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1.
Int J Dev Neurosci ; 74: 1-10, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30753937

RESUMO

The aim of this study was to examine the spatio-temporal appearance of different neuronal cell subtypes by analyzing expression patterns of several neuronal markers (calretinin, neurofilament 200 (NF200), vanilloid receptor 1(VR1) and calcitonin gene-related peptide (CGRP)) of the embryonic human spinal cord (SC). Developing human SCs from 11 human conceptuses beetwen 5-10 developmental weeks (DW) were examined by light and electron microscopy and immunofluorescence. Light and electron microscopy revealed different embryonic stages of recognizable structure of the SC. NF200, CGRP and VR1 positive cells were observed in SCs during 5th-6th DW. NF200 was predominantly expressed in the ventral part, indicating presence of motoneurons. As development advanced, NF200 was mainly expressed in the marginal zone. Expression of CGRP was intense during all of the investigated periods, predominantly during the 5th-6th DW pointing to neural sensory differentiation, as opposed to the last DW when reduced expression of CGRP in the marginal layer indicated the terminations of the sensory afferents. Expression of VR1 was highest in the intermediate zone, at the beginning and at the end of the investigated periods, pointing to VR1 spatial pattern in the visceral afferents in the grey matter, while the first signs of calretinin were found in the 9th-10th DW ventrally. Delineating the relationships between factors involved in processes of neuronal differentiation as well as spatial and temporal arrangement of SC interrelated neurons can provide a useful information about normal SC development as well as the insight in possible causes of anomalies and disorders during embryonic life.


Assuntos
Biomarcadores/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Medula Espinal/citologia , Medula Espinal/embriologia , Fatores Etários , Calbindina 2/metabolismo , Calbindina 2/ultraestrutura , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Idade Gestacional , Humanos , Microscopia Eletrônica de Transmissão , Proteínas do Tecido Nervoso/ultraestrutura , Proteínas de Neurofilamentos/metabolismo , Proteínas de Neurofilamentos/ultraestrutura , Neurônios/classificação , Neurônios/ultraestrutura , Canais de Cátion TRPV/metabolismo , Canais de Cátion TRPV/ultraestrutura
2.
Neuropathology ; 34(6): 589-95, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24673472

RESUMO

Basophilic inclusions (BIs) and neuronal intermediate filament inclusions (NIFIs) are key structures of basophilic inclusion body disease and neuronal intermediate filament inclusion disease (NIFID), respectively. BIs are sharply-defined, oval or crescent neuronal intracytoplasmic inclusions that appear pale blue-gray in color with HE staining and purple in color with Nissl but are stained poorly with silver impregnation techniques. Immunohistochemically BIs are negative for tau, trans-activation response DNA 43 (TDP-43), α-synuclein, neurofilament (NF) and α-internexin, positive for p62, and variably ubiquitinated. Noticeably, BIs are consistently fused in sarcoma (FUS) positive. NIFIs are by definition immuno-positive for class IV IFs including three NF triplet subunit proteins and α-internexin but negative for tau, TDP-43, and α-synuclein. In NIFID cases several types of inclusions have been identified. Among them, hyaline conglomerate-like inclusions are the only type that meets the above immunohistochemical features of NIFIs. This type of inclusion appears upon HE staining as multilobulated, faintly eosinophilic or pale amphophilic spherical masses with a glassy appearance. These hyaline conglomerates appear strongly argyrophilic, and robustly and consistently immuno-positive for IFs. In contrast, this type of inclusion shows no or only occasional dot-like FUS immunoreactivity. Therefore, BIs and NIFIs are distinct from each other in terms of morphological, tinctorial and immunohistochemical features. However, basophilic inclusion body disease (BIBD) and NIFID are difficult to differentiate clinically. Moreover, Pick body-like inclusions, the predominant type of inclusions seen in NIFID, are considerably similar to the BIs of BIBD in that this type of inclusion is basophilic, poorly argyrophilic, negative for IFs and intensely immuno-positive for FUS. As BIBD and NIFID share FUS accumulation as the most prominent molecular pathology, whether these two diseases are discrete entities or represent a pathological continuum remains a question to be answered.


