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Tau is a cytosolic protein that has also been observed in the nucleus, where it has multiple proposed functions that are regulated by phosphorylation. However, the mechanism underlying the nuclear import of tau is unclear, as is the contribution of nuclear tau to the pathology of tauopathies. We have previously generated a pathological form of tau, PH-tau (pseudophosphorylation mutants S199E, T212E, T231E, and S262E) that mimics AD pathological behavior in cells, Drosophila, and a mouse model. Here, we demonstrated that PH-tau translocates into the nucleus of transiently transfected HEK-293 cells, but wildtype tau does not. We identified a putative importin binding site in the tau sequence, and showed that disruption of this site prevents tau from entering the nucleus. We further showed that this nuclear translocation is prevented by inhibitors of both importin-α and importin-ß. In addition, expression of PH-tau resulted in an enlarged population of dying cells, which is prevented by blocking its entry into the nucleus. PH-tau-expressing cells also exhibited disruption of the nuclear lamina and mislocalization of TDP-43 to the cytoplasm. We found that PH-tau does not bundle microtubules, and this effect is independent of nuclear translocation. These results demonstrate that tau translocates into the nucleus through the importin-α/ß pathway, and that PH-tau exhibits toxicity after its nuclear translocation. We propose a model where hyperphosphorylated tau not only disrupts the microtubule network, but also translocates into the nucleus and interferes with cellular functions, such as nucleocytoplasmic transport, inducing mislocalization of proteins like TDP-43 and, ultimately, cell death.
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Biological 3D models have a multitude of applications in both research and academic settings, however the generation of such models at an ultrastructural scale has remained a daunting task. Here our group presents a method by which ultrastructural 3D models can be generated using tandem scanning electron microscopy (SEM) and photogrammetry. Our methods include a novel technique for mounting specimens for SEM which allowed our group to capture images from all angles around the specimen. Our results demonstrate that using our technique is adequate for the construction of an interactive, ultrastructural 3D model that can be viewed from all orientations. We ultimately see use for these models in educational settings and research when the 3D analysis of ultrastructural anatomy is necessary. HIGHLIGHTS: Scanning electron microscopy (SEM) was used in tandem with photogrammetry to generate an ultrastructural three-dimensional model of a whole zebrafish brain. A novel mounting technique for samples in SEM was used to view the sample from all angles. This technique can be applied to a wide range of samples for 3D model generation for use in several settings, including research and academia.
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Imageamento Tridimensional , Peixe-Zebra , Animais , Imageamento Tridimensional/métodos , Microscopia Eletrônica de Varredura , Fotogrametria/métodos , SoftwareRESUMO
Onchocerciasis and lymphatic filariasis are neglected tropical diseases caused by infection with filarial worms. Annual or biannual mass drug administration with microfilaricidal drugs that kill the microfilarial stages of the parasites has helped reduce infection rates and thus prevent transmission of both infections. However, success depends on high population coverage that is maintained for the duration of the adult worm's lifespan. Given that these filarial worms can live up to 14 years in their human hosts, a macrofilaricidal drug would vastly accelerate elimination efforts. Here, we have evaluated the repurposed drug pyrvinium pamoate as well as newly synthesized analogs of pyrvinium for their efficacy against filarial worms in vitro and in vivo. We found that pyrvinium pamoate, tetrahydropyrvinium and one of the analogs were highly potent in inhibiting worms in in vitro whole-worm screening assays, and that all three compounds reduced female worm fecundity and inhibited embryogenesis in the Brugia pahangi-gerbil in vivo model of infection.
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The SARS-CoV-2 coronavirus is an enveloped, positive-sense single-stranded RNA virus that is responsible for the COVID-19 pandemic. The spike is a class I viral fusion glycoprotein that extends from the viral surface and is responsible for viral entry into the host cell and is the primary target of neutralizing antibodies. The receptor binding domain (RBD) of the spike samples multiple conformations in a compromise between evading immune recognition and searching for the host-cell surface receptor. Using atomistic simulations of the glycosylated wild-type spike in the closed and 1-up RBD conformations, we map the free energy landscape for RBD opening and identify interactions in an allosteric pocket that influence RBD dynamics. The results provide an explanation for experimental observation of increased antibody binding for a clinical variant with a substitution in this pocket. Our results also suggest the possibility of allosteric targeting of the RBD equilibrium to favor open states via binding of small molecules to the hinge pocket. In addition to potential value as experimental probes to quantify RBD conformational heterogeneity, small molecules that modulate the RBD equilibrium could help explore the relationship between RBD opening and S1 shedding.
