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1.
J Vis Exp ; (207)2024 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-38884464

RESUMO

Intracameral injection is a standard administration routine in ophthalmology. The application of intracameral injection in rodents for research is challenging due to the limiting dimensions and anatomy of the eye, including the small aqueous humor volume, the lens curvature, and lens thickness. Potential damage during intracameral injections introduces adverse effects and experimental variability. This protocol describes a procedure for intracameral injection in rats, allowing precision and reproducibility. Sprague-Dawley rats were used as experimental models. Since the lens position in rats protrudes into the anterior chamber, injecting from the periphery, as done in humans, is unfavorable. Therefore, an incision is created in the central corneal region using a 31 gauge 0.8 mm stiletto blade to form a self-sealing tunnel into the anterior chamber. An incision at an angle close to the flat allows to create a long tunnel, which minimizes the loss of aqueous humor and shallowing of the anterior chamber. A 34 gauge nanoneedle is inserted into the tunnel for injection. This enables penetration with minimal friction resistance and avoids touching the lens. Injection of trypan-blue allows visualization by slit microscopy the presence of the dye in the anterior chamber and exclude leakage. Bioavailability to the corneal endothelial layer is demonstrated by injection of Hoechst dye, which stained the nuclei of corneal endothelial cells after injection. In conclusion, this protocol implements a procedure for accurate intracameral injection in rats. This procedure may be used for intracameral delivery of various drugs and compounds in experimental rat models, increasing the efficiency and reproducibility of ophthalmic research.


Assuntos
Câmara Anterior , Injeções Intraoculares , Ratos Sprague-Dawley , Animais , Ratos , Injeções Intraoculares/métodos , Câmara Anterior/efeitos dos fármacos , Injeção Intracameral
2.
Invest Ophthalmol Vis Sci ; 65(6): 14, 2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38848077

RESUMO

Purpose: The integrity of the corneal epithelium is essential in maintaining normal corneal function. Conditions disrupting the corneal epithelial layer range from chemical burns to dry eye disease and may result in impairment of both corneal transparency and sensation. Identifying factors that regulate corneal wound healing is key for the development of new treatment strategies. Here, we investigated a direct role of mitochondria in corneal wound healing via mitochondria transplantation. Methods: Human corneal epithelial cells (hCECs) were isolated from human corneas and incubated with mitochondria which were isolated from human ARPE-19 cells. We determined the effect of mitochondria transplantation on wound healing and proliferation of hCECs. In vivo, we used a mouse model of corneal chemical injury. Mitochondria were isolated from mouse livers and topically applied to the ocular surface following injury. We evaluated the time of wound repair, corneal re-epithelization, and stromal abnormalities. Results: Mitochondria transplantation induced the proliferation and wound healing of primary hCECs. Further, mitochondria transplantation promoted wound healing in vivo. Specifically, mice receiving mitochondria recovered twice as fast as control mice following corneal injury, presenting both enhanced and improved repair. Corneas treated with mitochondria demonstrated the re-epithelization of the wound area to a multi-layer appearance, compared to thinning and complete loss of the epithelium in control mice. Mitochondria transplantation also prevented the thickening and disorganization of the corneal stromal lamella, restoring normal corneal dehydration. Conclusions: Mitochondria promote corneal re-epithelization and wound healing. Augmentation of mitochondria levels via mitochondria transplantation may serve as an effective treatment for inducing the rapid repair of corneal epithelial defects.


Assuntos
Proliferação de Células , Modelos Animais de Doenças , Epitélio Corneano , Mitocôndrias , Cicatrização , Animais , Camundongos , Cicatrização/fisiologia , Humanos , Proliferação de Células/fisiologia , Queimaduras Químicas/cirurgia , Queimaduras Químicas/fisiopatologia , Camundongos Endogâmicos C57BL , Lesões da Córnea , Células Cultivadas , Queimaduras Oculares/induzido quimicamente
3.
J Vis Exp ; (190)2022 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-36533821