Assuntos
Esclerose Lateral Amiotrófica/patologia , Degeneração Lobar Frontotemporal/patologia , Corpos de Inclusão/ultraestrutura , Proteínas de Neurofilamentos/ultraestrutura , Neurônios/ultraestrutura , Esclerose Lateral Amiotrófica/metabolismo , Proteínas de Ligação a DNA/metabolismo , Degeneração Lobar Frontotemporal/metabolismo , Humanos , Filamentos Intermediários/metabolismo , Filamentos Intermediários/ultraestrutura , Proteínas de Neurofilamentos/metabolismo , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo
3.
J Neurosci ; 32(18): 6209-19, 2012 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-22553027

RESUMO

Maturation of the peripheral nervous system requires specification of axonal diameter, which, in turn, has a significant influence on nerve conduction velocity. Radial axonal growth initiates with myelination, and is dependent upon the C terminus of neurofilament medium (NF-M). Molecular phylogenetic analysis in mammals suggested that expanded NF-M C termini correlated with larger-diameter axons. We used gene targeting and computational modeling to test this new hypothesis. Increasing the length of NF-M C terminus in mice increased diameter of motor axons without altering neurofilament subunit stoichiometry. Computational modeling predicted that an expanded NF-M C terminus extended farther from the neurofilament core independent of lysine-serine-proline (KSP) phosphorylation. However, expansion of NF-M C terminus did not affect the distance between adjacent neurofilaments. Increased axonal diameter did not increase conduction velocity, possibly due to a failure to increase myelin thickness by the same proportion. Failure of myelin to compensate for larger axonal diameters suggested a lack of plasticity during the processes of myelination and radial axonal growth.


Assuntos
Axônios/fisiologia , Axônios/ultraestrutura , Bainha de Mielina/metabolismo , Bainha de Mielina/ultraestrutura , Condução Nervosa/fisiologia , Proteínas de Neurofilamentos/metabolismo , Proteínas de Neurofilamentos/ultraestrutura , Animais , Células Cultivadas , Camundongos , Camundongos Transgênicos , Conformação Proteica
4.
IEEE Trans Med Imaging ; 31(1): 117-30, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21859599

RESUMO

Neurofilaments are long flexible cytoplasmic protein polymers that are transported rapidly but intermittently along the axonal processes of nerve cells. Current methods for studying this movement involve manual tracking of fluorescently tagged neurofilament polymers in videos acquired by time-lapse fluorescence microscopy. Here, we describe an automated tracking method that uses particle filtering to implement a recursive Bayesian estimation of the filament location in successive frames of video sequences. To increase the efficiency of this approach, we take advantage of the fact that neurofilament movement is confined within the boundaries of the axon. We use piecewise cubic spline interpolation to model the path of the axon and then we use this model to limit both the orientation and location of the neurofilament in the particle tracking algorithm. Based on these two spatial constraints, we develop a prior dynamic state model that generates significantly fewer particles than generic particle filtering, and we select an adequate observation model to produce a robust tracking method. We demonstrate the efficacy and efficiency of our method by performing tracking experiments on real time-lapse image sequences of neurofilament movement, and we show that the method performs well compared to manual tracking by an experienced user. This spatially constrained particle filtering approach should also be applicable to the movement of other axonally transported cargoes.


Assuntos
Axônios/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Proteínas de Neurofilamentos/metabolismo , Algoritmos , Animais , Animais Recém-Nascidos , Axônios/ultraestrutura , Teorema de Bayes , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Camundongos , Proteínas de Neurofilamentos/ultraestrutura , Imagem com Lapso de Tempo
5.
J Comp Neurol ; 519(18): 3657-71, 2011 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-21618230

RESUMO

In mammals neurofilaments (NF) are formed by coassembly of three subunits: NFL, NFM, and NFH (light, medium, and heavy). It had been believed that lampreys have only one subunit, NF180. However, a previous study showed that NF180 could not self-assemble but could coassemble with rat NFL, suggesting the existence of additional NF subunits in lamprey. More recently, we cloned three additional NF subunits. These new subunits and NF180 have now been transfected in combinations into SW13cl.2Vim(-) cells, which lack endogenous cytoplasmic intermediate filaments. None of the subunits could self-assemble. No combination of NF subunits could form filaments in the absence of lamprey NFL (L-NFL). Assembly occurred at 28°C, but not at 37°C. L-NFL could form thick NF bundles with NF180 but not with NF132 and NF95, which formed only fine filamentous arrays. To determine which parts of the NF subunits are required for filament or bundle formation, we constructed deletion mutants of NF180 and cotransfected them with L-NFL. As with mammalian NF, only constructs with intact head and core domains could form filaments with L-NFL. However, the full length of NF180 was required to form NF bundles. As with NF180, in situ hybridization indicated that mRNA for L-NFL and NF132 was downregulated in identified reticulospinal neurons by 5 weeks after spinal cord transection, but was reexpressed at 10 weeks selectively in those neurons whose axons have a high probability of regenerating. This is consistent with a possible role of NFs in the mechanism of axon regeneration.


