RESUMO
Axonal death disrupts functional connectivity of neural circuits and is a critical feature of many neurodegenerative disorders. Pathological axon degeneration often occurs independently of known programmed death pathways, but the underlying molecular mechanisms remain largely unknown. Using traumatic injury as a model, we systematically investigate mitogen-activated protein kinase (MAPK) families and delineate a MAPK cascade that represents the early degenerative response to axonal injury. The adaptor protein Sarm1 is required for activation of this MAPK cascade, and this Sarm1-MAPK pathway disrupts axonal energy homeostasis, leading to ATP depletion before physical breakdown of damaged axons. The protective cytoNmnat1/Wld(s) protein inhibits activation of this MAPK cascade. Further, MKK4, a key component in the Sarm1-MAPK pathway, is antagonized by AKT signaling, which modulates the degenerative response by limiting activation of downstream JNK signaling. Our results reveal a regulatory mechanism that integrates distinct signals to instruct pathological axon degeneration.
Assuntos
Axônios/patologia , Sistema de Sinalização das MAP Quinases , Trifosfato de Adenosina/metabolismo , Animais , Proteínas do Domínio Armadillo/genética , Proteínas do Domínio Armadillo/metabolismo , Morte Celular , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , MAP Quinase Quinase 4/metabolismo , Camundongos , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologiaRESUMO
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has affected approximately 800 million people since the start of the Coronavirus Disease 2019 (COVID-19) pandemic. Because of the high rate of mutagenesis in SARS-CoV-2, it is difficult to develop a sustainable approach for prevention and treatment. The Envelope (E) protein is highly conserved among human coronaviruses. Previous studies reported that SARS-CoV-1 E deficiency reduced viral propagation, suggesting that E inhibition might be an effective therapeutic strategy for SARS-CoV-2. Here, we report inhibitory peptides against SARS-CoV-2 E protein named iPep-SARS2-E. Leveraging E-induced alterations in proton homeostasis and NFAT/AP-1 pathway in mammalian cells, we developed screening platforms to design and optimize the peptides that bind and inhibit E protein. Using Vero-E6 cells, human-induced pluripotent stem cell-derived branching lung organoid and mouse models with SARS-CoV-2, we found that iPep-SARS2-E significantly inhibits virus egress and reduces viral cytotoxicity and propagation in vitro and in vivo. Furthermore, the peptide can be customizable for E protein of other human coronaviruses such as Middle East Respiratory Syndrome Coronavirus (MERS-CoV). The results indicate that E protein can be a potential therapeutic target for human coronaviruses.
Assuntos
COVID-19 , SARS-CoV-2 , Camundongos , Animais , Chlorocebus aethiops , Humanos , Linhagem Celular , Células Vero , Peptídeos/farmacologia , MamíferosRESUMO
The cognitive mechanisms underlying attention-deficit hyperactivity disorder (ADHD), a highly heritable disorder with an array of candidate genes and unclear genetic architecture, remain poorly understood. We previously demonstrated that mice overexpressing CK1δ (CK1δ OE) in the forebrain show hyperactivity and ADHD-like pharmacological responses to D-amphetamine. Here, we demonstrate that CK1δ OE mice exhibit impaired visual attention and a lack of D-amphetamine-induced place preference, indicating a disruption of the dopamine-dependent reward pathway. We also demonstrate the presence of abnormalities in the frontostriatal circuitry, differences in synaptic ultra-structures by electron microscopy, as well as electrophysiological perturbations of both glutamatergic and GABAergic transmission, as observed by altered frequency and amplitude of mEPSCs and mIPSCs. Furthermore, gene expression profiling by next-generation sequencing alone, or in combination with bacTRAP technology to study specifically Drd1a versus Drd2 medium spiny neurons, revealed that developmental CK1δ OE alters transcriptional homeostasis in the striatum, including specific alterations in Drd1a versus Drd2 neurons. These results led us to perform a fine molecular characterization of targeted gene networks and pathway analysis. Importantly, a large fraction of 92 genes identified by GWAS studies as associated with ADHD in humans are significantly altered in our mouse model. The multiple abnormalities described here might be responsible for synaptic alterations and lead to complex behavioral abnormalities. Collectively, CK1δ OE mice share characteristics typically associated with ADHD and should represent a valuable model to investigate the disease in vivo.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade , Caseína Quinase Idelta/genética , Animais , Transtorno do Deficit de Atenção com Hiperatividade/genética , Corpo Estriado , Dopamina , Camundongos , Neurônios , Receptores de Dopamina D2/genéticaRESUMO
Ever since Stephen Paget's 1889 hypothesis, metastatic organotropism has remained one of cancer's greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6ß4 and α6ß1 were associated with lung metastasis, while exosomal integrin αvß5 was linked to liver metastasis. Targeting the integrins α6ß4 and αvß5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.