RESUMO

Establishing experimental choroidal melanoma models is challenging in terms of the ability to induce tumors at the correct localization. In addition, difficulties in observing posterior choroidal melanoma in vivo limit tumor location and growth evaluation in real-time. The approach described here optimizes techniques for establishing choroidal melanoma in mice via a multi-step sub-choroidal B16LS9 cell injection procedure. To enable precision in injecting into the small dimensions of the mouse uvea, the complete procedure is performed under a microscope. First, a conjunctival peritomy is formed in the dorsal-temporal area of the eye. Then, a tract into the sub-choroidal space is created by inserting a needle through the exposed sclera. This is followed by the insertion of a blunt needle into the tract and the injection of melanoma cells into the choroid. Immediately after injection, noninvasive optical coherence tomography (OCT) imaging is utilized to determine tumor location and progress. Retinal detachment is evaluated as a predictor of tumor site and size. The presented method enables the reproducible induction of choroid-localized melanoma in mice and the live imaging of tumor growth evaluation. As such, it provides a valuable tool for studying intraocular tumors.


Assuntos
Neoplasias da Coroide , Melanoma , Camundongos , Animais , Tomografia de Coerência Óptica/métodos , Corioide/diagnóstico por imagem , Neoplasias da Coroide/diagnóstico por imagem , Neoplasias da Coroide/patologia , Melanoma/diagnóstico por imagem , Melanoma/patologia
4.
Sci Rep ; 12(1): 5122, 2022 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-35332189

RESUMO

Ocular cells are highly dependent on mitochondrial function due to their high demand of energy supply and their constant exposure to oxidative stress. Indeed, mitochondrial dysfunction is highly implicated in various acute, chronic, and genetic disorders of the visual system. It has recently been shown that mitochondrial transplantation (MitoPlant) temporarily protects retinal ganglion cells (RGCs) from cell death during ocular ischemia. Here, we characterized MitoPlant dynamics in retinal ganglion precursor-like cells, in steady state and under oxidative stress. We developed a new method for detection of transplanted mitochondria using qPCR, based on a difference in the mtDNA sequence of C57BL/6 and BALB/c mouse strains. Using this approach, we show internalization of exogenous mitochondria already three hours after transplantation, and a decline in mitochondrial content after twenty four hours. Interestingly, exposure of target cells to moderate oxidative stress prior to MitoPlant dramatically enhanced mitochondrial uptake and extended the survival of mitochondria in recipient cells by more than three fold. Understanding the factors that regulate the exogenous mitochondrial uptake and their survival may promote the application of MitoPlant for treatment of chronic and genetic mitochondrial diseases.


Assuntos
Doenças Mitocondriais , Células Ganglionares da Retina , Animais , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Estresse Oxidativo , Células Ganglionares da Retina/metabolismo
5.
Exp Eye Res ; 204: 108431, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33406396

RESUMO

Uveal melanoma (UM) and conjunctival melanoma (CM) are ocular malignancies that give rise to life-threatening metastases. Although local disease can often be treated successfully, it is often associated with significant vision impairment and treatments are often not effective against metastatic disease. Novel treatment modalities that preserve vision may enable elimination of small tumors and may prevent subsequent metastatic spread. Very few mouse models of metastatic CM and UM are available for research and for development of novel therapies. One of the challenges is to follow tumor growth in-vivo and to determine the right size for treatment, mainly of the posterior, choroidal melanoma. Hence, the purpose of this study was to establish a simple, noninvasive imaging tool that will simplify visualization and tumor follow-up in mouse models of CM and UM. Tumors were induced by inoculation of murine B16LS9 cells into the sub-conjunctival or the choroidal space of a C57BL/6 mouse eye under a surgical microscope. Five to ten days following injection, tumor size was assessed by Phoenix MicronIV™ image-guided Optical Coherence Tomography (OCT) imaging, which included a real-time camera view and OCT scan of the conjunctiva and the retina. In addition, tumor size was evaluated by ultrasound and histopathological examination of eye sections. Tumor growth was observed 5-9 days following sub-conjunctival or sub-retinal injection of seven-thousand or seventy-thousand cells, respectively. A clear tumor mass was detected at these regions using the MicronIV™ imaging system camera and OCT scans. Histology of eye sections confirmed the presence of tumor tissue. OCT allowed an accurate measurement of tumor size in the UM model and a qualitative assessment of tumor size in the CM model. Moreover, OCT enabled assessing the success rate of the choroidal tumor induction and importantly, predicted final tumor size already on the day of cell inoculation. In conclusion, by using a simple, non-invasive imaging tool, we were able to follow intraocular tumor growth of both CM and UM, and to define, already at the time of cell inoculation, a grading scale to evaluate tumor size. This tool may be utilized for evaluation of new mouse models for CM and UM, as well as for testing new therapies for these diseases.


