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2.
Micron ; 132: 102851, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32092694

RESUMO

Kupffer cells are liver-resident macrophages that play an important role in mediating immune-related functions in mammals and humans. They are well-known for their capacity to phagocytose large amounts of waste complexes, cell debris, microbial particles and even malignant cells. Location, appearance and functional aspects are important features used to identify these characteristic cells of the liver sinusoid. To-date, there is limited information on the occurrence of macrophages in zebrafish liver. Therefore, we aimed to characterise the ultrastructural and functional aspects of liver-associated macrophages in the zebrafish model by taking advantage of the latest advances in zebrafish genetics and multimodal correlative imaging. Herein, we report on the occurrence of macrophages within the zebrafish liver exhibiting conventional ultrastructural features (e.g. presence of pseudopodia, extensive lysosomal apparatus, a phagolysosome and making up ∼3% of the liver volume). Intriguingly, these cells were not located within the sinusoidal vascular bed of hepatic tissue but instead resided between hepatocytes and lacked phagocytic function. While our results demonstrated the presence and structural similarities with liver macrophages from other experimental models, their functional characteristics were distinctly different from Kupffer cells that have been described in rodents and humans. These findings illustrate that the innate immune system of the zebrafish liver has some distinctly different characteristics compared to other animal experimental models. This conclusion underpins our call for future studies in order to have a better understanding of the physiological role of macrophages residing between the parenchymal cells of the zebrafish liver.

3.
Cell Mol Life Sci ; 76(13): 2499-2510, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30919022

RESUMO

Ubiquitin ligases play an integral role in fine-tuning signaling cascades necessary for normal cell function. Aberrant regulation of ubiquitin ligases has been implicated in several neurodegenerative diseases, generally, due to mutations within the E3 ligase itself. Several proteomic-based methods have recently emerged to facilitate the rapid identification of ligase-substrate pairs-a previously challenging feat due to the transient nature of ligase-substrate interactions. These novel methods complement standard immunoprecipitations (IPs) and include proximity-dependent biotin identification (BioID), ubiquitin ligase-substrate trapping, tandem ubiquitin-binding entities (TUBEs), and a molecular trapping unit known as the NEDDylator. The implementation of these techniques is expected to facilitate the rapid identification of novel substrates of E3 ubiquitin ligases, a process that is likely to enhance our understanding of neurodegenerative diseases and highlight novel therapeutic targets for the treatment of neurodegenerative diseases.


Assuntos
Terapia de Alvo Molecular , Doenças Neurodegenerativas/metabolismo , Proteômica/métodos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Animais , Humanos , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/patologia , Especificidade por Substrato , Ubiquitinação
5.
Exp Cell Res ; 374(1): 162-171, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30496757

RESUMO

Although liver transport routes have been extensively studied in rodents, live imaging under in situ and in vivo conditions of large volumes is still proven to be difficult. In this study, we took advantage of the optical transparency of zebrafish and their small size to explore their usefulness for correlative imaging studies and liver transport experimentations. First, we assessed the micro-architecture of the zebrafish liver and compared its fine structure to the rodent and humans' literature. Next, we investigated the transport routes and cellular distribution of albumin using combined and correlative microscopy approaches. These methods permitted us to track the injected proteins at different time points through the process of liver uptake and clearance of albumin. We demonstrate strong structural and functional resemblance between the zebrafish liver and its rodents and humans' counterparts. In as short as 5 min post-injection, albumin rapidly accumulated within the LSECs. Furthermore, albumin entered the space of Disse where it initially accumulated then subsequently was taken up by the hepatocytes. We propose the zebrafish as a viable alternative experimental model for hepatic transport studies, allowing swift multimodal imaging and direct quantification on the hepatic distribution of supramolecular complexes of interest.