Assuntos
Encéfalo/metabolismo , Exossomos/metabolismo , Integrinas/metabolismo , Fígado/metabolismo , Pulmão/metabolismo , Metástase Neoplásica/patologia , Metástase Neoplásica/prevenção & controle , Tropismo , Animais , Biomarcadores/metabolismo , Encéfalo/citologia , Linhagem Celular Tumoral , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Genes src , Humanos , Integrina alfa6beta1/metabolismo , Integrina alfa6beta4/antagonistas & inibidores , Integrina alfa6beta4/metabolismo , Cadeias beta de Integrinas/metabolismo , Integrina beta4/metabolismo , Integrinas/antagonistas & inibidores , Células de Kupffer/citologia , Células de Kupffer/metabolismo , Fígado/citologia , Pulmão/citologia , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Fosforilação , Receptores de Vitronectina/antagonistas & inibidores , Receptores de Vitronectina/metabolismo , Proteínas S100/genéticaRESUMO
The horizontal transfer of mtDNA and its role in mediating resistance to therapy and an exit from dormancy have never been investigated. Here we identified the full mitochondrial genome in circulating extracellular vesicles (EVs) from patients with hormonal therapy-resistant (HTR) metastatic breast cancer. We generated xenograft models of HTR metastatic disease characterized by EVs in the peripheral circulation containing mtDNA. Moreover, these human HTR cells had acquired host-derived (murine) mtDNA promoting estrogen receptor-independent oxidative phosphorylation (OXPHOS). Functional studies identified cancer-associated fibroblast (CAF)-derived EVs (from patients and xenograft models) laden with whole genomic mtDNA as a mediator of this phenotype. Specifically, the treatment of hormone therapy (HT)-naive cells or HT-treated metabolically dormant populations with CAF-derived mtDNAhi EVs promoted an escape from metabolic quiescence and HTR disease both in vitro and in vivo. Moreover, this phenotype was associated with the acquisition of EV mtDNA, especially in cancer stem-like cells, expression of EV mtRNA, and restoration of OXPHOS. In summary, we have demonstrated that the horizontal transfer of mtDNA from EVs acts as an oncogenic signal promoting an exit from dormancy of therapy-induced cancer stem-like cells and leading to endocrine therapy resistance in OXPHOS-dependent breast cancer.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , DNA Mitocondrial/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Exossomos/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , DNA Mitocondrial/genética , Feminino , Fibroblastos/patologia , Transferência Genética Horizontal , Genoma Mitocondrial/genética , Humanos , Células MCF-7 , NADH Desidrogenase/genética , Fosforilação Oxidativa , Receptores de Estrogênio/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The nuclear pore complex (NPC) is responsible for nucleocytoplasmic transport and constitutes a hub for control of gene expression. The components of NPCs from several eukaryotic lineages have been determined, but only the yeast and vertebrate NPCs have been extensively characterized at the quaternary level. Significantly, recent evidence indicates that compositional similarity does not necessarily correspond to homologous architecture between NPCs from different taxa. To address this, we describe the interactome of the trypanosome NPC, a representative, highly divergent eukaryote. We identify numerous new NPC components and report an exhaustive interactome, allowing assignment of trypanosome nucleoporins to discrete NPC substructures. Remarkably, despite retaining similar protein composition, there are exceptional architectural dissimilarities between opisthokont (yeast and vertebrates) and excavate (trypanosomes) NPCs. Whilst elements of the inner core are conserved, numerous peripheral structures are highly divergent, perhaps reflecting requirements to interface with divergent nuclear and cytoplasmic functions. Moreover, the trypanosome NPC has almost complete nucleocytoplasmic symmetry, in contrast to the opisthokont NPC; this may reflect divergence in RNA export processes at the NPC cytoplasmic face, as we find evidence supporting Ran-dependent mRNA export in trypanosomes, similar to protein transport. We propose a model of stepwise acquisition of nucleocytoplasmic mechanistic complexity and demonstrate that detailed dissection of macromolecular complexes provides fuller understanding of evolutionary processes.
