular/fisiopatologia , Receptores Proteína Tirosina Quinases/agonistas , Esclerose Lateral Amiotrófica/patologia , Animais , Diafragma/patologia , Modelos Animais de Doenças , Ativação Enzimática/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios Motores/patologia , Junção Neuromuscular/patologia , Superóxido Dismutase-1/genéticaRESUMO
TNF superfamily death ligands are expressed on the surface of immune cells and can trigger apoptosis in susceptible cancer cells by engaging cognate death receptors. A recombinant soluble protein comprising the ectodomain of Apo2 ligand/TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) has shown remarkable preclinical anticancer activity but lacked broad efficacy in patients, possibly owing to insufficient exposure or potency. We observed that antibody cross-linking substantially enhanced cytotoxicity of soluble Apo2L/TRAIL against diverse cancer cell lines. Presentation of the ligand on glass-supported lipid bilayers enhanced its ability to drive receptor microclustering and apoptotic signaling. Furthermore, covalent surface attachment of Apo2L/TRAIL onto liposomes--synthetic lipid-bilayer nanospheres--similarly augmented activity. In vivo, liposome-displayed Apo2L/TRAIL achieved markedly better exposure and antitumor activity. Thus, covalent synthetic-membrane attachment of a cell-surface ligand enhances efficacy, increasing therapeutic potential. These findings have translational implications for liposomal approaches as well as for Apo2L/TRAIL and other clinically relevant TNF ligands.
Assuntos
Antineoplásicos/química , Membrana Celular/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Apoptose , Biotinilação , Ligante CD27/metabolismo , Caspase 8/metabolismo , Caspases/metabolismo , Linhagem Celular Tumoral , Epitopos/química , Proteína Ligante Fas/metabolismo , Humanos , Sistema Imunitário , Imunoterapia/métodos , Concentração Inibidora 50 , Ligantes , Lipossomos/química , Camundongos , Camundongos Nus , Microscopia de Fluorescência , Transplante de Neoplasias , Neoplasias/imunologia , Neoplasias/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
Axon degeneration is a programed process that takes place during development, in response to neuronal injury, and as a component of neurodegenerative disease pathology, yet the molecular mechanisms that drive this process remain poorly defined. In this study, we have developed a semi-automated, 384-well format axon degeneration assay in rat dorsal root ganglion (DRG) neurons using a trophic factor withdrawal paradigm. Using this setup, we have screened a library of known drugs and bioactives to identify several previously unappreciated regulators of axon degeneration, including lipoxygenases. Multiple structurally distinct lipoxygenase inhibitors as well as mouse DRG neurons lacking expression of 12/15-lipoxygenase display protection of axons in this context. Retinal ganglion cell axons from 12/15-lipoxygenase-null mice were similarly protected from degeneration following nerve crush injury. Through additional mechanistic studies, we demonstrate that lipoxygenases act cell autonomously within neurons to regulate degeneration, and are required for mitochondrial permeabilization and caspase activation in the axon. These findings suggest that these enzymes may represent an attractive target for treatment of neuropathies and provide a potential mechanism for the neuroprotection observed in various settings following lipoxygenase inhibitor treatment.
Assuntos
Araquidonato 12-Lipoxigenase/metabolismo , Araquidonato 15-Lipoxigenase/metabolismo , Axônios/patologia , Degeneração Neural/enzimologia , Algoritmos , Animais , Araquidonato 12-Lipoxigenase/genética , Araquidonato 15-Lipoxigenase/genética , Axônios/metabolismo , Células Cultivadas , Técnicas de Cocultura , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Feminino , Gânglios Espinais/citologia , Biblioteca Gênica , Masculino , Camundongos , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Degeneração Neural/diagnóstico , Degeneração Neural/tratamento farmacológico , Degeneração Neural/etiologia , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Doenças do Nervo Óptico/complicações , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
In polarized, migrating cells, stress fibers are a highly dynamic network of contractile acto-myosin structures composed of bundles of actin filaments held together by actin cross-linking proteins such as α-actinins. As such, α-actinins influence actin cytoskeleton organization and dynamics and focal adhesion maturation. In response to environmental signals, α-actinins are tyrosine phosphorylated and this affects their binding to actin stress fibers; however, the cellular role of α-actinin tyrosine phosphorylation remains largely unknown. We found that non-muscle α-actinin1/4 are critical for the establishment of dorsal stress fibers and maintenance of transverse arc stress fibers. Analysis of cells genetically depleted of α-actinin1 and 4 reveals two distinct modes for focal adhesion maturation. An α-actinin1 or 4 dependent mode that uses dorsal stress fiber precursors as a template for establishing focal adhesions and their maturation, and an α-actinin-independent manner that uses transverse arc precursors to establish focal adhesions at both ends. Focal adhesions formed in the absence of α-actinins are delayed in their maturation, exhibit altered morphology, have decreased amounts of Zyxin and VASP, and reduced adhesiveness to extracellular matrix. Further rescue experiments demonstrate that the tyrosine phosphorylation of α-actinin1 at Y12 and α-actinin4 at Y265 is critical for dorsal stress fiber establishment, transverse arc maintenance and focal adhesion maturation.