Assuntos
Albuminas/metabolismo , Fígado/metabolismo , Imagem Molecular , Peixe-Zebra/metabolismo , Animais , Fluorescência , Larva/metabolismo , Fígado/ultraestrutura , Modelos Biológicos , Transporte Proteico
6.
Redox Biol ; 19: 226-234, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30193184

RESUMO

Generation of reactive oxygen species (ROS) has been shown to be important for many physiological processes, ranging from cell differentiation to apoptosis. With the development of the genetically encoded photosensitiser KillerRed (KR) it is now possible to efficiently produce ROS dose-dependently in a specific cell type upon green light illumination. Zebrafish are the ideal vertebrate animal model for these optogenetic methods because of their transparency and efficient transgenesis. Here we describe a zebrafish model that expresses membrane-targeted KR selectively in motor neurons. We show that KR-activated neurons in the spinal cord undergo stress and cell death after induction of ROS. Using single-cell resolution and time-lapse confocal imaging, we selectively induced neurodegeneration in KR-expressing neurons leading to characteristic signs of apoptosis and cell death. We furthermore illustrate a targeted microglia response to the induction site as part of a physiological response within the zebrafish spinal cord. Our data demonstrate the successful implementation of KR mediated ROS toxicity in motor neurons in vivo and has important implications for studying the effects of ROS in a variety of conditions within the central nervous system, including aging and age-related neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis.


Assuntos
Neurônios Motores/patologia , Estresse Oxidativo , Análise de Célula Única/métodos , Medula Espinal/patologia , Animais , Apoptose , Morte Celular , Neurônios Motores/citologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Optogenética/métodos , Espécies Reativas de Oxigênio/metabolismo , Medula Espinal/citologia , Medula Espinal/metabolismo , Peixe-Zebra
7.
Acta Neuropathol ; 136(3): 445-459, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29943193

RESUMO

Transactivating DNA-binding protein-43 (TDP-43) deposits represent a typical finding in almost all ALS patients, more than half of FTLD patients and patients with several other neurodegenerative disorders. It appears that perturbation of nucleo-cytoplasmic transport is an important event in these conditions but the mechanistic role and the fate of TDP-43 during neuronal degeneration remain elusive. We have developed an experimental system for visualising the perturbed nucleocytoplasmic transport of neuronal TDP-43 at the single-cell level in vivo using zebrafish spinal cord. This approach enabled us to image TDP-43-expressing motor neurons before and after experimental initiation of cell death. We report the formation of mobile TDP-43 deposits within degenerating motor neurons, which are normally phagocytosed by microglia. However, when microglial cells were depleted, injury-induced motor neuron degeneration follows a characteristic process that includes TDP-43 redistribution into the cytoplasm, axon and extracellular space. This is the first demonstration of perturbed TDP-43 nucleocytoplasmic transport in vivo, and suggests that impairment in microglial phagocytosis of dying neurons may contribute towards the formation of pathological TDP-43 presentations in ALS and FTLD.


Assuntos
Axônios/metabolismo , Proteínas de Ligação a DNA/metabolismo , Microglia/metabolismo , Neurônios Motores/metabolismo , Degeneração Neural/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Axônios/patologia , Microglia/patologia , Neurônios Motores/patologia , Degeneração Neural/patologia , Transporte Proteico , Peixe-Zebra
8.
Am J Pathol ; 188(6): 1447-1456, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29577934

RESUMO

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing and fatal disease characterized by muscular atrophy because of loss of upper and lower motor neurons. Histopathologically, most patients with ALS have abnormal cytoplasmic accumulation and aggregation of the nuclear RNA-regulating protein TAR DNA-binding protein 43 (TDP-43). Pathogenic mutations in the TARDBP gene that encode TDP-43 have been identified in familial ALS. We have previously reported transgenic mice with neuronal expression of human TDP-43 carrying the pathogenic A315T mutation (iTDP-43A315T mice), presenting with early-onset motor deficits in adolescent animals. Here, we analyzed aged iTDP-43A315T mice, focusing on the spatiotemporal profile and progression of neurodegeneration in upper and lower motor neurons. Magnetic resonance imaging and histologic analysis revealed a differential loss of upper motor neurons in a hierarchical order as iTDP-43A315T mice aged. Furthermore, we report progressive gait problems, profound motor deficits, and muscle atrophy in aged iTDP-43A315T mice. Despite these deficits and TDP-43 pathologic disorders in lower motor neurons, stereological analysis did not show cell loss in spinal cords. Taken together, neuronal populations in aging iTDP-43A315T mice show differential susceptibility to the expression of human TDP-43A315T.