Assuntos
Lampreias/fisiologia , Regeneração Nervosa/fisiologia , Proteínas de Neurofilamentos/metabolismo , Subunidades Proteicas/classificação , Traumatismos da Medula Espinal/metabolismo , Animais , Citoesqueleto/metabolismo , Humanos , Imuno-Histoquímica , Proteínas de Neurofilamentos/ultraestrutura , Conformação Proteica , Processamento de Proteína Pós-Traducional , Subunidades Proteicas/metabolismo , Deleção de Sequência , Medula Espinal/metabolismo , Temperatura , Transfecção/métodos , Células Tumorais Cultivadas
6.
J Neurosci Res ; 89(3): 310-9, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21259318

RESUMO

The polarized domains of myelinated axons are specifically organized to maximize the efficiency of saltatory conduction. The paranodal region is directly adjacent to the node of Ranvier and contains specialized septate-like junctions that provide adhesion between axons and glial cells and that constitute a lateral diffusion barrier for nodal components. To complement and extend earlier studies on the peripheral nervous system, electron tomography was used to image paranodal regions from the central nervous system (CNS). Our three-dimensional reconstructions revealed short filamentous linkers running directly from the septate-like junctions to neurofilaments, microfilaments, and organelles within the axon. The intercellular spacing between axons and glia was measured to be 7.4 ± 0.6 nm, over twice the value previously reported in the literature (2.5-3.0 nm). Averaging of individual junctions revealed a bifurcated structure in the intercellular space that is consistent with a dimeric complex of cell adhesion molecules composing the septate-like junction. Taken together, these findings provide new insight into the structural organization of CNS paranodes and suggest that, in addition to providing axo-glial adhesion, cytoskeletal linkage to the septate-like junctions may be required to maintain axonal domains and to regulate organelle transport in myelinated axons.


Assuntos
Axônios/ultraestrutura , Sistema Nervoso Central/citologia , Citoesqueleto/ultraestrutura , Tomografia com Microscopia Eletrônica/métodos , Junções Intercelulares/ultraestrutura , Neuroglia/citologia , Animais , Axônios/metabolismo , Citoesqueleto/metabolismo , Camundongos , Proteínas de Neurofilamentos/metabolismo , Proteínas de Neurofilamentos/ultraestrutura , Neuroglia/ultraestrutura
7.
J Neurochem ; 112(5): 1147-55, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19968758

RESUMO

Acute axonal shear and stretch in the brain induces an evolving form of axonopathy and is a major cause of ongoing motor, cognitive and emotional dysfunction. We have utilized an in vitro model of mild axon bundle stretch injury, in cultured primary cortical neurons, to determine potential early critical cellular alterations leading to secondary axonal degeneration. We determined that transient axonal stretch injury induced an initial acute increase in intracellular calcium, principally derived from intracellular stores, which was followed by a delayed increase in calcium over 48 h post-injury (PI). This progressive and persistent increase in intracellular calcium was also associated with increased frequency of spontaneous calcium fluxes as well as cytoskeletal abnormalities. Additionally, at 48 h post-injury, stretch-injured axon bundles demonstrated filopodia-like sprout formation that preceded secondary axotomy and degeneration. Pharmacological inhibition of the calcium-activated phosphatase, calcineurin, resulted in reduced secondary axotomy (p < 0.05) and increased filopodial sprout length. In summary, these results demonstrate that stretch injury of axons induced an initial substantial release of calcium from intracellular stores with elevated intracellular calcium persisting over 2 days. These long-lasting calcium alterations may provide new insight into the earliest neuronal abnormalities that follow traumatic brain injury as well as the key cellular changes that lead to the development of diffuse axonal injury and secondary degeneration.


Assuntos
Cálcio/metabolismo , Espaço Extracelular/metabolismo , Neurônios/fisiologia , Neurônios/ultraestrutura , Animais , Axotomia/métodos , Calcineurina/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Espaço Extracelular/efeitos dos fármacos , Imunossupressores/farmacologia , Microscopia Eletrônica de Varredura/métodos , Proteínas de Neurofilamentos/metabolismo , Proteínas de Neurofilamentos/ultraestrutura , Neurônios/efeitos dos fármacos , Ratos , Ratos Wistar , Estresse Mecânico , Tacrolimo/farmacologia , Tapsigargina/farmacologia , Fatores de Tempo , Tubulina (Proteína)/metabolismo
8.
Biochemistry ; 47(40): 10526-39, 2008 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-18783251