Assuntos
Neoplasias da Túnica Conjuntiva/diagnóstico por imagem , Modelos Animais de Doenças , Melanoma/diagnóstico por imagem , Tomografia de Coerência Óptica , Ultrassonografia , Neoplasias Uveais/diagnóstico por imagem , Animais , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Neoplasias da Túnica Conjuntiva/metabolismo , Neoplasias da Túnica Conjuntiva/patologia , Imuno-Histoquímica , Antígeno MART-1/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Antígenos Específicos de Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Monofenol Mono-Oxigenase/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Uveais/metabolismo , Neoplasias Uveais/patologia
6.
J Ophthalmol ; 2017: 1606854, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29109865

RESUMO

PURPOSE: This study aimed to investigate the effect of OM-101 on the fibrotic response occurring in proliferative vitreoretinopathy (PVR) in an animal model. METHODS: Antifibrotic effect of OM-101 was investigated in vivo. As control, eight weeks old c57black mice underwent intravitreal injection with Hepes (group A) or dispase (0.3 units), to induce retinal detachment (RD) and PVR. The dispase-injected mice were randomly divided into two groups B and C (N = 25 mice); in group C, the eyes were treated with intravitreal injection of OM-101 (3 µl), and group B with PBS, as a control. After additional five days, mice were injected with the same initial treatment. Three days later, mice were euthanized, and the eyes were enucleated and processed for histological analysis. RESULTS: Intravitreal injection of dispase caused RD in 64% of the mice in group B, and 93% of those mice had PVR. Only 32% of mice treated with OM-101 and dispase (group C) developed RD, and only 25% of those developed PVR. CONCLUSIONS: OM-101 was found effective in reducing the incidence of RD and PVR maintaining the normal architecture of the retina. This study suggests that OM-101 is a potentially effective and safe drug for the treatment of PVR patients.

7.
PLoS Genet ; 12(12): e1006486, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27997532

RESUMO

Familial Dysautonomia (FD) is a neurodegenerative disease in which aberrant tissue-specific splicing of IKBKAP exon 20 leads to reduction of IKAP protein levels in neuronal tissues. Here we generated a conditional knockout (CKO) mouse in which exon 20 of IKBKAP is deleted in the nervous system. The CKO FD mice exhibit developmental delays, sensory abnormalities, and less organized dorsal root ganglia (DRGs) with attenuated axons compared to wild-type mice. Furthermore, the CKO FD DRGs show elevated HDAC6 levels, reduced acetylated α-tubulin, unstable microtubules, and impairment of axonal retrograde transport of nerve growth factor (NGF). These abnormalities in DRG properties underlie neuronal degeneration and FD symptoms. Phosphatidylserine treatment decreased HDAC6 levels and thus increased acetylation of α-tubulin. Further PS treatment resulted in recovery of axonal outgrowth and enhanced retrograde axonal transport by decreasing histone deacetylase 6 (HDAC6) levels and thus increasing acetylation of α-tubulin levels. Thus, we have identified the molecular pathway that leads to neurodegeneration in FD and have demonstrated that phosphatidylserine treatment has the potential to slow progression of neurodegeneration.