Assuntos
Sistema Nervoso Central/patologia , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Transtornos Motores/patologia , Atrofia Muscular/patologia , Doenças Neurodegenerativas/patologia , Envelhecimento , Animais , Sistema Nervoso Central/metabolismo , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transtornos Motores/genética , Transtornos Motores/metabolismo , Atrofia Muscular/genética , Atrofia Muscular/metabolismo , Mutação , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Análise Espaço-Temporal
9.
Chem Commun (Camb) ; 54(29): 3609-3612, 2018 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-29570195

RESUMO

A superior biocompatible spherical nucleic acid (SNA) conjugate was fabricated by grafting siRNA onto the surface of a core composed of a spherical DNA nanostructure that we have termed a DNA nanoclew (DC). After uptake by cultured cancer cells, SNA nanoparticles release engrafted siRNAs by cleavage of the intracellular Dicer enzyme. Moreover, in vitro experiments reveal that such SNAs demonstrate potent gene knockdown at both mRNA and protein levels, while with negligible cytotoxicity.


Assuntos
DNA/química , Sistemas de Liberação de Medicamentos/métodos , Técnicas de Silenciamento de Genes/métodos , Nanopartículas/química , RNA Interferente Pequeno/administração & dosagem , Inativação Gênica , Células HeLa , Humanos , Nanopartículas/toxicidade , Hibridização de Ácido Nucleico , Tamanho da Partícula , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/toxicidade , Ribonuclease III/química
10.
Sci Rep ; 8(1): 4685, 2018 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-29549349

RESUMO

Cannabinoids exert dynamic control over many physiological processes including memory formation, cognition and pain perception. In the central nervous system endocannabinoids mediate negative feedback of quantal transmitter release following postsynaptic depolarization. The influence of cannabinoids in the peripheral nervous system is less clear and might have broad implications for the therapeutic application of cannabinoids. We report a novel cannabinoid effect upon the mouse neuromuscular synapse: acutely increasing synaptic vesicle volume and raising the quantal amplitudes. In a mouse model of myasthenia gravis the cannabinoid receptor agonist WIN 55,212 reversed fatiguing failure of neuromuscular transmission, suggesting future therapeutic potential. Our data suggest an endogenous pathway by which cannabinoids might help to regulate transmitter release at the neuromuscular junction.


Assuntos
Endocanabinoides/administração & dosagem , Miastenia Gravis/tratamento farmacológico , Junção Neuromuscular/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Animais , Benzoxazinas/farmacologia , Modelos Animais de Doenças , Endocanabinoides/metabolismo , Endocanabinoides/farmacologia , Potenciais Evocados/efeitos dos fármacos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Potenciais Pós-Sinápticos em Miniatura/efeitos dos fármacos , Morfolinas/farmacologia , Miastenia Gravis/etiologia , Miastenia Gravis/metabolismo , Naftalenos/farmacologia , Junção Neuromuscular/efeitos dos fármacos
11.
Cell Mol Life Sci ; 75(23): 4269-4285, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29468257

RESUMO

Aurora kinase B (AurkB) is a serine/threonine protein kinase with a well-characterised role in orchestrating cell division and cytokinesis, and is prominently expressed in healthy proliferating and cancerous cells. However, the role of AurkB in differentiated and non-dividing cells has not been extensively explored. Previously, we have described a significant upregulation of AurkB expression in cultured cortical neurons following an experimental axonal transection. This is somewhat surprising, as AurkB expression is generally associated only with dividing cells Frangini et al. (Mol Cell 51:647-661, 2013); Hegarat et al. (J Cell Biol 195:1103-1113, 2011); Lu et al. (J Biol Chem 283:31785-31790, 2008); Trakala et al. (Cell Cycle 12:1030-1041, 2014). Herein, we present the first description of a role for AurkB in terminally differentiated neurons. AurkB was prominently expressed within post-mitotic neurons of the zebrafish brain and spinal cord. The expression of AurkB varied during the development of the zebrafish spinal motor neurons. Utilising pharmacological and genetic manipulation to impair AurkB activity resulted in truncation and aberrant motor axon morphology, while overexpression of AurkB resulted in extended axonal outgrowth. Further pharmacological inhibition of AurkB activity in regenerating axons delayed their recovery following UV laser-mediated injury. Collectively, these results suggest a hitherto unreported role of AurkB in regulating neuronal development and axonal outgrowth.