Assuntos
Evolução Molecular , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/genética , Microscopia Imunoeletrônica , Poro Nuclear/química , Poro Nuclear/metabolismo , Estrutura Quaternária de Proteína , Trypanosoma brucei bruceiRESUMO
Hepatitis C virus (HCV) is a major cause of chronic liver disease, with an estimated 170 million people infected worldwide. Low yields, poor stability, and inefficient binding to conventional EM grids have posed significant challenges to the purification and structural analysis of HCV. In this report, we generated an infectious HCV genome with an affinity tag fused to the E2 envelope glycoprotein. Using affinity grids, previously described to isolate proteins and macromolecular complexes for single-particle EM, we were able to purify enveloped particles directly from cell culture media. This approach allowed for rapid in situ purification of virions and increased particle density that were instrumental for cryo-EM and cryoelectron tomography (cryo-ET). Moreover, it enabled ultrastructural analysis of virions produced by primary human hepatocytes. HCV appears to be the most structurally irregular member of the Flaviviridae family. Particles are spherical, with spike-like projections, and heterogeneous in size ranging from 40 to 100 nm in diameter. Exosomes, although isolated from unfractionated culture media, were absent in highly infectious, purified virus preparations. Cryo-ET studies provided low-resolution 3D structural information of highly infectious virions. In addition to apolipoprotein (apo)E, HCV particles also incorporate apoB and apoA-I. In general, host apolipoproteins were more readily accessible to antibody labeling than HCV glycoproteins, suggesting either lower abundance or masking by host proteins.
Assuntos
Hepacivirus/ultraestrutura , Proteínas do Envelope Viral/ultraestrutura , Vírion/ultraestrutura , Apolipoproteínas/metabolismo , Linhagem Celular Tumoral , Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica , Hepatócitos/virologia , Humanos , Imuno-Histoquímica , Marcação por Isótopo , Especificidade da Espécie , Proteínas do Envelope Viral/genética , Vírion/isolamento & purificaçãoRESUMO
Scanning electron microscopy (SEM) offers an unparalleled view of the membrane topography of mammalian cells by using a conventional osmium (OsO4) and ethanol-based tissue preparation. However, conventional SEM methods limit optimal resolution due to ethanol and lipid interactions and interfere with visualization of fluorescent reporter proteins. Therefore, SEM correlative light and electron microscopy (CLEM) has been hindered by the adverse effects of ethanol and OsO4 on retention of fluorescence signals. To overcome this technological gap in achieving high-resolution SEM and retain fluorescent reporter signals, we developed a freeze-drying method with gaseous nitrogen (FDGN). We demonstrate that FDGN preserves cyto-architecture to allow visualization of detailed membrane topography while retaining fluorescent signals and that FDGN processing can be used in conjunction with a variety of high-resolution imaging systems to enable collection and validation of unique, high-quality data from these approaches. In particular, we show that FDGN coupled with high resolution microscopy provided detailed insight into viral or tumor-derived extracellular vesicle (TEV)-host cell interactions and may aid in designing new approaches to intervene during viral infection or to harness TEVs as therapeutic agents.