Assuntos
Actinina/metabolismo , Adesões Focais/fisiologia , Proteínas Tirosina Quinases/metabolismo , Fibras de Estresse/metabolismo , Fibras de Estresse/fisiologia , Actinina/genética , Actinina/fisiologia , Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Quinase 1 de Adesão Focal/metabolismo , Adesões Focais/química , Adesões Focais/genética , Adesões Focais/metabolismo , Células HEK293 , Humanos , Proteínas dos Microfilamentos/metabolismo , Paxilina/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/genética , Fosforilação/fisiologia , Proteínas Tirosina Quinases/fisiologia , Tirosina/genética , Tirosina/metabolismo , Zixina/metabolismoRESUMO
While epidermal growth factor receptor (EGFR) has been shown to be important in the entry process for multiple viruses, including hepatitis C virus (HCV), the molecular mechanisms by which EGFR facilitates HCV entry are not well understood. Using the infectious cell culture HCV model (HCVcc), we demonstrate that the binding of HCVcc particles to human hepatocyte cells induces EGFR activation that is dependent on interactions between HCV and CD81 but not claudin 1. EGFR activation can also be induced by antibody mediated cross-linking of CD81. In addition, EGFR ligands that enhance the kinetics of HCV entry induce EGFR internalization and colocalization with CD81. While EGFR kinase inhibitors inhibit HCV infection primarily by preventing EGFR endocytosis, antibodies that block EGFR ligand binding or inhibitors of EGFR downstream signaling have no effect on HCV entry. These data demonstrate that EGFR internalization is critical for HCV entry and identify a hitherto-unknown association between CD81 and EGFR.
Assuntos
Receptores ErbB/metabolismo , Hepacivirus/metabolismo , Tetraspanina 28/metabolismo , Internalização do Vírus , Linhagem Celular Tumoral , Claudina-1/metabolismo , Receptores ErbB/antagonistas & inibidores , Humanos , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , RNA Interferente Pequeno , RNA ViralRESUMO
Inhibiting angiogenesis has become an important therapeutic strategy for cancer treatment but, like other current targeted therapies, benefits experienced for late-stage cancers can be curtailed by inherent refractoriness or by acquired drug resistance, requiring a need for better mechanistic understanding of such effects. Numerous preclinical studies have demonstrated that VEGF pathway inhibitors suppress primary tumour growth and metastasis. However, it has been recently reported that short-term VEGF and VEGFR inhibition can paradoxically accelerate tumour invasiveness and metastasis in certain models. Here we comprehensively compare the effects of both antibody and small molecule receptor tyrosine kinase (RTK) inhibitors targeting the VEGF-VEGFR pathway, using short-term therapy in various mouse models of metastasis. Our findings demonstrate that antibody inhibition of VEGF pathway molecules does not promote metastasis, in contrast to selected small molecule RTK inhibitors at elevated-therapeutic drug dosages. In particular, a multi-targeted RTK inhibitor, sunitinib, which most profoundly potentiated metastasis, also increased lung vascular permeability and promoted tumour cell extravasation. Mechanistically, sunitinib, but not anti-VEGF treatment, attenuated endothelial barrier function in culture and caused a global inhibition of protein tyrosine phosphorylation, including molecules important for maintaining endothelial cell-cell junctions. Together these findings indicate that, rather than a specific consequence of inhibiting the VEGF signalling pathway, pharmacological inhibitors of the VEGF pathway can have dose- and drug class-dependent side-effects on the host vasculature. These findings also advocate for the continued identification of mechanisms of resistance to anti-angiogenics and for therapy development to overcome it.