Assuntos
Aurora Quinase B/metabolismo , Axônios/fisiologia , Neurônios Motores/metabolismo , Regeneração Nervosa/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Aurora Quinase B/antagonistas & inibidores , Aurora Quinase B/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Organofosfatos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Medula Espinal/citologia , Medula Espinal/embriologia , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/fisiopatologia , Peixe-Zebra , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética
12.
Ann N Y Acad Sci ; 1412(1): 54-61, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29125188

RESUMO

While the majority of myasthenia gravis patients express antibodies targeting the acetylcholine receptor, the second most common cohort instead displays autoantibodies against muscle-specific kinase (MuSK). MuSK is a transmembrane tyrosine kinase found in the postsynaptic membrane of the neuromuscular junction. During development, MuSK serves as a signaling hub, coordinating the alignment of the pre- and postsynaptic components of the synapse. Adult mice that received repeated daily injections of IgG from anti-MuSK+ myasthenia gravis patients developed muscle weakness, associated with neuromuscular transmission failure. MuSK autoantibodies are predominantly of the IgG4 type. They suppress the kinase activity of MuSK and the phosphorylation of target proteins in the postsynaptic membrane. Loss of postsynaptic acetylcholine receptors is the primary cause of neuromuscular transmission failure. MuSK autoantibodies also disrupt the capacity of the motor nerve terminal to adaptively increase acetylcholine release in response to the reduced postsynaptic responsiveness to acetylcholine. The passive IgG transfer model of MuSK myasthenia gravis has been used to test candidate treatments. Pyridostigmine, a first-line cholinesterase inhibitor drug, exacerbated the disease process, while 3,4-diaminopyridine and albuterol were found to be beneficial in this mouse model.


Assuntos
Miastenia Gravis Autoimune Experimental/etiologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/imunologia , Animais , Inibidores da Colinesterase/farmacologia , Feminino , Humanos , Imunização Passiva , Camundongos , Proteínas Musculares/metabolismo , Miastenia Gravis Autoimune Experimental/imunologia , Miastenia Gravis Autoimune Experimental/fisiopatologia , Receptores Proteína Tirosina Quinases/fisiologia , Receptores Colinérgicos/imunologia , Receptores Colinérgicos/metabolismo , Sinapses/imunologia , Sinapses/fisiologia
13.
Cell Mol Life Sci ; 75(2): 335-354, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28852778

RESUMO

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that have common molecular and pathogenic characteristics, such as aberrant accumulation and ubiquitylation of TDP-43; however, the mechanisms that drive this process remain poorly understood. We have recently identified CCNF mutations in familial and sporadic ALS and FTD patients. CCNF encodes cyclin F, a component of an E3 ubiquitin-protein ligase (SCFcyclin F) complex that is responsible for ubiquitylating proteins for degradation by the ubiquitin-proteasome system. In this study, we examined the ALS/FTD-causing p.Ser621Gly (p.S621G) mutation in cyclin F and its effect upon downstream Lys48-specific ubiquitylation in transfected Neuro-2A and SH-SY5Y cells. Expression of mutant cyclin FS621G caused increased Lys48-specific ubiquitylation of proteins in neuronal cells compared to cyclin FWT. Proteomic analysis of immunoprecipitated Lys48-ubiquitylated proteins from mutant cyclin FS621G-expressing cells identified proteins that clustered within the autophagy pathway, including sequestosome-1 (p62/SQSTM1), heat shock proteins, and chaperonin complex components. Examination of autophagy markers p62, LC3, and lysosome-associated membrane protein 2 (Lamp2) in cells expressing mutant cyclin FS621G revealed defects in the autophagy pathway specifically resulting in impairment in autophagosomal-lysosome fusion. This finding highlights a potential mechanism by which cyclin F interacts with p62, the receptor responsible for transporting ubiquitylated substrates for autophagic degradation. These findings demonstrate that ALS/FTD-causing mutant cyclin FS621G disrupts Lys48-specific ubiquitylation, leading to accumulation of substrates and defects in the autophagic machinery. This study also demonstrates that a single missense mutation in cyclin F causes hyper-ubiquitylation of proteins that can indirectly impair the autophagy degradation pathway, which is implicated in ALS pathogenesis.