RESUMO
Human stem cell-derived hepatocyte-like cells (HLCs) offer an attractive platform to study liver biology. Despite their numerous advantages, HLCs lack critical in vivo characteristics, including cell polarity. Here, we report a stem cell differentiation protocol that uses transwell filters to generate columnar polarized HLCs with clearly defined basolateral and apical membranes separated by tight junctions. We show that polarized HLCs secrete cargo directionally: Albumin, urea, and lipoproteins are secreted basolaterally, whereas bile acids are secreted apically. Further, we show that enterically transmitted hepatitis E virus (HEV) progeny particles are secreted basolaterally as quasi-enveloped particles and apically as naked virions, recapitulating essential steps of the natural infectious cycle in vivo. We also provide proof-of-concept that polarized HLCs can be used for pharmacokinetic and drug-drug interaction studies. This novel system provides a powerful tool to study hepatocyte biology, disease mechanisms, genetic variation, and drug metabolism in a more physiologically relevant setting.
Assuntos
Técnicas de Cultura de Células/métodos , Polaridade Celular , Hepatócitos/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Antivirais/farmacologia , Diferenciação Celular , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos/métodos , Interações Medicamentosas , Vírus da Hepatite A Humana/fisiologia , Vírus da Hepatite E/fisiologia , Hepatócitos/ultraestrutura , Hepatócitos/virologia , Humanos , Fígado/citologia , Fígado/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Microscopia Eletrônica de Transmissão , Estudo de Prova de Conceito , Vírion/metabolismo , Liberação de Vírus , Replicação ViralRESUMO
Transgenic (Tg) mice overexpressing human wild-type alpha-synuclein in oligodendrocytes under the control of the 2,' 3'-cyclic nucleotide 3'-phosphodiesterase (CNP) promoter are shown here to recapitulate features of multiple system atrophy (MSA), including the accumulation of filamentous human alpha-synuclein aggregates in oligodendrocytes linked to their degeneration and autophagocytosis of myelin. Significantly, endogenous mouse alpha-synuclein also accumulated in normal and degenerating axons and axon terminals in association with oligodendroglia and neuron loss and slowly progressive motor impairments. Our studies demonstrate that overexpression of alpha-synuclein in oligodendrocytes of mice results in MSA-like degeneration in the CNS and that alpha-synuclein inclusions in oligodendrocytes participate in the degeneration of neurons in MSA.
Assuntos
Atrofia de Múltiplos Sistemas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/patologia , Neurônios/patologia , Oligodendroglia/metabolismo , Degeneração Walleriana/metabolismo , 2',3'-Nucleotídeo Cíclico 3'-Fosfodiesterase , Animais , Axônios/metabolismo , Axônios/patologia , Axônios/ultraestrutura , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Sistema Nervoso Central/fisiopatologia , Modelos Animais de Doenças , Humanos , Corpos de Inclusão/genética , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Atrofia de Múltiplos Sistemas/genética , Atrofia de Múltiplos Sistemas/fisiopatologia , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Bainha de Mielina/ultraestrutura , Proteínas do Tecido Nervoso/genética , Oligodendroglia/patologia , Oligodendroglia/ultraestrutura , Fagocitose/genética , Diester Fosfórico Hidrolases/genética , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/patologia , Terminações Pré-Sinápticas/ultraestrutura , Regiões Promotoras Genéticas/genética , Sinucleínas , Degeneração Walleriana/genética , Degeneração Walleriana/fisiopatologia , alfa-SinucleínaRESUMO
alpha-Synuclein (SYN) is the major component of Lewy bodies, the neuropathological hallmarks of Parkinson's disease (PD). Missense mutations and multiplications of the SYN gene cause autosomal dominant inherited PD. Thus, SYN is implicated in the pathogenesis of PD. However, the mechanism whereby SYN promotes neurodegeneration remains unclear. Familial PD with SYN gene mutations are rare because the majority of PD is sporadic and emerging evidence indicates that sporadic PD may result from genetic and environmental risk factors including neuroinflammation. Hence, we examined the relationship between SYN dysfunction and neuroinflammation in mediating