RESUMO

The abnormal aggregation of the microtubule-associated protein Tau into paired helical filaments (PHFs) is one of the hallmarks of Alzheimer disease (AD). Tau in solution behaves as a natively unfolded or intrinsically disordered protein while its aggregation is based on the partial structural transition from random coil to beta-structure. Our aim is to understand in more detail the unfolded nature of Tau, to investigate the aggregation of Tau under different conditions and the molecular interactions of Tau in filaments. We show that soluble Tau remains natively unfolded even when its net charge is minimized, in contrast to other unfolded proteins. The CD signature of the random-coil character of Tau shows no major change over wide variations in charge (pH), ionic strength, solvent polarity, and denaturation. Thus there is no indication of a hydrophobicity-driven collapse, neither in the microtubule-binding repeat domain constructs nor in full-length Tau. This argues that the lack of hydrophobic residues but not the net charge accounts for unfolded nature of soluble Tau. The aggregation of the Tau repeat domain (that forms the core of PHFs) in the presence of nucleating polyanionic cofactors (heparin) is efficient in a range of buffers and pH values between approximately 5 and 10 but breaks down beyond that range, presumably because the pattern of charged interactions disappears. Similarly, elevated ionic strength attenuates aggregation, and the temperature dependence is bell-shaped with an optimum around 50 degrees C. Reporter dyes ThS and ANS record the aggregation process but sense different states (cross-beta-structure vs hydrophobic pockets) with different kinetics. Preformed PHFs are surprisingly labile and can be disrupted by denaturants at rather low concentration ( approximately 1.0 M GdnHCl), much less than required to denature globular proteins. Partial disaggregation of Tau filaments at extreme pH values monitored by CD and EM indicate the importance of salt bridges in filament formation. In contrast, Tau filaments are remarkably resistant to high temperature and high ionic strength. Overall, the stability of PHFs appears to depend mainly on directed salt bridges with contributions from hydrophobic interactions as well, consistent with a recent structural model of the PHF core derived from solid state NMR (Andronesi, O. C., von Bergen, M., Biernat, J., Seidel, K., Griesinger, C., Mandelkow, E., and Baldus, M. (2008) Characterization of Alzheimer's-like paired helical filaments from the core domain of tau protein using solid-state NMR spectroscopy.


Assuntos
Doença de Alzheimer/metabolismo , Proteínas de Neurofilamentos/química , Proteínas tau/química , Dicroísmo Circular , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Microscopia Eletrônica de Varredura , Modelos Biológicos , Proteínas de Neurofilamentos/ultraestrutura , Dobramento de Proteína , Solventes/química , Temperatura , Proteínas tau/ultraestrutura
9.
Clin J Pain ; 24(8): 717-24, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18806537

RESUMO

OBJECTIVES: To compare the neuroablative effects of pulsed radiofrequency (PRF) and conventional radiofrequency (CRF) techniques on the sciatic nerve, a peripheral nerve that includes motor, sensory, and autonomous fibers. METHODS: The study consisted of 5 groups of 6 adult male Wistar rats. In the control group, no procedure was performed. In the sham group, electrode placement was the same as the other groups, but radiofrequency energy was not given to the rats. In the CRF40 group, 40 degrees C CRF was applied to the rats for 90 seconds. In the CRF80 group, 80 degrees C CRF was applied for 90 seconds. In the PRF group, the rats received 45 V PRF, which did not exceed 42 degrees C for 240 seconds. Two days later, sciatic nerve samples were taken. All specimens were evaluated both with light and electron microscopy. Sciatic nerve morphology was analyzed to compare the effects of CRF and PRF. Kruskal-Wallis and Mann-Whitney U tests were used for comparing the means. RESULTS: Minimal damage was observed in the control group, but damage increased in the sham group and became increasingly more distinct in the PRF, CRF40, and CRF80 groups. DISCUSSION: Nerve tissues can be affected during any type of procedure, even during surgical applications. Our results suggest that PRF is less destructive than CRF for the peripheral nerves. However, this idea should also be investigated at the molecular level, and safety analysis should be performed for routine clinical practice.


Assuntos
Ablação por Cateter/efeitos adversos , Ondas de Rádio/efeitos adversos , Nervo Isquiático/patologia , Nervo Isquiático/efeitos da radiação , Animais , Relação Dose-Resposta à Radiação , Masculino , Microscopia Eletrônica de Transmissão/métodos , Proteínas de Neurofilamentos/metabolismo , Proteínas de Neurofilamentos/ultraestrutura , Ratos , Ratos Wistar , Nervo Isquiático/ultraestrutura
10.
Neurol Res ; 30(9): 990-4, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18662498