Assuntos
Esclerose Amiotrófica Lateral/genética , Autofagia/genética , Ciclinas/genética , Demência Frontotemporal/genética , Ubiquitinação/genética , Esclerose Amiotrófica Lateral/complicações , Células Cultivadas , Demência Frontotemporal/complicações , Células HEK293 , Humanos , Lisina/metabolismo , Mutação de Sentido Incorreto/fisiologia
14.
Open Biol ; 7(10)2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-29021214

RESUMO

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that is characterized by progressive weakness, paralysis and muscle loss often resulting in patient death within 3-5 years of diagnosis. Recently, we identified disease-linked mutations in the CCNF gene, which encodes the cyclin F protein, in cohorts of patients with familial and sporadic ALS and frontotemporal dementia (FTD) (Williams KL et al 2016 Nat. Commun.7, 11253. (doi:10.1038/ncomms11253)). Cyclin F is a part of a Skp1-Cul-F-box (SCF) E3 ubiquitin-protein ligase complex and is responsible for ubiquitylating proteins for degradation by the proteasome. In this study, we investigated the phosphorylation status of cyclin F and the effect of the serine to glycine substitution at site 621 (S621G) on E3 ligase activity. This specific mutation (S621G) was found in a multi-generational Australian family with ALS/FTD. We identified seven phosphorylation sites on cyclin F, of which five are newly reported including Ser621. These phosphorylation sites were mostly identified within the PEST (proline, glutamic acid, serine and threonine) sequence located at the C-terminus of cyclin F. Additionally, we determined that casein kinase II (CK2) can phosphorylate Ser621 and thereby regulate the E3 ligase activity of the SCF(cyclin F) complex. Furthermore, the S621G mutation in cyclin F prevents phosphorylation by CK2 and confers elevated Lys48-ubiquitylation activity, a hallmark of ALS/FTD pathology. These findings highlight the importance of phosphorylation in regulating the activity of the SCF(cyclin F) E3 ligase complex that can affect downstream processes and may lead to defective motor neuron development, neuron degeneration and ultimately ALS and FTD.


Assuntos
Caseína Quinase II/metabolismo , Ciclinas/metabolismo , Complexos Multiproteicos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida , Ativação Enzimática , Células HEK293 , Humanos , Lisina , Espectrometria de Massas , Modelos Moleculares , Fosfatidilserinas , Fosforilação , Ligação Proteica , Ubiquitinação
15.
Front Neurosci ; 11: 476, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28912673

RESUMO

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease affecting the upper and lower motor neurons in the motor cortex and spinal cord. Abnormal accumulation of mutant superoxide dismutase I (SOD1) in motor neurons is a pathological hallmark of some forms of the disease. We have shown that the orderly progression of the disease may be explained by misfolded SOD1 cell-to-cell propagation, which is reliant upon its active endogenous synthesis. Reducing the levels of SOD1 is therefore a promising therapeutic approach. Antisense oligonucleotides (ASOs) can efficiently silence proteins with gain-of-function mutations. However, naked ASOs have a short circulation half-life and are unable to cross the blood brain barrier (BBB) warranting the use of a drug carrier for effective delivery. In this study, calcium phosphate lipid coated nanoparticles (CaP-lipid NPs) were developed for delivery of SOD1 ASO to motor neurons. The most promising nanoparticle formulation (Ca/P ratio of 100:1), had a uniform spherical core-shell morphology with an average size of 30 nm, and surface charge (ζ-potential) of -4.86 mV. The encapsulation efficiency of ASO was 48% and stability studies found the particle to be stable over a period of 20 days. In vitro experiments demonstrated that the negatively charged ASO-loaded CaP-lipid NPs could effectively deliver SOD1-targeted ASO into a mouse motor neuron-like cell line (NSC-34) through endocytosis and significantly down-regulated SOD1 expression in HEK293 cells. The CaP-lipid NPs exhibited a pH-dependant dissociation, suggesting that that the acidification of lysosomes is the likely mechanism responsible for facilitating intracellular ASO release. To demonstrate tissue specific delivery and localization of these NPs we performed in vivo microinjections into zebrafish. Successful delivery of these NPs was confirmed for the zebrafish brain, the blood stream, and the spinal cord. These results suggest that CaP-lipid NPs could be an effective and safe delivery system for the improved delivery of SOD1 ASOs to motor neurons. Further in vivo evaluation in transgenic mouse models of SOD1 ALS are therefore warranted.