dopaminergic neurodegeneration in mice and dopaminergic neuronal cultures derived from wild-type SYN and mutant A53T SYN transgenic mice in a murine SYN-null (SYNKO) background (M7KO and M83KO, respectively). Stereotaxic injection of an inflammagen, lipopolysaccharide, into substantia nigra of these SYN genetically engineered mice induced similar inflammatory reactions. In M7KO and M83KO, but not in SYNKO mice, the neuroinflammation was associated with dopaminergic neuronal death and the accumulation of insoluble aggregated SYN as cytoplasmic inclusions in nigral neurons. Nitrated/oxidized SYN was detected in these inclusions and abatement of microglia-derived nitric oxide and superoxide provided significant neuroprotection in neuron-glia cultures from M7KO mice. These data suggest that nitric oxide and superoxide released by activated microglia may be mediators that link inflammation and abnormal SYN in mechanisms of PD neurodegeneration. This study advances understanding of the role of neuroinflammation and abnormal SYN in the pathogenesis of PD and opens new avenues for the discovery of more effective therapies for PD.
Assuntos
Dopamina/metabolismo , Doenças Neurodegenerativas/complicações , Doenças Neurodegenerativas/genética , Óxido Nítrico Sintase/metabolismo , Estresse Oxidativo/fisiologia , alfa-Sinucleína/genética , Análise de Variância , Animais , Contagem de Células , Células Cultivadas , Técnicas de Cocultura/métodos , Embrião de Mamíferos , Humanos , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Lipopolissacarídeos/efeitos adversos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neurônios/fisiologia , Neurônios/ultraestrutura , alfa-Sinucleína/deficiênciaRESUMO
TAR DNA-binding protein (TDP-43) has been recently described as a major pathological protein in both frontotemporal dementia with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis. However, little is known about the relative abundance and distribution of different pathological TDP-43 species, which include hyperphosphorylated, ubiquitinated, and N-terminally cleaved TDP-43. Here, we developed novel N-terminal (N-t) and C-terminal (C-t)-specific TDP-43 antibodies and performed biochemical and immunohistochemical studies to analyze cortical, hippocampal, and spinal cord tissue from frontotemporal dementia with ubiquitin-positive inclusions and amyotrophic lateral sclerosis cases. C-t-specific TDP-43 antibodies revealed similar abundance, morphology, and distribution of dystrophic neurites and neuronal cytoplasmic inclusions in cortex and hippocampus compared with previously described pan-TDP-43 antibodies. By contrast, N-t-specific TDP-43 antibodies only detected a small subset of these lesions. Biochemical studies confirmed the presence of C-t TDP-43 fragments but not extreme N-t fragments. Surprisingly, immunohistochemical analysis of inclusions in spinal cord motor neurons in both diseases showed that they are N-t and C-t positive. TDP-43 inclusions in Alzheimer's disease brains also were examined, and similar enrichment in C-t TDP-43 fragments was observed in cortex and hippocampus. These results show that the composition of the inclusions in brain versus spinal cord tissues differ, with an increased representation of C-t TDP-43 fragments in cortical and hippocampal regions. Therefore, regionally different pathogenic processes may underlie the development of abnormal TDP-43 proteinopathies.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Encéfalo/patologia , Proteínas de Ligação a DNA/metabolismo , Demência/patologia , Corpos de Inclusão/metabolismo , Medula Espinal/patologia , Esclerose Lateral Amiotrófica/metabolismo , Encéfalo/metabolismo , Proteínas de Ligação a DNA/química , Demência/metabolismo , Imunofluorescência , Humanos , Immunoblotting , Imuno-Histoquímica , Corpos de Inclusão/patologia , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Medula Espinal/metabolismoRESUMO