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SARS-CoV-2/química , Glicoproteína da Espícula de Coronavírus/química , Sítio Alostérico , Simulação de Dinâmica Molecular , Domínios Proteicos , TermodinâmicaRESUMO
Classical electron microscopic morphological studies provide detailed ultrastructural information, which may lend insights into cellular functions. As a follow-up to our morphological investigation of the adult zebrafish (Danio rerio) optic tectum, in this study, we have analyzed the ependymal structures lining the surfaces of the tectal ventricle: the torus, tegmental surface of the valvula cerebelli and the periventricular gray zone of the optic tectal cortex. We used toluidine blue stained plastic (semithin) sections for light microscopy and scanning electron microscopy. Our morphological findings of gated entrances and/or egresses indicate that, at least in the adult zebrafish brain, there may be a bidirectional direct flow communication between the ventricular cerebrospinal fluid and the parenchymal interstitial fluid.
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Encéfalo/fisiologia , Epêndima/ultraestrutura , Hidrodinâmica , Colículos Superiores/ultraestrutura , Peixe-Zebra/anatomia & histologia , Animais , Líquido Cefalorraquidiano/fisiologia , Epêndima/anatomia & histologia , Líquido Extracelular/fisiologia , Feminino , Masculino , Microscopia , Microscopia Eletrônica de Varredura , Colículos Superiores/citologiaRESUMO
Tau is a neuronal microtubule associated protein whose main biological functions are to promote microtubule self-assembly by tubulin and to stabilize those already formed. Tau also plays an important role as an axonal microtubule protein. Tau is an amazing protein that plays a key role in cognitive processes, however, deposits of abnormal forms of tau are associated with several neurodegenerative diseases, including Alzheimer disease (AD), the most prevalent, and Chronic Traumatic Encephalopathy (CTE) and Traumatic Brain Injury (TBI), the most recently associated to abnormal tau. Tau post-translational modifications (PTMs) are responsible for its gain of toxic function. Alonso et al. (1996) were the first to show that the pathological tau isolated from AD brains has prion-like properties and can transfer its toxic function to the normal molecule. Furthermore, we reported that the pathological changes are associated with tau phosphorylation at Ser199 and 262 and Thr212 and 231. This pathological version of tau induces subcellular mislocalization in cultured cells and neurons, and translocates into the nucleus or accumulated in the perinuclear region of cells. We have generated a transgenic mouse model that expresses pathological human tau (PH-Tau) in neurons at two different concentrations (4% and 14% of the total endogenous tau). In this model, PH-Tau causes cognitive decline by at least two different mechanisms: one that involves the cytoskeleton with axonal disruption (at high concentration), and another in which the apparent neuronal morphology is not grossly affected, but the synaptic terminals are altered (at lower concentration). We will discuss the putative involvement of tau in proteostasis under these conditions. Understanding tau's biological activity on and off the microtubules will help shed light to the mechanism of neurodegeneration and of normal neuronal function.
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INTRODUCTION: Accumulation of hyperphosphorylated tau and the disruption of microtubules are correlated with synaptic loss and pathology of Alzheimer's disease (AD). Impaired cognitive function and pathology of AD is correlated with this lesion. This review looks at the mechanism of neurodegeneration, the prion-like behavior of tau in its interaction with normal MAPs in correlation with tau hyperphosphorylation. METHODS: We reviewed our work in the field as well as current literature that pertains to tau phosphorylation and the biological effects. RESULTS: Hyperphosphorylation of tau in AD, in vitro, in cells, or in animal models converts this protein into a prion-like protein that is able to propagate the altered conformation. DISCUSSION: These findings suggest that phosphorylation of tau is a critical event in neurodegeneration. The combination of phosphorylation sites can generate a gain of toxic function for tau. The mechanism of tau toxicity might involve not only the microtubule system but also interference with other cellular compartments such as the nucleus and the actin cytoskeleton.