RESUMO

Amyotrophic lateral sclerosis (ALS) is a progressive neurological disorder. A pathologic hallmark of ALS is accumulation of neurofilaments in proximal axons of affected motor neurones. As the neurofilaments involved in immune-mediated spinal cord ventral horn motor neuron degeneration and loss, we developed immune-mediated motor neuron injury animal model by inoculating Lewis rats with swine spinal cord homogenate and investigated the ultrastructural features of neurofilament accumulation using transmission electron microscopy. Our results showed that there was aberrant accumulation of neurofilaments in perikarya and processes of remaining motor neurons in recipient animals, which is similar to those observed in ALS patients. These findings suggest that immune-mediated motor neuron injury may share a common pathogenesis with ALS.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Doença dos Neurônios Motores/metabolismo , Neurônios Motores/metabolismo , Proteínas de Neurofilamentos/metabolismo , Esclerose Lateral Amiotrófica/imunologia , Esclerose Lateral Amiotrófica/patologia , Animais , Modelos Animais de Doenças , Masculino , Microscopia Eletrônica de Transmissão , Doença dos Neurônios Motores/imunologia , Doença dos Neurônios Motores/patologia , Neurônios Motores/patologia , Neurônios Motores/ultraestrutura , Proteínas de Neurofilamentos/ultraestrutura , Ratos , Ratos Endogâmicos Lew , Medula Espinal/metabolismo , Medula Espinal/patologia , Medula Espinal/ultraestrutura , Suínos , Extratos de Tecidos/metabolismo
11.
FEBS Lett ; 582(15): 2303-2308, 2008 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-18519042

RESUMO

Neurofilaments are synthesised in neuronal cell bodies and then transported through axons. Damage to neurofilament transport is seen in amyotrophic lateral sclerosis (ALS). Here, we show that PKN1, a neurofilament head-rod domain kinase is cleaved and activated in SOD1G93A transgenic mice that are a model of ALS. Moreover, we demonstrate that glutamate, a proposed toxic mechanism in ALS leads to caspase cleavage and disruption of PKN1 in neurons. Finally, we demonstrate that a cleaved form of PKN1 but not wild-type PKN1 disrupts neurofilament organisation and axonal transport. Thus, deregulation of PKN1 may contribute to the pathogenic process in ALS.


Assuntos
Esclerose Lateral Amiotrófica/enzimologia , Transporte Axonal , Proteínas de Neurofilamentos/metabolismo , Proteínas de Neurofilamentos/ultraestrutura , Proteína Quinase C/metabolismo , Esclerose Lateral Amiotrófica/etiologia , Esclerose Lateral Amiotrófica/genética , Animais , Transporte Axonal/genética , Caspase 3/metabolismo , Modelos Animais de Doenças , Ácido Glutâmico/toxicidade , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Neurônios/ultraestrutura , Proteína Quinase C/genética , Ratos , Medula Espinal/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase-1
12.
J Neurosci ; 27(36): 9573-84, 2007 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-17804618

RESUMO

In the vertebrate nervous system, axon calibers correlate positively with myelin sheath dimensions and electrophysiological parameters including action potential amplitude and conduction velocity. Neurofilaments, a prominent component of the neuronal cytoskeleton, are required by axons to support their normal radial growth. To distinguish between fiber features that arise in response to absolute axon caliber and those that are under autonomous control, we investigated transgenic mice in which neurofilaments are sequestered in neuronal cell bodies. The neurofilament deficient axons in such mice achieve mature calibers only 50% of normal and have altered conduction properties. We show here that this primary axonal defect also induces multiple changes in myelin sheath composition and radial dimensions. Remarkably, other fundamental fiber features, including internodal spacing and the architecture and composition of nodes of Ranvier, remain unaltered. Thus, many fiber characteristics are controlled through mechanisms operating independently of absolute axon caliber and the neurofilament cytoskeleton.


Assuntos
Axônios/metabolismo , Proteínas de Neurofilamentos/metabolismo , Neurônios/metabolismo , Nós Neurofibrosos/metabolismo , Nós Neurofibrosos/ultraestrutura , Animais , Axônios/ultraestrutura , Células Cultivadas , Sistema Nervoso Central/fisiologia , Sistema Nervoso Central/ultraestrutura , Óperon Lac , Camundongos , Camundongos Transgênicos , Bainha de Mielina/química , Bainha de Mielina/metabolismo , Fibras Nervosas Mielinizadas/química , Fibras Nervosas Mielinizadas/fisiologia , Fibras Nervosas Mielinizadas/ultraestrutura , Condução Nervosa/genética , Condução Nervosa/fisiologia , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/ultraestrutura , Neurônios/ultraestrutura , Sistema Nervoso Periférico/fisiologia , Sistema Nervoso Periférico/ultraestrutura , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Transgenes
13.
J Neurosci Methods ; 161(2): 199-204, 2007 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-17157386

RESUMO

Neurofilaments (NFs) are heteropolymers composed of light (NF-L), middle (NF-M), and heavy (NF-H) subunits, present in most neurons. NF-L polymerizes on its own to provide a scaffold on which regular NFs form via the cross-bridging of NF-M or NF-H. To clarify the mechanism of regulation of NF-L self-assembly, we developed an assay using truncated mutant NF-L fused to glutathione-S transferase (GST). Western immunoblotting data show that the GST-fused head-rod domains of NF-L are necessary and sufficient for detecting assembled NF-L. The levels of self-assembled NF-L subunits detected using GST fusion proteins were consistent with those detected by electron microscopy and turbidity assay. Our results collectively imply that GST-fused head-rod domains of NF-L are critical tools for analyzing NF-L self-assembly in vitro.


