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1.
Adv Mater ; 32(24): e2000416, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32374446

RESUMO

Small interfering RNA (siRNA) has been considered as a highly promising therapeutic agent for human cancer treatment including glioblastoma (GBM), which is a fatal disease without effective therapy methods. However, siRNA-based GBM therapy is seriously hampered by a number of challenges in siRNA brain delivery including poor stability, short blood circulation, low blood-brain barrier (BBB) penetration, and tumor accumulation, as well as inefficient siRNA intracellular release. Herein, an Angiopep-2 (Ang) functionalized intracellular-environment-responsive siRNA nanocapsule (Ang-NCss (siRNA)) is successfully developed as a safe and efficient RNAi agent to boost siRNA-based GBM therapy. The experimental results demonstrate that the developed Ang-NCss (siRNA) displays long circulation in plasma, efficient BBB penetration capability, and GBM accumulation and retention, as well as responsive intracellular siRNA release due to the unique design of small size (25 nm) with polymeric shell for siRNA protection, Ang functionalization for BBB crossing and GBM targeting, and disulfide bond as a linker for intracellular-environment-responsive siRNA release. Such superior properties of Ang-NCss (siRNA) result in outstanding growth inhibition of orthotopic U87MG xenografts without causing adverse effects, achieving remarkably improved survival benefits. The developed siRNA nanocapsules provide a new strategy for RNAi therapy of GBM and beyond.

2.
Cell Mol Life Sci ; 2020 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-32130428

RESUMO

Metallomics is a rapidly evolving field of bio-metal research that integrates techniques and perspectives from other "-omics" sciences (e.g. genomics, proteomics) and from research vocations further afield. Perhaps the most esoteric of this latter category has been the recent coupling of biomedicine with element and isotope geochemistry, commonly referred to as isotope metallomics. Over the course of less than two decades, isotope metallomics has produced numerous benchmark studies highlighting the use of stable metal isotope distribution in developing disease diagnostics-e.g. cancer, neurodegeneration, osteoporosis-as well as their utility in deciphering the underlying mechanisms of such diseases. These pioneering works indicate an enormous wealth of potential and provide a call to action for researchers to combine and leverage expertise and resources to create a clear and meaningful path forward. Doing so with efficacy and impact will require not only building on existing research, but also broadening collaborative networks, bolstering and deepening cross-disciplinary channels, and establishing unified and realizable objectives. The aim of this review is to briefly summarize the field and its underpinnings, provide a directory of the state of the art, outline the most encouraging paths forward, including their limitations, outlook and speculative upcoming breakthroughs, and finally to offer a vision of how to cultivate isotope metallomics for an impactful future.

3.
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.

4.
Eur J Neurosci ; 2020 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-31954073

RESUMO

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, characterised by the degeneration of motor neurons innervating skeletal muscle. The mechanisms underlying neurodegeneration in ALS are not yet fully elucidated, and with current therapeutics only able to extend lifespan by a matter of months there is a clear need for novel therapies to increase lifespan and patient quality of life. Here, we evaluated whether moderate-intensity treadmill exercise and/or treatment with metallothionein-2 (MT2), a neuroprotective protein, could improve survival, behavioural or neuropathological outcomes in SOD1G93A familial ALS mice. Six-week-old female SOD1G93A mice were allocated to one of four treatment groups: MT2 injection, i.m.; moderate treadmill exercise; neither MT2 nor exercise; or both MT2 and exercise. MT2-treated mice survived around 3% longer than vehicle-treated mice, with this mild effect reaching statistical significance in Cox proportional hazards analysis once adjusted for potential confounders. Mixed model body weight trajectories over time indicated that MT2-treated mice, with or without exercise, reached maximum body weight at a later age, suggesting a delay in disease onset of around 4% compared to saline-treated mice. Exercise alone did not significantly increase survival or delay disease onset, and neither exercise nor MT2 substantially ameliorated gait abnormalities or muscle strength loss. We conclude that neither exercise nor MT2 treatment was detrimental in female SOD1G93A mice, and further study could determine whether the mild effect of peripheral MT2 administration on disease onset and survival could be improved via direct administration of MT2 to the central nervous system.