Pathologic TAR-DNA-binding protein 43 (TDP-43) is a disease protein in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis. We studied the presence, frequency, and distribution of TDP-43 pathology by immunohistochemistry and biochemistry in a series of clinically well-characterized tauopathy patient brains, including 182 Alzheimer disease (AD), 39 corticobasal degeneration, 77 progressive supranuclear palsy, and 12 Pick disease cases and investigated the clinical impact of concomitant TDP-43 pathology in these cases. TAR-DNA-binding protein 43 pathology was found in 25.8% of AD cases. It was restricted to the dentate gyrus and entorhinal cortex in approximately 75% of cases; approximately 25% showed more widespread TDP-43 pathology in frontal and temporal cortices, resembling the FTLD-U subtype associated with progranulin mutations. TAR-DNA-binding protein 43 pathology in AD was associated with significantly longer disease duration, but there was no association with the clinical presentation (148 cases diagnosed as AD and 34 cases diagnosed as frontotemporal lobar degeneration). Progressive supranuclear palsy and Pick disease cases showed no TDP-43 inclusions and no biochemical alterations of TDP-43. There was, however, a unique, predominantly glial TDP-43 pathology with staining of astrocytic plaque-like structures and coiled bodies in 15.4% of corticobasal degeneration cases; this was associated with biochemical TDP-43 changes similar to those in FTLD-U. These findings provide further insight into the burden and clinical significance of TDP-43 pathology in disorders other than FTLD-U and amyotrophic lateral sclerosis.
Assuntos
Doença de Alzheimer/patologia , Encéfalo/patologia , Proteínas de Ligação a DNA/metabolismo , Neurônios/patologia , Tauopatias/patologia , Idoso , Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Feminino , Humanos , Immunoblotting , Imuno-Histoquímica , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Masculino , Neurônios/metabolismo , Tauopatias/metabolismoRESUMO
This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (Aß) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). Aß, which forms neurotoxic amyloid aggregates in Alzheimer's disease (AD), has been shown to interact with fibrinogen. This Aß-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. Aß-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the Aß-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of Aß and Aß-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of Aß-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate Aß-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified Aß-fibrinogen interaction inhibitor.
Assuntos
Peptídeos beta-Amiloides/efeitos adversos , Fibrina/metabolismo , Microscopia Eletrônica de Varredura/métodos , Análise Espectral/métodos , Fibrina/análise , HumanosRESUMO
BACKGROUND: Peripheral artery disease (PAD), a diffuse manifestation of atherothrombosis, is a major cardiovascular threat. Although platelets are primary mediators of atherothrombosis, their role in the pathogenesis of PAD remains unclear. OBJECTIVES: The authors sought to investigate the role of platelets in a cohort of symptomatic PAD. METHODS: The authors profiled platelet activity, mRNA, and effector roles in patients with symptomatic PAD and in healthy controls. Patients with PAD and carotid artery stenosis were recruited into ongoing studies (NCT02106429 and NCT01897103) investigating platelet activity, platelet RNA, and cardiovascular disease. RESULTS: Platelet RNA sequence profiling mapped a robust up-regulation of myeloid-related protein (MRP)-14 mRNA, a potent calcium binding protein heterodimer, in PAD. Circulating activated platelets were enriched with MRP-14 protein, which augmented the expression of the adhesion mediator, P-selectin, thereby promoting monocyte-platelet aggregates. Electron microscopy confirmed the firm interaction of platelets with monocytes in vitro and colocalization of macrophages with MRP-14 confirmed their cross talk in atherosclerotic manifestations of PAD in vivo. Platelet-derived MRP-14 was channeled to monocytes, thereby fueling their expression of key PAD lesional hallmarks and increasing their directed locomotion, which were both suppressed in the presence of antibody-mediated blockade. Circulating MRP-14 was heightened in the setting of PAD, significantly correlated with PAD severity, and was associated with incident limb events. CONCLUSIONS: The authors identified a heightened platelet activity profile and unraveled a novel immunomodulatory effector role of platelet-derived MRP-14 in reprograming monocyte activation in symptomatic PAD. (Platelet Activity in Vascular Surgery and Cardiovascular Events [PACE]; NCT02106429; and Platelet Activity in Vascular Surgery for Thrombosis and Bleeding [PIVOTAL]; NCT01897103).