Assuntos
Transporte Axonal/efeitos dos fármacos , Disautonomia Familiar/genética , Histona Desacetilases/genética , Fosfatidilserinas/administração & dosagem , Tubulina (Proteína)/genética , Processamento Alternativo/genética , Animais , Transporte Axonal/genética , Axônios/efeitos dos fármacos , Modelos Animais de Doenças , Disautonomia Familiar/tratamento farmacológico , Disautonomia Familiar/patologia , Éxons/genética , Gânglios Espinais/crescimento & desenvolvimento , Gânglios Espinais/patologia , Desacetilase 6 de Histona , Histona Desacetilases/biossíntese , Humanos , Camundongos , Camundongos Knockout , Degeneração Neural/tratamento farmacológico , Degeneração Neural/genética , Degeneração Neural/patologia , Fator de Crescimento Neural/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Fosfatidilserinas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
8.
Biochem Cell Biol ; 94(2): 188-96, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26928052

RESUMO

Autophagy is an evolutionarily conserved mechanism for degrading long-lived or malfunctioning proteins and organelles, such as those resulting from oxidative stress. Several publications have demonstrated the importance of the autophagy process in the pathophysiology of dry age-related macular degeneration (AMD). Still, the mechanism underlying this process and its involvement in dry AMD are not fully characterized. Investigating the autophagy process in retinal pigment epithelial (RPE) cells, we identified transforming growth factor ß activated kinase 1 (TAK1) as a key player in the process. We found increased TAK1 phosphorylation in ARPE-19 and D407 cells treated with different inducers of autophagy, such as oxidative stress and rapamycin. Moreover, utilizing TAK1 specific inhibitor prior to oxidative stress or rapamycin treatment, we found significant reduction in LC3A/B-II expression. These results point at the involvement of TAK1 in the regulation of autophagy in RPE cells. This study suggests that aberrant activity of this kinase impairs autophagy and subsequently leads to alterations in the vitality of RPE cells. Proper activity of TAK1 may be essential for efficient autophagy, and crucial for the ability of RPE cells to respond to stress and dispose of damaged organelles, thus preventing or delaying retinal pathologies.


Assuntos
Autofagia , MAP Quinase Quinase Quinases/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Células Cultivadas , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , Epitélio Pigmentado da Retina/patologia
9.
Eur J Cell Biol ; 95(2): 69-88, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26689471

RESUMO

Molecular communication between the motoneuron and the muscle is vital for neuromuscular junction (NMJ) formation and maintenance. Disruption in the structure and function of NMJs is a hallmark of various neurodegenerative processes during both development and pathological events. Still due to the complexity of this process, it is very difficult to elucidate the cellular mechanisms underlying it, generating a keen interest for developing better tools for investigating it. Here we describe a simplified method to study mechanisms of NMJs formation, maintenance and disruption. A spinal cord explant from mice expressing the Hb9::GFP motoneuron marker is plated on one side of a compartmental chamber, and myotubes derived from muscle satellite progenitor cells are plated on the other. The GFP labeled motoneurons extend their axons via microgrooves in the chamber to innervate the muscle cells and to form functional in-vitro NMJs. Next we provide procedures to measure axon growth and to reliably quantify NMJ activity using imaging of both muscle contractions and fast intracellular calcium changes. This platform allows precise control, monitoring and manipulation of subcellular microenvironments. Specifically, it enables to distinguish local from retrograde signaling mechanisms and allows restricted experimental intervention in local compartments along the muscle-neuron route.


Assuntos
Técnicas de Cultura de Células/métodos , Microfluídica/métodos , Junção Neuromuscular/metabolismo , Animais , Sinalização do Cálcio , Células Cultivadas , Camundongos , Microscopia de Fluorescência/métodos , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Neurônios Motores/fisiologia , Junção Neuromuscular/crescimento & desenvolvimento , Junção Neuromuscular/fisiologia , Medula Espinal/citologia
10.
J Cell Sci ; 128(6): 1241-52, 2015 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-25632161

RESUMO

Bidirectional molecular communication between the motoneuron and the muscle is vital for neuromuscular junction (NMJ) formation and maintenance. The molecular mechanisms underlying such communication are of keen interest and could provide new targets for intervention in motoneuron disease. Here, we developed a microfluidic platform with motoneuron cell bodies on one side and muscle cells on the other, connected by motor axons extending through microgrooves to form functional NMJs. Using this system, we were able to differentiate between the proximal and distal effects of oxidative stress and glial-derived neurotrophic factor (GDNF), demonstrating a dying-back degeneration and retrograde transmission of pro-survival signaling, respectively. Furthermore, we show that GDNF acts differently on motoneuron axons versus soma, promoting axonal growth and innervation only when applied locally to axons. Finally, we track for the first time the retrograde transport of secreted GDNF from muscle to neuron. Thus, our data suggests spatially distinct effects of GDNF--facilitating growth and muscle innervation at axon terminals and survival pathways in the soma.