Assuntos
Adenocarcinoma/metabolismo , Adenocarcinoma/ultraestrutura , Western Blotting/métodos , Proteínas de Neurofilamentos/metabolismo , Proteínas de Neurofilamentos/ultraestrutura , Animais , Linhagem Celular Tumoral , Humanos , Mutagênese Sítio-Dirigida , Células PC12 , Ratos , Relação Estrutura-Atividade
14.
J Neurosci ; 26(39): 10006-19, 2006 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-17005864

RESUMO

Alpha-internexin, a neuronal intermediate filament protein implicated in neurodegenerative disease, coexists with the neurofilament (NF) triplet proteins (NF-L, NF-M, and NF-H) but has an unknown function. The earlier peak expression of alpha-internexin than the triplet during brain development and its ability to form homopolymers, unlike the triplet, which are obligate heteropolymers, have supported a widely held view that alpha-internexin and neurofilament triplet form separate filament systems. Here, we demonstrate, however, that despite a postnatal decline in expression, alpha-internexin is as abundant as the triplet in the adult CNS and exists in a relatively fixed stoichiometry with these subunits. Alpha-internexin exhibits transport and turnover rates identical to those of triplet proteins in optic axons and colocalizes with NF-M on single neurofilaments by immunogold electron microscopy. Alpha-internexin also coassembles with all three neurofilament proteins into a single network of filaments in quadruple-transfected SW13vim(-) cells. Genetically deleting NF-M alone or together with NF-H in mice dramatically reduces alpha-internexin transport and content in axons throughout the CNS. Moreover, deleting alpha-internexin potentiates the effects of NF-M deletion on NF-H and NF-L transport. Finally, overexpressing a NF-H-LacZ fusion protein in mice induces alpha-internexin and neurofilament triplet to aggregate in neuronal perikarya and greatly reduces their transport and content selectively in axons. Our data show that alpha-internexin and the neurofilament proteins are functionally interdependent. The results strongly support the view that alpha-internexin is a fourth subunit of neurofilaments in the adult CNS, providing a basis for its close relationship with neurofilaments in CNS diseases associated with neurofilament accumulation.


Assuntos
Axônios/química , Proteínas de Filamentos Intermediários/fisiologia , Filamentos Intermediários/química , Proteínas de Neurofilamentos/fisiologia , Animais , Axônios/ultraestrutura , Cruzamentos Genéticos , Feminino , Proteínas de Filamentos Intermediários/análise , Proteínas de Filamentos Intermediários/deficiência , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/ultraestrutura , Filamentos Intermediários/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Complexos Multiproteicos , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Proteínas de Neurofilamentos/análise , Proteínas de Neurofilamentos/deficiência , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/ultraestrutura , Mapeamento de Interação de Proteínas , Transporte Proteico , Ratos , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/fisiologia , Células Ganglionares da Retina/química , Células Ganglionares da Retina/ultraestrutura , Medula Espinal/química , Medula Espinal/ultraestrutura , Relação Estrutura-Atividade , Transfecção
15.
Neurobiol Aging ; 27(9): 1258-68, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16054268

RESUMO

Here we have tested whether tau modification either by point mutation or by hyperphosphorylation can exert maximal pathogenic effects or if, on the contrary, both types of tau modifications can act synergistically to induce neuropathology. For this, we have combined transgenic mice overexpressing the enzyme GSK-3beta (Tet/GSK-3beta mice), with transgenic mice expressing Tau with a triple FTDP-17 mutation which develop prefibrillar tau-aggregates (VLW mice). Tet/GSK-3beta/VLW transgenic mice show tau hyperphosphorylation in hippocampal neurons. This is accompanied by thioflavin-S staining, and formation of filaments similar in width to those found in tauophaties. Finally, the atrophy of the hippocampal dentate gyrus observed in Tet/GSK-3beta mice develops much faster in Tet/GSK-3beta/VLW mice. All these morphological and biochemical data demonstrate that there is a synergistic contribution of both types of tau modifications and that the potential of GSK-3 inhibitors for AD therapeutics also extends to tauopathies caused by point mutations in tau gene.


Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Degeneração Neural/diagnóstico , Proteínas tau/metabolismo , Animais , Benzotiazóis , Biomarcadores/metabolismo , Western Blotting/métodos , Giro Denteado/patologia , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Imuno-Histoquímica/métodos , Camundongos , Camundongos Transgênicos , Proteínas de Neurofilamentos/metabolismo , Proteínas de Neurofilamentos/ultraestrutura , Fosforilação , Tiazóis , Proteínas tau/genética
16.
J Mol Biol ; 354(3): 569-77, 2005 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-16257415

RESUMO

Intermediate filaments (IFs) are structural elements of eukaryotic cells with distinct mechanical properties. Tissue integrity is severely impaired, in particular in skin and muscle, when IFs are either absent or malfunctioning due to mutations. Our knowledge on the mechanical properties of IFs is mainly based on tensile testing of macroscopic fibers and on the rheology of IF networks. At the single filament level, the only piece of data available is a measure of the persistence length of vimentin IFs. Here, we have employed an atomic force microscopy (AFM) based protocol to directly probe the mechanical properties of single cytoplasmic IFs when adsorbed to a solid support in physiological buffer environment. Three IF types were studied in vitro: recombinant murine desmin, recombinant human keratin K5/K14 and neurofilaments isolated from rat brains, which are composed of the neurofilament triplet proteins NF-L, NF-M and NF-H. Depending on the experimental conditions, the AFM tip was used to laterally displace or to stretch single IFs on the support they had been adsorbed to. Upon applying force, IFs were stretched on average 2.6-fold. The maximum stretching that we encountered was 3.6-fold. A large reduction of the apparent filament diameter was observed concomitantly. The observed mechanical properties therefore suggest that IFs may indeed function as mechanical shock absorbers in vivo.


Assuntos
Filamentos Intermediários/metabolismo , Filamentos Intermediários/ultraestrutura , Animais , Desmina/química , Desmina/ultraestrutura , Humanos , Filamentos Intermediários/química , Queratinas/química , Queratinas/ultraestrutura , Camundongos , Microscopia de Força Atômica , Nanotecnologia , Proteínas de Neurofilamentos/química , Proteínas de Neurofilamentos/ultraestrutura , Ratos , Fatores de Tempo
17.
J Neurotrauma ; 22(10): 1081-91, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16238485

RESUMO

We report a new model of transient axonal stretch injury involving pressurized fluid deflection of bundles of axons, resulting in a transient 1-6% increase in original axon length to investigate the slow progression of axonal alterations that are characteristic of diffuse axonal injury (DAI). We found no discernable difference in axon bundle morphology or cytoskeletal neurofilament protein arrangement between unstretched and stretched axonal bundles at 24 h post-injury. However, by 48 h post-injury, there was a stereotypical response of stretched axons involving characteristic neurofilament alterations that bear similarities to in vivo neuronal responses associated with DAI that have been reported previously. For instance, neurofilament protein immunoreactivity (SMI-312) was increased in axons contained within 51% of all injured axon bundles at 48 h compared to surrounding unstretched axon bundles, suggestive of neurofilament compaction. Furthermore, axonal bundle derangement occurred in 25% of injured axon bundles, with individual fibres segregating from each other and becoming undulating and wavy. By 72 h post-stretch, 70% of injured axon bundles underwent secondary axotomy, becoming completely severed at the site of initial stretch injury. While these results suggest a temporal series of stereotypical responses of axons to injury, we were able to distinguish very clear differences between mildly (100-103% increase in original axonal length) injured and strongly injured (106%+) axons. For instance, mildly injured axons developed increased neurofilament immunoreactivtity (SMI-312) within 48 h, and the marked development of ring-like neurofilament immunoreactive structures within axonal bundles, which were rarely axotomized. Conversely, at more severe strain levels increased neurofilament immunoreactivity was less apparent, while axons often became distorted and disorganised within axonal bundles and eventually became completely disconnected. Almost no ring-like neurofilament structures were observed in these severely injured axonal bundles. This suggests that axons do not respond in a stereotypical manner to a transient stretch insult, and indeed that variable degrees of stretch injury activate different responses within axons, with dramatically different outcomes. Hence, it is possible that the cytoskeletal characteristics that we have used in this study may be useful parameters for discriminating between mildly and severely injured axons following TBI.


Assuntos
Lesões Encefálicas/patologia , Lesão Axonal Difusa/patologia , Proteínas de Neurofilamentos/ultraestrutura , Animais , Axotomia , Células Cultivadas , Modelos Animais de Doenças , Imageamento Tridimensional , Técnicas In Vitro , Microscopia Eletrônica de Varredura , Ratos , Ratos Wistar
18.
Biochem Biophys Res Commun ; 334(4): 979-86, 2005 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-16039605