Assuntos
Inibidores da Angiogênese/classificação , Inibidores da Angiogênese/uso terapêutico , Antineoplásicos/classificação , Antineoplásicos/uso terapêutico , Metástase Neoplásica/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Animais , Anticorpos Monoclonais/uso terapêutico , Benzamidas , Benzenossulfonatos/uso terapêutico , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/uso terapêutico , Feminino , Mesilato de Imatinib , Técnicas In Vitro , Indóis/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Niacinamida/análogos & derivados , Compostos de Fenilureia , Piperazinas/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Piridinas/uso terapêutico , Pirimidinas/uso terapêutico , Pirróis/uso terapêutico , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/imunologia , Sorafenibe , Sunitinibe , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/imunologiaRESUMO
Tumor progression locus 2 (TPL2) (MAP3K8) is a central signaling node in the inflammatory response of peripheral immune cells. We find that TPL2 kinase activity modulates microglial cytokine release and is required for microglia-mediated neuron death in vitro. In acute in vivo neuroinflammation settings, TPL2 kinase activity regulates microglia activation states and brain cytokine levels. In a tauopathy model of chronic neurodegeneration, loss of TPL2 kinase activity reduces neuroinflammation and rescues synapse loss, brain volume loss, and behavioral deficits. Single-cell RNA sequencing analysis indicates that protection in the tauopathy model was associated with reductions in activated microglia subpopulations as well as infiltrating peripheral immune cells. Overall, using various models, we find that TPL2 kinase activity can promote multiple harmful consequences of microglial activation in the brain including cytokine release, iNOS (inducible nitric oxide synthase) induction, astrocyte activation, and immune cell infiltration. Consequently, inhibiting TPL2 kinase activity could represent a potential therapeutic strategy in neurodegenerative conditions.
Assuntos
MAP Quinase Quinase Quinases , Tauopatias , Animais , Humanos , Camundongos , Encéfalo/patologia , Células Cultivadas , Espinhas Dendríticas/patologia , Lipopolissacarídeos , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Camundongos Knockout , Microglia/metabolismo , Doenças Neuroinflamatórias/patologia , Análise de Sequência de RNA , Análise de Célula Única , Proteínas tau/genética , Proteínas tau/metabolismo , Tauopatias/metabolismo , Tauopatias/patologia , Tauopatias/fisiopatologiaRESUMO
Antisense oligonucleotide (ASO) therapeutics are being investigated for a broad range of neurological diseases. While ASOs have been effective in the clinic, improving productive ASO internalization into target cells remains a key area of focus in the field. Here, we investigated how the delivery of ASO-loaded lipid nanoparticles (LNPs) affects ASO activity, subcellular trafficking, and distribution in the brain. We show that ASO-LNPs increase ASO activity up to 100-fold in cultured primary brain cells as compared to non-encapsulated ASO. However, in contrast to the widespread ASO uptake and activity observed following free ASO delivery in vivo, LNP-delivered ASOs did not downregulate mRNA levels throughout the brain after intracerebroventricular injection. This lack of activity was likely due to ASO accumulation in cells lining the ventricles and blood vessels. Furthermore, we reveal a formulation-dependent activation of the immune system post dosing, suggesting that LNP encapsulation cannot mask cellular ASO backbone-mediated toxicities. Together, these data provide insights into how LNP encapsulation affects ASO distribution as well as activity in the brain, and a foundation that enables future optimization of brain-targeting ASO-LNPs.
RESUMO
Loss-of-function mutations in Nav1.7, a voltage-gated sodium channel, cause congenital insensitivity to pain (CIP) in humans, demonstrating that Nav1.7 is essential for the perception of pain. However, the mechanism by which loss of Nav1.7 results in insensitivity to pain is not entirely clear. It has been suggested that loss of Nav1.7 induces overexpression of enkephalin, an endogenous opioid receptor agonist, leading to opioid-dependent analgesia. Using behavioral pharmacology and single-cell RNA-seq analysis, we find that overexpression of enkephalin occurs only in cLTMR neurons, a subclass of sensory neurons involved in low-threshold touch detection, and that this overexpression does not play a role in the analgesia observed following genetic removal of Nav1.7. Furthermore, we demonstrate using laser speckle contrast imaging (LSCI) and in vivo electrophysiology that Nav1.7 function is required for the initiation of C-fiber action potentials (APs), which explains the observed insensitivity to pain following genetic removal or inhibition of Nav1.7.