Assuntos
Axônios/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Microfluídica , Neurônios Motores/metabolismo , Músculo Esquelético/metabolismo , Junção Neuromuscular/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Técnicas de Cocultura , Técnicas Imunoenzimáticas , Microscopia de Fluorescência , Neurônios Motores/citologia , Músculo Esquelético/citologia , Estresse Oxidativo , Fosforilação , Medula Espinal/citologia , Medula Espinal/metabolismo
11.
J Biol Chem ; 288(39): 27812-24, 2013 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-23960070

RESUMO

Cytoplasmic dynein is well characterized as an organelle motor, but dynein also acts to tether and stabilize dynamic microtubule plus-ends in vitro. Here we identify a novel and direct interaction between dynein and the 180-kDa isoform of the neural cell adhesion molecule (NCAM). Optical trapping experiments indicate that dynein bound to beads via the NCAM180 interaction domain can tether projecting microtubule plus-ends. Live cell assays indicate that the NCAM180-dependent recruitment of dynein to the cortex leads to the selective stabilization of microtubules projecting to NCAM180 patches at the cell periphery. The dynein-NCAM180 interaction also enhances cell-cell adhesion in heterologous cell assays. Dynein and NCAM180 co-precipitate from mouse brain extract and from synaptosomal fractions, consistent with an endogenous interaction in neurons. Thus, we examined microtubule dynamics and synaptic density in primary cortical neurons. We find that depletion of NCAM, inhibition of the dynein-NCAM180 interaction, or dampening of microtubule dynamics with low dose nocodazole all result in significantly decreased in synaptic density. Based on these observations, we propose a working model for the role of dynein at the synapse, in which the anchoring of the motor to the cortex via binding to an adhesion molecule mediates the tethering of dynamic microtubule plus-ends to potentiate synaptic stabilization.


Assuntos
Dineínas/química , Microtúbulos/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Neurônios/metabolismo , Sinapses/metabolismo , Animais , Células COS , Chlorocebus aethiops , Citoplasma/metabolismo , Dineínas do Citoplasma/química , Células HeLa , Humanos , Camundongos , Ligação Proteica , Sinaptossomos/metabolismo , Técnicas do Sistema de Duplo-Híbrido
12.
Hum Mol Genet ; 22(23): 4720-5, 2013 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-23836781

RESUMO

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder of motor neurons. Although most cases of ALS are sporadic (sALS) and of unknown etiology, there are also inherited familial ALS (fALS) cases that share a phenotype similar to sALS pathological and clinical phenotype. In this study, we have identified two new potential genetic ALS biomarkers in human bone marrow mesenchymal stem cells (hMSC) obtained from sALS patients, namely the TDP-43 (TAR DNA-binding protein 43) and SLPI (secretory leukocyte protease inhibitor). Together with the previously discovered ones-CyFIP2 and RbBP9, we investigated whether these four potential ALS biomarkers may be differentially expressed in tissues obtained from mutant SOD1(G93A) transgenic mice, a model that is relevant for at least 20% of the fALS cases. Quantitative real-time PCR analysis of brain, spinal cord and muscle tissues of the mSOD1(G93A) and controls at various time points during the progression of the neurological disease showed differential expression of the four identified biomarkers in correlation with (i) the tissue type, (ii) the stage of the disease and (iii) the gender of the animals, creating thus a novel spatiotemporal molecular signature of ALS. The biomarkers detected in the fALS animal model were homologous to those that were identified in hMSC of our sALS cases. These results support the possibility of a molecular link between sALS and fALS and may indicate common pathogenetic mechanisms involved in both types of ALS. Moreover, these results may pave the path for using the mSOD1(G93A) mouse model and these biomarkers as molecular beacons to evaluate the effects of novel drugs/treatments in ALS.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Esclerose Lateral Amiotrófica/patologia , Biomarcadores/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Inibidor Secretado de Peptidases Leucocitárias/genética , Superóxido Dismutase/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adulto , Esclerose Lateral Amiotrófica/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Músculos/metabolismo , Músculos/patologia , Inibidor Secretado de Peptidases Leucocitárias/metabolismo , Medula Espinal/metabolismo , Medula Espinal/patologia , Superóxido Dismutase/metabolismo , Adulto Jovem
13.
Dev Neurobiol ; 73(3): 247-56, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23055261