RESUMO

Complications of diabetes mellitus within the nervous system are peripheral and central neuropathy. In peripheral neuropathy, defects in neurofilament and microtubules have been demonstrated. In this study, we examined the effects of insulin deficiency within the brain in insulin knockout mice (I-/-). The I-/- exhibited hyperphosphorylation of tau, at threonine 231, and neurofilament. In addition, we showed hyperphosphorylation of c-Jun N-terminal kinase (JNK) and glycogen synthase kinase 3 beta (GSK-3 beta) at serine 9. Extracellular signal-regulated kinase 1 (ERK 1) showed decrease in phosphorylation, whereas ERK 2 showed no changes. Ultrastructural examination demonstrated swollen mitochondria, endoplasmic reticulum, and Golgi apparatus, and dispersion of the nuclear chromatin. Microtubules showed decrease in the number of intermicrotubule bridges and neurofilament presented as bunches. Thus, lack of insulin brain stimulation induces JNK hyperphosphorylation followed by hyperphosphorylation of tau and neurofilament, and ultrastructural cellular damage, that over time may induce decrease in cognition and learning disabilities.


Assuntos
Encéfalo/metabolismo , Encéfalo/ultraestrutura , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Quinase 3 da Glicogênio Sintase/metabolismo , Insulina/deficiência , Proteínas de Neurofilamentos/metabolismo , Proteínas tau/metabolismo , Animais , Animais Recém-Nascidos , Camundongos , Camundongos Knockout , Proteínas de Neurofilamentos/ultraestrutura , Proteínas tau/ultraestrutura
19.
Acta Neuropathol ; 110(1): 48-56, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15920660

RESUMO

Impaired axonal transport of the fast or slow component has been reported in patients with sporadic amyotrophic lateral sclerosis (ALS), animal models for ALS, and familial ALS-linked mutant Cu/Zn superoxide dismutase (SOD1) transgenic mice. However, little is known about the impairment of axonal transport in mutant SOD1 transgenic mice. This is the first electron microscopic investigation of the axon hillock (AH) and the initial segment (IS) of anterior horn cells in the spinal cord of transgenic mice expressing the G93A mutant human SOD1, and it was launched with a view toward examining whether the axonal transport is impaired in this region. Six transgenic mice were killed at ages ranging from the presymptomatic to symptomatic stages. Six age-matched non-transgenic wild-type mice served as controls. In the non-transgenic mice, 91 AH and IS were observed, but those with increased neurofilaments or mitochondria were rarely found. In the transgenic mice, 95 AH and IS directly emanating from normal-looking large anterior horn cells were seen. AH and IS with increased neurofilaments or, to a lesser extent, increased mitochondria, and round-shaped mitochondria in particular, were more frequently observed, even at the early presymptomatic stage, than in the controls, and the frequency increased with time through the presymptomatic stages. On the other hand, the somata of large motor neurons directly connected with the axons did not exhibit any abnormal accumulation of neurofilaments or mitochondria. These findings suggest that both the slow axonal transport of neurofilaments and the fast axonal transport of mitochondria are impaired in AH and IS before the onset of disease in this animal model.


Assuntos
Células do Corno Anterior/metabolismo , Transporte Axonal/fisiologia , Axônios/metabolismo , Superóxido Dismutase/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Células do Corno Anterior/ultraestrutura , Axônios/ultraestrutura , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Mitocôndrias/ultraestrutura , Mutação , Proteínas de Neurofilamentos/ultraestrutura , Superóxido Dismutase-1
20.
Biochem Biophys Res Commun ; 324(2): 489-96, 2004 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-15474454

RESUMO

Recent studies have advanced the notion that the axonal organization of neurofilaments (NFs) is based on mutual steric repulsion between the unstructured "sidearm" domains of adjacent NFs. Here, we present experimental evidence that these repulsive forces are modulated by the degree of sidearm phosphorylation. When NFs are sedimented into a gelatinous pellet, pellet volume falls with increasing ionic strength and enzymatic dephosphorylation; sedimentation of phosphorylated NFs in the presence of divalent cations also dramatically reduces pellet volume. Further, atomic force microscopy imaging of isolated mammalian NFs reveals robust exclusion of colloidal particles from the NF backbone that is reduced at high ionic strength and attenuated when the filaments are enzymatically dephosphorylated. Phosphate-phosphate repulsion on the NF sidearm appears to modulate NF excluded volume in a graded fashion, thereby controlling axonal NF organization through interfilament forces.


Assuntos
Proteínas de Neurofilamentos/química , Proteínas de Neurofilamentos/ultraestrutura , Esclerose Lateral Amiotrófica/metabolismo , Animais , Axônios/metabolismo , Cálcio/metabolismo , Cálcio/farmacologia , Cátions , Bovinos , Células Cultivadas , Relação Dose-Resposta a Droga , Concentração de Íons de Hidrogênio , Íons , Magnésio/farmacologia , Microscopia de Força Atômica , Fosforilação , Conformação Proteica , Sais/farmacologia , Medula Espinal/citologia , Medula Espinal/patologia
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