5.
Commun Biol ; 2: 198, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31149642

RESUMO

Precise genome editing is limited by the inefficiency of homology-directed repair (HDR) compared to the non-homologous end-joining (NHEJ) of double strand breaks (DSBs). The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 system generates precise, locus-specific DSBs that can serve as substrates for HDR. We developed an in vivo visual reporter assay to quantify HDR-mediated events at single-cell resolution in zebrafish and used this system to identify small-molecule modulators that shift the DNA repair equilibrium in favor of HDR. By further optimizing the reaction environment and repair template, we achieved dramatic enhancement of HDR-mediated repair efficiency in zebrafish. Accordingly, under optimized conditions, inhibition of NHEJ with NU7441 enhanced HDR-mediated repair up to 13.4-fold. Importantly, we demonstrate that the increase in somatic HDR events correlates directly with germline transmission, permitting the efficient recovery of large seamlessly integrated DNA fragments in zebrafish.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Genótipo , Proteínas de Fluorescência Verde/metabolismo , RNA/metabolismo , Reparo de DNA por Recombinação
6.
Cells ; 8(2)2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30791479

RESUMO

The formation of cytotoxic intracellular protein aggregates is a pathological signature of multiple neurodegenerative diseases. The principle aggregating protein in Parkinson's disease (PD) and atypical Parkinson's diseases is α-synuclein (α-syn), which occurs in neural cytoplasmic inclusions. Several factors have been found to trigger α-syn aggregation, including raised calcium, iron, and copper. Transcriptional inducers have been explored to upregulate expression of endogenous metal-binding proteins as a potential neuroprotective strategy. The vitamin-D analogue, calcipotriol, induced increased expression of the neuronal vitamin D-dependent calcium-binding protein, calbindin-D28k, and this significantly decreased the occurrence of α-syn aggregates in cells with transiently raised intracellular free Ca, thereby increasing viability. More recently, the induction of endogenous expression of the Zn and Cu binding protein, metallothionein, by the glucocorticoid analogue, dexamethasone, gave a specific reduction in Cu-dependent α-syn aggregates. Fe accumulation has long been associated with PD. Intracellularly, Fe is regulated by interactions between the Fe storage protein ferritin and Fe transporters, such as poly(C)-binding protein 1. Analysis of the transcriptional regulation of Fe binding proteins may reveal potential inducers that could modulate Fe homoeostasis in disease. The current review highlights recent studies that suggest that transcriptional inducers may have potential as novel mechanism-based drugs against metal overload in PD.


Assuntos
Metais/metabolismo , Doença de Parkinson/metabolismo , Sequência de Aminoácidos , Homeostase , Humanos , Neuroproteção , Ligação Proteica , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo
8.
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
9.
Int J Mol Sci ; 19(12)2018 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-30572656

RESUMO

Multiple system atrophy, characterized by atypical Parkinsonism, results from central nervous system (CNS) cell loss and dysfunction linked to aggregates of the normally pre-synaptic α-synuclein protein. Mostly cytoplasmic pathological α-synuclein inclusion bodies occur predominantly in oligodendrocytes in affected brain regions and there is evidence that α-synuclein released by neurons is taken up preferentially by oligodendrocytes. However, extracellular α-synuclein has also been shown to interact with other neural cell types, including astrocytes and microglia, as well as extracellular factors, mediating neuroinflammation, cell-to-cell spread and other aspects of pathogenesis. Here, we review the current evidence for how α-synuclein present in the extracellular milieu may act at the cell surface to drive components of disease progression. A more detailed understanding of the important extracellular interactions of α-synuclein with neuronal and non-neuronal cell types both in the brain and periphery may provide new therapeutic targets to modulate the disease process.


Assuntos
Espaço Extracelular/metabolismo , Atrofia de Múltiplos Sistemas/metabolismo , alfa-Sinucleína/metabolismo , Animais , Astrócitos/metabolismo , Humanos , Microglia/metabolismo , Neurônios/metabolismo , Oligodendroglia/metabolismo
10.
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
11.
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
12.
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
13.
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
14.
J Neuroinflammation ; 15(1): 56, 2018 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-29471847