RESUMO

Automated analyses of neuronal morphology are important for quantifying connectivity and circuitry in vivo, as well as in high content imaging of primary neuron cultures. The currently available tools for quantification of neuronal morphology either are highly expensive commercial packages or cannot provide automated image quantifications at single cell resolution. Here, we describe a new software package called WIS-NeuroMath, which fills this gap and provides solutions for automated measurement of neuronal processes in both in vivo and in vitro preparations. Diverse image types can be analyzed without any preprocessing, enabling automated and accurate detection of neurites followed by their quantification in a number of application modules. A cell morphology module detects cell bodies and attached neurites, providing information on neurite length, number of branches, cell body area, and other parameters for each cell. A neurite length module provides a solution for images lacking cell bodies, such as tissue sections. Finally, a ganglion explant module quantifies outgrowth by identifying neurites at different distances from the ganglion. Quantification of a diverse series of preparations with WIS-NeuroMath provided data that were well matched with parallel analyses of the same preparations in established software packages such as MetaXpress or NeuronJ. The capabilities of WIS-NeuroMath are demonstrated in a range of applications, including in dissociated and explant cultures and histological analyses on thin and whole-mount sections. WIS-NeuroMath is freely available to academic users, providing a versatile and cost-effective range of solutions for quantifying neurite growth, branching, regeneration, or degeneration under different experimental paradigms.


Assuntos
Algoritmos , Ensaios de Triagem em Larga Escala , Processamento de Imagem Assistida por Computador/métodos , Neurônios/ultraestrutura , Software , Animais , Automação , Humanos
14.
EMBO J ; 31(6): 1350-63, 2012 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-22246183

RESUMO

Retrograde axonal injury signalling stimulates cell body responses in lesioned peripheral neurons. The involvement of importins in retrograde transport suggests that transcription factors (TFs) might be directly involved in axonal injury signalling. Here, we show that multiple TFs are found in axons and associate with dynein in axoplasm from injured nerve. Biochemical and functional validation for one TF family establishes that axonal STAT3 is locally translated and activated upon injury, and is transported retrogradely with dynein and importin α5 to modulate survival of peripheral sensory neurons after injury. Hence, retrograde transport of TFs from axonal lesion sites provides a direct link between axon and nucleus.


Assuntos
Axônios/metabolismo , Gânglios Espinais/metabolismo , Traumatismos dos Nervos Periféricos/metabolismo , Células Receptoras Sensoriais/metabolismo , Fatores de Transcrição/metabolismo , Animais , Transporte Axonal/fisiologia , Núcleo Celular/metabolismo , Dineínas/metabolismo , Carioferinas/metabolismo , Masculino , Camundongos , Transporte Proteico/fisiologia , Ratos , Ratos Wistar , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/fisiologia
15.
Sci Signal ; 3(130): ra53, 2010 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-20628157

RESUMO

Retrograde signaling from axon to soma activates intrinsic regeneration mechanisms in lesioned peripheral sensory neurons; however, the links between axonal injury signaling and the cell body response are not well understood. Here, we used phosphoproteomics and microarrays to implicate approximately 900 phosphoproteins in retrograde injury signaling in rat sciatic nerve axons in vivo and approximately 4500 transcripts in the in vivo response to injury in the dorsal root ganglia. Computational analyses of these data sets identified approximately 400 redundant axonal signaling networks connected to 39 transcription factors implicated in the sensory neuron response to axonal injury. Experimental perturbation of individual overrepresented signaling hub proteins, including Abl, AKT, p38, and protein kinase C, affected neurite outgrowth in sensory neurons. Paradoxically, however, combined perturbation of Abl together with other hub proteins had a reduced effect relative to perturbation of individual proteins. Our data indicate that nerve injury responses are controlled by multiple regulatory components, and suggest that network redundancies provide robustness to the injury response.