Assuntos
Plaquetas/fisiologia , Calgranulina B/imunologia , Monócitos/fisiologia , Doença Arterial Periférica , Adulto , Reprogramação Celular/imunologia , Feminino , Hemostasia , Humanos , Macrófagos/fisiologia , Masculino , Pessoa de Meia-Idade , Selectina-P/imunologia , Doença Arterial Periférica/sangue , Doença Arterial Periférica/fisiopatologia , Ativação Plaquetária/imunologiaRESUMO
Vertebrate cells can initiate ciliogenesis from centrioles at the cell center, near the Golgi, forming primary cilia confined or submerged in a deep narrow pit created by membrane invagination. How or why cells maintain submerged cilia is unclear. Here, by characterizing centriole subdistal appendages (sDAP) in cells exclusively growing submerged cilia, we found that a group of sDAP components localize to the centriole proximal end through the cohesion factor C-Nap1 and that sDAP function redundantly with C-Nap1 for submerged cilia maintenance. Loss of sDAP and C-Nap1 has no effect on cilia assembly, but it disrupts stable Golgi-cilia association and allows normally submerged cilia to fully surface, losing the deep membrane invagination. Intriguingly, unlike submerged cilia (stationary), surfaced cilia actively respond to mechanical stimuli with motions and can ectopically recruit Hedgehog signaling components in the absence of agonist. We propose that spatial control of ciliogenesis uncouples or specifies sensory properties of cilia.
Assuntos
Cílios/metabolismo , Morfogênese , Autoantígenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Centríolos/metabolismo , Centríolos/ultraestrutura , Centrossomo/metabolismo , Centrossomo/ultraestrutura , Cílios/ultraestrutura , Técnicas de Inativação de Genes , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Proteínas Hedgehog/metabolismo , Humanos , Centro Organizador dos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/ultraestrutura , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Movimento (Física) , Mutação/genética , Reologia , Sensação , Transdução de SinaisRESUMO
Enhanced secretion of tumorigenic effector proteins is a feature of malignant cells. The molecular mechanisms underlying this feature are poorly defined. We identify PITPNC1 as a gene amplified in a large fraction of human breast cancer and overexpressed in metastatic breast, melanoma, and colon cancers. Biochemical, molecular, and cell-biological studies reveal that PITPNC1 promotes malignant secretion by binding Golgi-resident PI4P and localizing RAB1B to the Golgi. RAB1B localization to the Golgi allows for the recruitment of GOLPH3, which facilitates Golgi extension and enhanced vesicular release. PITPNC1-mediated vesicular release drives metastasis by increasing the secretion of pro-invasive and pro-angiogenic mediators HTRA1, MMP1, FAM3C, PDGFA, and ADAM10. We establish PITPNC1 as a PI4P-binding protein that enhances vesicular secretion capacity in malignancy.