16.
Nanoscale ; 9(36): 13683-13692, 2017 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-28876356

RESUMO

There is considerable interest in developing diagnostic nanotools for early detection and delivery of various therapeutic agents for treatment of neurodegenerative diseases. However, a key challenge remains in the selection of suitable surfaces to overcome the nano-bio interface issue, namely that many nanoparticle surfaces demonstrate instability when administered into biological environments and show substantial cytotoxicity to the central nervous system. In this study, we fabricated an evaluation platform for bio-nano surface selection based on the combination of upconversion nanoparticles (UCNPs), cultured neural cells and zebra fish, and systemically demonstrated how it can evaluate the suitability of nanoparticle surfaces for applications in the central nervous system. Firstly, we fabricated highly lanthanide-doped UCNPs, which generate the strongest tissue penetrable emission at 800 nm. We then functionalized these UCNPs with four popular surfaces for evaluation. Next, we systematically evaluated the spectral emission properties, biophysical stability, cytotoxicity and cell uptake capability of these surface-functionalized UCNPs in biological solutions or with cultured NSC-34 cells. Through these studies, PEG-COOH proved to be the superior surface modification. Accordingly, we further confirmed the bioavailability of unmodified and surface modified UCNPs in the spinal cord of living zebrafish. As predicted, PEG-UCNPs displayed excellent dispersal and uptake into spinal motor neurons in living zebrafish. Collectively, this study developed a versatile upconversion platform for systematic evaluation of nanoparticle surfaces, which can provide valuable information via systemic surface evaluation in vitro and in vivo for future construction of multifunctional nanosystems for theranostic applications in neurodegenerative diseases.


Assuntos
Sistema Nervoso Central/efeitos dos fármacos , Elementos da Série dos Lantanídeos/química , Nanopartículas/química , Neurônios/efeitos dos fármacos , Animais , Linhagem Celular , Camundongos , Peixe-Zebra
17.
Exp Physiol ; 102(7): 773-778, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28497900

RESUMO

NEW FINDINGS: What is the central question of this study? Non-invasive muscle function tests have not been validated for use in the study of muscle performance in high-fat-fed mice. What is the main finding and its importance? This study shows that grip strength, hang wire and four-limb hanging tests are able to discriminate the muscle performance between chow-fed and high-fat-fed mice at different time points, with grip strength being reliable after 5, 10 and 20 weeks of dietary intervention. Non-invasive tests are commonly used for assessing muscle function in animal models. The value of these tests in obesity, a condition where muscle strength is reduced, is unclear. We investigated the utility of three non-invasive muscle function tests, namely grip strength (GS), hang wire (HW) and four-limb hanging (FLH), in C57BL/6 mice fed chow (chow group, n = 48) or a high-fat diet (HFD group, n = 48) for 20 weeks. Muscle function tests were performed at 5, 10 and 20 weeks. After 10 and 20 weeks, HFD mice had significantly reduced GS (in newtons; mean ± SD: 10 weeks chow, 1.89 ± 0.1 and HFD, 1.79 ± 0.1; 20 weeks chow, 1.99 ± 0.1 and HFD, 1.75 ± 0.1), FLH [in seconds per gram body weight; median (interquartile range): 10 weeks chow, 2552 (1337-4964) and HFD, 1230 (749-1994); 20 weeks chow, 2048 (765-3864) and HFD, 1036 (717-1855)] and HW reaches [n; median (interquartile range): 10 weeks chow, 4 (2-5) and HFD, 2 (1-3); 20 weeks chow, 3 (1-5) and HFD, 1 (0-2)] and higher falls [n; median (interquartile range): 10 weeks chow, 0 (0-2) and HFD, 3 (1-7); 20 weeks chow, 1 (0-4) and HFD, 8 (5-10)]. Grip strength was reliable in both dietary groups [intraclass correlation coefficient (ICC) = 0.5-0.8; P < 0.05], whereas FLH showed good reliability in chow (ICC = 0.7; P < 0.05) but not in HFD mice after 10 weeks (ICC < 0.5). Our data demonstrate that non-invasive muscle function tests are valuable and reliable tools for assessment of muscle strength and function in high-fat-fed mice.