Assuntos
Redes Reguladoras de Genes/fisiologia , Regeneração Nervosa , Traumatismos dos Nervos Periféricos , Degeneração Retrógrada , Transdução de Sinais/fisiologia , Animais , Gânglios Espinais/lesões , Neuritos , Neurônios/metabolismo , Neurônios/patologia , Fosfoproteínas/análise , Proteômica/métodos , RNA Mensageiro/análise , Ratos , Nervo Isquiático/lesões
16.
Results Probl Cell Differ ; 48: 327-38, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19582413

RESUMO

The cell body of a lesioned neuron must receive accurate and timely information on the site and extent of axonal damage, in order to mount an appropriate response. Specific mechanisms must therefore exist to transmit such information along the length of the axon from the lesion site to the cell body. Three distinct types of signals have been postulated to underlie this process, starting with injury-induced discharge of axon potentials, and continuing with two distinct types of retrogradely transported macromolecular signals. The latter includes, on the one hand, an interruption of the normal supply of retrogradely transported trophic factors from the target, and, on the other hand, activated proteins originating from the injury site. This chapter reviews the progress on understanding the different mechanistic aspects of the axonal response to injury, and how the information is conveyed from the injury site to the cell body to initiate regeneration.


Assuntos
Axônios , Eletrofisiologia , Carioferinas/fisiologia , Degeneração Retrógrada , Animais , Axônios/patologia , Axônios/fisiologia , Humanos , Fatores de Crescimento Neural/fisiologia
17.
Neuron ; 59(2): 241-52, 2008 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-18667152

RESUMO

Peripheral sensory neurons respond to axon injury by activating an importin-dependent retrograde signaling mechanism. How is this mechanism regulated? Here, we show that Ran GTPase and its associated effectors RanBP1 and RanGAP regulate the formation of importin signaling complexes in injured axons. A gradient of nuclear RanGTP versus cytoplasmic RanGDP is thought to be fundamental for the organization of eukaryotic cells. Surprisingly, we find RanGTP in sciatic nerve axoplasm, distant from neuronal cell bodies and nuclei, and in association with dynein and importin-alpha. Following injury, localized translation of RanBP1 stimulates RanGTP dissociation from importins and subsequent hydrolysis, thereby allowing binding of newly synthesized importin-beta to importin-alpha and dynein. Perturbation of RanGTP hydrolysis or RanBP1 blockade at axonal injury sites reduces the neuronal conditioning lesion response. Thus, neurons employ localized mechanisms of Ran regulation to control retrograde injury signaling in peripheral nerve.


Assuntos
Axônios/enzimologia , Traumatismos dos Nervos Periféricos , Nervos Periféricos/enzimologia , Degeneração Retrógrada/enzimologia , Transdução de Sinais/fisiologia , Proteína ran de Ligação ao GTP/metabolismo , Animais , Axônios/patologia , Células Cultivadas , Carioferinas/biossíntese , Carioferinas/metabolismo , Carioferinas/fisiologia , Masculino , Nervos Periféricos/patologia , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Degeneração Retrógrada/patologia , Neuropatia Ciática/enzimologia , Neuropatia Ciática/patologia , Proteína ran de Ligação ao GTP/fisiologia
18.
J Mol Biol ; 364(5): 938-44, 2006 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-17046786

RESUMO

Cleavage fragments of de novo synthesized vimentin were recently reported to interact with phosphorylated Erk1 and Erk2 MAP kinases (pErk) in injured sciatic nerve, thus linking pErk to a signaling complex retrogradely transported on importins and dynein. Here we clarify the structural basis for this interaction, which explains how pErk is protected from dephosphorylation while bound to vimentin. Pull-down and ELISA experiments revealed robust calcium-dependent binding of pErk to the second coiled-coil domain of vimentin, with observed affinities of binding increasing from 180 nM at 0.1 microM calcium to 15 nM at 10 microM calcium. In contrast there was little or no binding of non-phosphorylated Erk to vimentin under these conditions. Geometric and electrostatic complementarity docking generated a number of solutions wherein vimentin binding to pErk occludes the lip containing the phosphorylated residues in the kinase. Binding competition experiments with Erk peptides confirmed a solution in which vimentin covers the phosphorylation lip in pErk, interacting with residues above and below the lip. The same peptides inhibited pErk binding to the dynein complex in sciatic nerve axoplasm, and interfered with protection from phosphatases by vimentin. Thus, a soluble intermediate filament fragment interacts with a signaling kinase and protects it from dephosphorylation by calcium-dependent steric hindrance.


Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Vimentina/metabolismo , Animais , Cálcio/metabolismo , Cricetinae , Citosol/metabolismo , Ativação Enzimática , Ensaio de Imunoadsorção Enzimática , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Imunoprecipitação , Mesocricetus , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/genética , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Vimentina/genética
19.
Mol Cell Neurosci ; 29(3): 381-93, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15890528

RESUMO

'Protective autoimmunity' refers to a well-controlled anti-self response that helps the body resist neurodegeneration. The response is mediated by autoimmune T cells, which produce cytokines and growth factors. Using an in vitro assay of hippocampal slices, we show that the cytokines interferon-gamma and (especially) interleukin-4, characteristic of pro-inflammatory and anti-inflammatory T cells, respectively, can make microglia neuroprotective. Aggregated beta-amyloid, like bacterial cell wall-derived lipopolysaccharide, rendered the microglia cytotoxic. Cytotoxicity was correlated with a signal transduction pathway that down-regulates expression of class-II major histocompatibility proteins (MHC-II) through the MHC-II-transactivator and the invariant chain. Protection by interleukin-4 was attributed to down-regulation of tumor necrosis factor-alpha and up-regulation of insulin-like growth factor I. These findings suggest that beneficial or harmful expression of the local immune response in the damaged CNS depends on how microglia interpret the threat, and that a well-regulated T-cell-mediated response enables microglia to alleviate rather than exacerbate stressful situations in the CNS.


Assuntos
Encefalite/imunologia , Gliose/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Interferon gama/imunologia , Interleucina-4/imunologia , Microglia/imunologia , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/imunologia , Peptídeos beta-Amiloides/farmacologia , Animais , Animais Recém-Nascidos , Autoimunidade/efeitos dos fármacos , Autoimunidade/imunologia , Linhagem Celular , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , Encefalite/fisiopatologia , Encefalite/terapia , Gliose/fisiopatologia , Gliose/terapia , Hipocampo/efeitos dos fármacos , Hipocampo/imunologia , Hipocampo/fisiopatologia , Antígenos de Histocompatibilidade Classe II/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/imunologia , Interferon gama/farmacologia , Interleucina-4/farmacologia , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Microglia/efeitos dos fármacos , Doenças Neurodegenerativas/imunologia , Doenças Neurodegenerativas/fisiopatologia , Doenças Neurodegenerativas/terapia , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/fisiologia , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/imunologia
20.
Neuron ; 45(5): 715-26, 2005 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-15748847

RESUMO

How are phosphorylated kinases transported over long intracellular distances, such as in the case of axon to cell body signaling after nerve injury? Here, we show that the MAP kinases Erk1 and Erk2 are phosphorylated in sciatic nerve axoplasm upon nerve injury, concomitantly with the production of soluble forms of the intermediate filament vimentin by local translation and calpain cleavage in axoplasm. Vimentin binds phosphorylated Erks (pErk), thus linking pErk to the dynein retrograde motor via direct binding of vimentin to importin beta. Injury-induced Elk1 activation and neuronal regeneration are inhibited or delayed in dorsal root ganglion neurons from vimentin null mice, and in rats treated with a MEK inhibitor or with a peptide that prevents pErk-vimentin binding. Thus, soluble vimentin enables spatial translocation of pErk by importins and dynein in lesioned nerve.


Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neuropatia Ciática/metabolismo , Vimentina/biossíntese , Sequência de Aminoácidos/genética , Animais , Transporte Axonal/efeitos dos fármacos , Transporte Axonal/fisiologia , Inibidores Enzimáticos/farmacologia , Masculino , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/genética , Ratos , Ratos Wistar , Neuropatia Ciática/genética , Vimentina/genética
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