Assuntos
Complexo de Golgi/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas rab1 de Ligação ao GTP/metabolismo , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Feminino , Humanos , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
Traumatic brain injury (TBI) increases susceptibility to Alzheimer's disease (AD), but it is not known how TBI contributes to the onset or progression of this common late life dementia. To address this question, we studied neuropathological and behavioral consequences of single versus repetitive mild TBI (mTBI) in transgenic (Tg) mice (Tg2576) that express mutant human Abeta precursor protein, and we demonstrate elevated brain Abeta levels and increased Abeta deposition. Nine-month-old Tg2576 and wild-type mice were subjected to single (n = 15) or repetitive (n = 39) mTBI or sham treatment (n = 37). At 2 d and 9 and 16 weeks after treatment, we assessed brain Abeta deposits and levels in addition to brain and urine isoprostanes generated by lipid peroxidation in these mice. A subset of mice also was studied behaviorally at 16 weeks after injury. Repetitive but not single mTBI increased Abeta deposition as well as levels of Abeta and isoprostanes only in Tg mice, and repetitive mTBI alone induced cognitive impairments but no motor deficits in these mice. This is the first experimental evidence linking TBI to mechanisms of AD by showing that repetitive TBI accelerates brain Abeta accumulation and oxidative stress, which we suggest could work synergistically to promote the onset or drive the progression of AD. Additional insights into the role of TBI in mechanisms of AD pathobiology could lead to strategies for reducing the risk of AD associated with previous episodes of brain trauma and for preventing progressive brain amyloidosis in AD patients.
Assuntos
Doença de Alzheimer/fisiopatologia , Amiloidose/fisiopatologia , Lesões Encefálicas/fisiopatologia , Transtornos Cognitivos/fisiopatologia , Modelos Animais de Doenças , Doença de Alzheimer/complicações , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/biossíntese , Precursor de Proteína beta-Amiloide/genética , Amiloidose/complicações , Amiloidose/patologia , Animais , Comportamento Animal , Biomarcadores/análise , Biomarcadores/urina , Encéfalo/patologia , Encéfalo/fisiopatologia , Lesões Encefálicas/complicações , Lesões Encefálicas/patologia , Transtornos Cognitivos/complicações , Transtornos Cognitivos/patologia , Progressão da Doença , Suscetibilidade a Doenças/fisiopatologia , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Traumatismos Cranianos Fechados/complicações , Traumatismos Cranianos Fechados/fisiopatologia , Humanos , Isoprostanos/análise , Isoprostanos/metabolismo , Peroxidação de Lipídeos , Masculino , Camundongos , Camundongos Transgênicos , Atividade Motora , Exame Neurológico , Tempo de ReaçãoRESUMO
Traumatic brain injury (TBI) is a risk factors for Alzheimer's disease (AD), and repetitive TBI (rTBI) may culminate in dementia pugilistica (DP), a syndrome characterized by progressive dementia, parkinsonism, and the hallmark brain lesions of AD, including neurofibrillary tangles (NFTs), formed by abnormal tau filaments and senile plaques (SPs) composed of Abeta fibrils. Previous study showed that mild rTBI (mrTBI) accelerated the deposition of Abeta in the brains of transgenic (Tg) mice (Tg2576) that over-express human Abeta precursor proteins with the familial AD Swedish mutations (APP695swe) and model of AD-like amyloidosis. Here, we report studies of the effects of mrTBI on AD-like tau pathologies in Tg mice expressing the shortest human tau isoform (T44) subjected to mrTBI, causing brain concussion without structural brain damage to simulate injuries linked to DP. Twelve-month-old Tg T44 (n = 18) and wild-type (WT; n = 24) mice were subjected to mrTBI (four times a day, 1 day per week, for 4 weeks; n = 24) or sham treatment (n = 18). Histopathological analysis of mice at 9 months after mrTBI revealed that one of the Tg T44 mice showed extensive telencephalic NFT and cerebral atrophy. Although statistical analysis of neurobehavioral tests at 6 months after mrTBI did not show any significant difference in any of groups of mice, the Tg T44 mouse with extensive NFT had an exceptionally low neurobehavioral score. The reasons for the augmentation of tau pathologies in only one T44 tau Tg mouse subjected to mrTBI remain to be elucidated.