RESUMO

BACKGROUND: The extracellular environment plays an important role in supporting the regeneration of axons after injury. Metallothionein-II (MTII) is a metal-binding protein known for its neuroprotective effect by directly stimulating the growth of axons after injury. Previous studies have shown that MTII also modulates the response of astrocytes and microglia after injury. However, a detailed analysis describing how MTII modulates the interaction between microglia and neurons is lacking. METHODS: We introduced fluorescently labelled MTII into the cortex at the time of needlestick injury to investigate the cellular uptake of MTII using immunohistochemistry with antibodies against cell-type-specific markers. The role of MTII in modulating the effect of microglia on axon outgrowth following an inflammatory response is further investigated using a co-culture model involving primary rodent microglia pre-treated with TNFα and primary rodent cortical neurons. The axon lengths were assessed 24 h after the plating of the neurons onto treated microglia. We also utilised siRNA to knockdown the expression of LRP1, which allows us to investigate the role of LRP1 receptors in the MTII-mediated effect of microglia on axon outgrowth. RESULTS: Fluorescently labelled MTII was found to be associated with neurons, astrocytes and microglia following injury in vivo. Microglia-neuron co-culture experiments demonstrated that exogenous MTII altered the response of microglia to TNFα. The neurons plated onto the TNFα-stimulated microglia pre-treated with MTII have shown a significantly longer axonal length compare to the TNFα-stimulated microglia without the MTII treatment. This suggested that MTII reduce cytokine-stimulated activation of microglia, which would ordinarily impair neurite outgrowth. This inhibitory effect of MTII on activated microglia was blocked by siRNA-mediated downregulation of LRP1 receptor expression in microglia, suggesting that MTII acts via the LRP1 receptor on microglia. CONCLUSIONS: This study demonstrates that exogenous MTII acts via the LRP1 receptor to alter the inflammatory response of microglia following TNFα stimulation, providing a more supportive environment for axon growth.


Assuntos
Córtex Cerebral/metabolismo , Metalotioneína/metabolismo , Microglia/metabolismo , Regeneração Nervosa/fisiologia , Neurônios/metabolismo , Fator de Necrose Tumoral alfa/toxicidade , Animais , Animais Recém-Nascidos , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Técnicas de Cocultura , Metalotioneína/farmacologia , Microglia/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Coelhos , Ratos , Ratos Sprague-Dawley
15.
Neurotox Res ; 33(2): 229-238, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29064068

RESUMO

Intracellular aggregates of α-synuclein are the pathological hallmark of Parkinson's disease (PD) and dementia with Lewy bodies (DLB), being linked to neurotoxicity. Multiple triggers of α-synuclein aggregation have been implicated, including raised copper. The potential protective role of the endogenous copper-/zinc-binding proteins, metallothioneins (MT), has been explored in relation to copper-induced α-synuclein aggregation. Up-regulated endogenous expression of MT was induced in SHSY-5Y cells by the synthetic glucocorticoid analogue, dexamethasone. After treatment to induce endogenous MT expression, immunofluorescence confocal microscopy was used to quantify protein aggregates in cells with/without copper treatment. MT induction resulted in significant (p < 0.01), dose-dependent up-regulation of MT expression and significant reduction in Cu-dependent α-synuclein intracellular aggregates (p < 0.01) that could be suppressed by MT-specific siRNA. Ubiquitous (MT-2) and brain-specific (MT-3) isoforms were investigated by transient transfection of the GFP-fusion proteins, observing equivalent α-synuclein aggregate suppression by each. These studies indicate MT induction could have potential in PD/DLB neuroprotective therapy by suppressing α-synuclein aggregation.


Assuntos
Cobre/farmacologia , Dexametasona/farmacologia , Metalotioneína/efeitos dos fármacos , alfa-Sinucleína/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Linhagem Celular Tumoral , Humanos , Doença por Corpos de Lewy/tratamento farmacológico , Metalotioneína/metabolismo , Doença de Parkinson/metabolismo , alfa-Sinucleína/metabolismo
16.
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
17.
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
18.
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.

19.
Int J Biochem Cell Biol ; 89: 216-220, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28652210

RESUMO

Cyclin F, encoded by CCNF, is the substrate recognition component of the Skp1-Cul1-F-box E3 ubiquitin ligase complex, SCFcyclin F. E3 ubiquitin ligases play a key role in ubiquitin-proteasome mediated protein degradation, an essential component of protein homeostatic mechanisms within the cell. By recognising and regulating the availability of several protein substrates, SCFcyclin F plays a role in regulating various cellular processes including replication and repair of DNA and cell cycle checkpoint control. Cyclin F dysfunction has been implicated in various forms of cancer and CCNF mutations were recently linked to familial and sporadic amyotrophic lateral sclerosis and frontotemporal dementia, offering a new lead to understanding the pathogenic mechanisms underlying neurodegeneration. In this review, we evaluate the current literature on the function of cyclin F with an emphasis on its roles in cancer and neurodegeneration.


Assuntos
Ciclinas/metabolismo , Neoplasias/metabolismo , Doenças Neurodegenerativas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Ciclinas/química , Ciclinas/genética , Regulação da Expressão Gênica , Humanos , Neoplasias/patologia , Doenças Neurodegenerativas/patologia
20.
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
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