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Microtúbulos , Doenças Priônicas , Tauopatias/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/patologia , Animais , Humanos , FosforilaçãoRESUMO
Tau is a microtubule-associated protein mainly found in neurons. The protein is associated with process of microtubule assembly, which plays an important role in intracellular transport and cell structure of the neuron. Tauopathies are a group of neurodegenerative diseases specifically associated with tau abnormalities. While a well-defined mechanism remains unknown, most facts point to tau as a prominent culprit in neurodegeneration. In most cases of Tauopathies, aggregates of hyperphosphorylated tau have been found. Two proposals are present when discussing tau toxicity, one being the aggregation of tau proteins and the other points toward a conformational change within the protein. Previous work we carried out showed tau hyperphosphorylation promotes tau to behave abnormally resulting in microtubule assembly disruption as well as a breakdown in tau self-assembly. We found that tau's N-terminal region has a putative site for ATP/GTP binding. In this paper we demonstrate that tau is able to bind ATP and not GTP, that this binding induces tau self-assembly into filaments. At 1 mM ATP the filaments are 4-7 nm in width, whereas at 10 mM ATP the filaments appeared to establish lateral interaction, bundling and twisting, forming filaments that resembled the Paired Helical Filaments (PHF) isolated from Alzheimer disease brain. ATP-induced self-assembly is not energy dependent because the nonhydrolysable analogue of the ATP induces the same assembly.
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Trifosfato de Adenosina/metabolismo , Desnaturação Proteica , Multimerização Proteica , Proteínas tau/metabolismo , Humanos , Ligação Proteica , Tauopatias/patologia , Tauopatias/fisiopatologiaRESUMO
Cellular composition of the adult zebrafish (Danio rerio) optic tectal cortex was examined in this study. Morphological techniques such as 1 µm thick serial plastic sections stained with osmium tetroxide and toluidine blue, modified rapid Golgi silver impregnation, GFAP immunohistochemistry, confocal microscopy, as well as scanning and transmission electron microscopy were used. Neuronal and glial components are described and the layers of the cortex are revisited. Specific neuronal arrangements as well as unique glial/ependymal cells are described. A three dimensional rendering of the astrocytic fiber arrangement in the marginal zone is presented and a composite drawing summarizes the cellular composition of the optic tectum.
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Colículos Superiores/citologia , Peixe-Zebra/anatomia & histologia , Animais , Imuno-Histoquímica , Microscopia Confocal , Microscopia EletrônicaRESUMO
Alzheimer's disease is a progressive neurodegenerative disease that is characterized histopathologically by the presence of plaques, mainly composed of Abeta amyloid and the tangles, mainly composed of hyperphosphorylated tau. To date, there is no treatment that can reverse the disease, and all the current therapeutics is directed to cope with the symptoms of the disease. Here we describe the efforts dedicated to attack the plaques and, in more detail, the process of neurofibrillary degeneration, linked to the presence of the hyperphosphorylated microtubule associated protein tau. We have identified the different putative targets for therapeutics and the current knowledge on them.
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Doença de Alzheimer/tratamento farmacológico , Terapia de Alvo Molecular , Proteínas tau/metabolismo , Precursor de Proteína beta-Amiloide/imunologia , Animais , Humanos , Modelos BiológicosRESUMO
The synthesis of a water/plasma soluble, noncytotoxic, "clicked" sugar-derivative of curcumin with amplified bioefficacy in modulating amyloid-ß and tau peptide aggregation is presented. Curcumin inhibits amyloid-ß and tau peptide aggregation at micromolar concentrations; the sugar-curcumin conjugate inhibits Aß and tau peptide aggregation at concentrations as low as 8 nM and 0.1 nM, respectively. In comparison to curcumin, this conveniently synthesized Alzheimer's drug candidate is a more powerful antioxidant.