Assuntos
Peso Corporal/fisiologia , Dieta Hiperlipídica , Obesidade/fisiopatologia , Animais , Insulina/metabolismo , Camundongos Endogâmicos C57BL , Modelos Animais , Músculo Esquelético/fisiologia , Reprodutibilidade dos Testes
18.
Methods Cell Biol ; 140: 215-244, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28528635

RESUMO

In this chapter the authors report on an automated hardware and software solution enabling swift correlative sample array mapping of fluorescently stained molecules within cells and tissues across length scales. Samples are first observed utilizing wide-field optical and fluorescence microscopy, followed by scanning electron microscopy, using calibration points on a dedicated sample-relocation holder. We investigated HeLa cells in vitro, fluorescently labeled for monosialoganglioside one (GM-1), across both imaging platforms within tens of minutes of initial sample preparation. This resulted in a high-throughput and high spatially resolved correlative fluorescence and electron microscopy analysis and allowed us to collect complementary nanoscopic information on the molecular and structural composition of two differently distinct HeLa cell populations expressing different levels of GM-1. Furthermore, using the small zebrafish animal model Danio rerio, we showed the versatility and relocation accuracy of the sample-relocation holder to locate fluo-tagged macromolecular complexes within large volumes using long ribbons of serial tissue sections. The subsequent electron microscopy imaging of the tissue arrays of interest enabled the generation of correlated information on the fine distribution of albumin within hepatic and kidney tissue. Our approach underpins the merits that an automated sample-relocation holder solution brings in support of results-driven research, where relevant biological questions can be answered, and high-throughput data can be generated in a rigorous statistical manner.


Assuntos
Microscopia Eletrônica de Varredura/métodos , Microscopia de Fluorescência/métodos , Animais , Células Endoteliais/ultraestrutura , Células HeLa , Humanos , Larva/ultraestrutura , Fígado/citologia , Peixe-Zebra/metabolismo
19.
Hum Mol Genet ; 26(14): 2616-2626, 2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-28444311

RESUMO

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, fatal neurodegenerative disease characterised by the death of upper and lower motor neurons. Approximately 10% of cases have a known family history of ALS and disease-linked mutations in multiple genes have been identified. ALS-linked mutations in CCNF were recently reported, however the pathogenic mechanisms associated with these mutations are yet to be established. To investigate possible disease mechanisms, we developed in vitro and in vivo models based on an ALS-linked missense mutation in CCNF. Proteomic analysis of the in vitro models identified the disruption of several cellular pathways in the mutant model, including caspase-3 mediated cell death. Transient overexpression of human CCNF in zebrafish embryos supported this finding, with fish expressing the mutant protein found to have increased levels of cleaved (activated) caspase-3 and increased cell death in the spinal cord. The mutant CCNF fish also developed a motor neuron axonopathy consisting of shortened primary motor axons and increased frequency of aberrant axonal branching. Importantly, we demonstrated a significant correlation between the severity of the CCNF-induced axonopathy and a reduced motor response to a light stimulus (photomotor response). This is the first report of an ALS-linked CCNF mutation in vivo and taken together with the in vitro model identifies the disruption of cell death pathways as a significant consequence of this mutation. Additionally, this study presents a valuable new tool for use in ongoing studies investigating the pathobiology of ALS-linked CCNF mutations.


Assuntos
Esclerose Amiotrófica Lateral/genética , Ciclinas/genética , Demência Frontotemporal/genética , Medula Espinal/patologia , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Animais Geneticamente Modificados , Axônios/patologia , Caspase 3/metabolismo , Morte Celular/genética , Ciclinas/biossíntese , Ciclinas/metabolismo , Modelos Animais de Doenças , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Humanos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Mutação de Sentido Incorreto , Medula Espinal/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Peixe-Zebra
20.
Small ; 13(20)2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28371073

RESUMO

Innovative nanoparticles hold promising potential for disease therapy as drug delivery systems. For brain-disease therapy, a drug delivery system that can sustainably control drug-release and monitor fluorescence of the drug cargos is highly desirable. In this study, a light-traceable and intracellular microenvironment-responsive drug delivery system was developed based on the combination of glutathione-responsive autoflurescent nanogel, dendrimer-like mesoporous silica nanoparticles, and gold nanoparticles. The resulting hybrid nanoparticles represent a new class of delivery system that can efficiently load, transport, and control multistage-release of sulfydryl-containing drugs into neurons, with light-traceable monitoring for future brain-disease therapy.


Assuntos
Microambiente Celular , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Espaço Intracelular/metabolismo , Nanopartículas Metálicas/química , Neurônios/metabolismo , Animais , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Linhagem Celular , Portadores de Fármacos/química , Fluorescência , Nanopartículas Metálicas/ultraestrutura , Camundongos , Porosidade , Dióxido de Silício/química , Propriedades de Superfície
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