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Peptídeos beta-Amiloides/metabolismo , Curcumina/síntese química , Curcumina/metabolismo , Galactose/síntese química , Galactose/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Animais , Antioxidantes/administração & dosagem , Antioxidantes/síntese química , Antioxidantes/metabolismo , Células Cultivadas , Curcumina/administração & dosagem , Galactose/administração & dosagem , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , CamundongosRESUMO
Abnormal hyperphosphorylation of the microtubule-associated protein Tau is a hallmark of Alzheimer disease and related diseases called tauopathies. As yet, the exact mechanism by which this pathology causes neurodegeneration is not understood. The present study provides direct evidence that Tau abnormal hyperphosphorylation causes its aggregation, breakdown of the microtubule network, and cell death and identifies phosphorylation sites involved in neurotoxicity. We generated pseudophosphorylated Tau proteins by mutating Ser/Thr to Glu and, as controls, to Ala. These mutations involved one, two, or three pathological phosphorylation sites by site-directed mutagenesis using as backbones the wild type or FTDP-17 mutant R406W Tau. Pseudophosphorylated and corresponding control Tau proteins were expressed transiently in PC12 and CHO cells. We found that a single phosphorylation site alone had little influence on the biological activity of Tau, except Thr(212), which, upon mutation to Glu in the R406W background, induced Tau aggregation in cells, suggesting phosphorylation at this site along with a modification on the C-terminal of the protein facilitates self-assembly of Tau. The expression of R406W Tau pseudophosphorylated at Thr(212), Thr(231), and Ser(262) triggered caspase-3 activation in as much as 85% of the transfected cells, whereas the corresponding value for wild type pseudophosphorylated Tau was 30%. Cells transfected with pseudophosphorylated Tau became TUNEL-positive.
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Doenças Neurodegenerativas/metabolismo , Proteínas tau/metabolismo , Substituição de Aminoácidos , Animais , Células CHO , Caspase 3/genética , Caspase 3/metabolismo , Cricetinae , Cricetulus , Ativação Enzimática/genética , Humanos , Mutação de Sentido Incorreto , Doenças Neurodegenerativas/genética , Células PC12 , Fosforilação/genética , Estrutura Terciária de Proteína , Ratos , Proteínas tau/genéticaRESUMO
BACKGROUND: The present patent deals with the generation of peptides derived from the activity-dependent peptide and tau mimetic to study its effect on microtubule stability, its ability to bind to tubulin and MAPs, as well as promoting cell survival. OBJECTIVE: To analyze these peptides and their effects as potential therapeutic elements for neurodegenerative diseases. METHODS: We review the action of the peptides described by Gozes and collaborators and compare the effectiveness with those already reported in the literature for Alzheimer's disease. CONCLUSION: The research of Dr. Gozes and collaborators has shown that the addition of picomolar concentration of the peptides promotes cell survival, by interacting with tubulin and stabilizing the microtubules. Based on the results, these peptides seem to be very attractive candidates for therapeutical intervention in neurodegenerative diseases.
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Doença de Alzheimer/tratamento farmacológico , Microtúbulos/metabolismo , Peptídeos/farmacologia , Doença de Alzheimer/fisiopatologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Humanos , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/fisiopatologia , Patentes como Assunto , Ligação Proteica , Tubulina (Proteína)/metabolismo , Proteínas tau/metabolismoRESUMO
In the absence of a fully effective herpes simplex virus (HSV) vaccine, topical microbicides represent an important strategy for preventing HSV transmission. (-)-Epigallocatechin gallate (EGCG) (molecular weight, 458.4) is the primary catechin in green tea. The present study shows that EGCG has greater anti-HSV activity than other green tea catechins and inactivates multiple clinical isolates of HSV type 1 (HSV-1) and HSV-2. EGCG reduced HSV-2 titers by >or=1,000-fold in 10 to 20 min and reduced HSV-1 titers by the same amount in 30 to 40 min. The anti-HSV activity of EGCG is due to a direct effect on the virion, and incubating Vero and CV1 cells with EGCG for 48 h prior to infection with HSV-1 and HSV-2, respectively, does not reduce HSV production. Electron microscopic (EM) studies showed that purified virions exposed to EGCG were damaged, and EM immunogold labeling of the envelope glycoproteins gB and gD was significantly reduced following EGCG treatment while capsid protein labeling was unchanged. When purified HSV-1 envelope glycoproteins gB and gD were incubated with EGCG and then examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, lower-molecular-weight gB and gD bands decreased and new higher-molecular-weight bands appeared, indicating the EGCG-dependent production of macromolecular complexes. gB and gD are essential for HSV infectivity, and these results suggest that EGCG could inactivate HSV virions by binding to gB, gD, or another envelope glycoprotein. EGCG is stable in the pH range found in the vagina and appears to be a promising candidate for use in a microbicide to reduce HSV transmission.