RESUMO
Information processing and storage in the brain rely on AMPA-receptors (AMPARs) and their context-dependent dynamics in synapses and extra-synaptic sites. We found that distribution and dynamics of AMPARs in the plasma membrane are controlled by Noelins, a three-member family of conserved secreted proteins expressed throughout the brain in a cell-type-specific manner. Noelin tetramers tightly assemble with the extracellular domains of AMPARs and interconnect them in a network-like configuration with a variety of secreted and membrane-anchored proteins including Neurexin1, Neuritin1, and Seizure 6-like. Knock out of Noelins1-3 profoundly reduced AMPARs in synapses onto excitatory and inhibitory (inter)neurons, decreased their density and clustering in dendrites, and abolished activity-dependent synaptic plasticity. Our results uncover an endogenous mechanism for extracellular anchoring of AMPARs and establish Noelin-organized networks as versatile determinants of constitutive and context-dependent neurotransmission.
Assuntos
Encéfalo , Proteínas de Membrana , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico , Proteínas de Membrana/genética , Transporte Biológico , Membrana Celular , Receptores de AMPARESUMO
Classically, the regulation of energy balance has been based on central and peripheral mechanisms sensing energy, nutrients, metabolites, and hormonal cues. Several cellular mechanisms at central level, such as hypothalamic AMP-activated protein kinase (AMPK), integrate this information to elicit counterregulatory responses that control feeding, energy expenditure, and glucose homeostasis, among other processes. Recent data have added more complexity to the homeostatic regulation of metabolism by introducing, for example, the key role of "traditional" senses and sensorial information in this complicated network. In this regard, current evidence is showing that olfaction plays a key and bidirectional role in energy homeostasis. Although nutritional status dynamically and profoundly impacts olfactory sensitivity, the sense of smell is involved in food appreciation and selection, as well as in brown adipose tissue (BAT) thermogenesis and substrate utilization, with some newly described actors, such as olfactomedin 2 (OLFM2), likely playing a major role. Thus, olfactory inputs are contributing to the regulation of both sides of the energy balance equation, namely, feeding and energy expenditure (EE), as well as whole body metabolism. Here, we will review the current knowledge and advances about the role of olfaction in the regulation of energy homeostasis.
Assuntos
Obesidade , Olfato , Humanos , Obesidade/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Tecido Adiposo Marrom/metabolismo , Metabolismo Energético/fisiologia , Hipotálamo/metabolismo , TermogêneseRESUMO
BACKGROUND AND AIMS: Olfactomedin 2 (OLFM2; also known as noelin 2) is a pleiotropic protein that plays a major role in olfaction and Olfm2 null mice exhibit reduced olfactory sensitivity, as well as abnormal motor coordination and anxiety-related behavior. Here, we investigated the possible metabolic role of OLFM2. METHODS: Olfm2 null mice were metabolically phenotyped. Virogenetic modulation of central OLFM2 was also performed. RESULTS: Our data showed that, the global lack of OLFM2 in mice promoted anorexia and increased energy expenditure due to elevated brown adipose tissue (BAT) thermogenesis and browning of white adipose tissue (WAT). This phenotype led to resistance to high fat diet (HFD)-induced obesity. Notably, virogenetic overexpression of Olfm2 in the lateral hypothalamic area (LHA) induced weight gain associated with decreased BAT thermogenesis. CONCLUSION: Overall, this evidence first identifies central OLFM2 as a new molecular actor in the regulation of whole-body energy homeostasis.
Assuntos
Tecido Adiposo Marrom , Termogênese , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Dieta Hiperlipídica/efeitos adversos , Metabolismo Energético/genética , Proteínas da Matriz Extracelular , Glicoproteínas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/metabolismo , Termogênese/genéticaRESUMO
miRNA are short non-coding RNA responsible for the knockdown of proteins through their targeting and silencing of complimentary mRNA sequences. The miRNA landscape of a cell thus affects the levels of its proteins and has significant consequences to its health. Deviations in this miRNA landscape have been implicated in a variety of neurodegenerative diseases and have also garnered interest as targets for treatment. Retinal ganglion cells are the sole projection neuron of the retina with their axons making up the optic nerve. They are a focus of study not only for their importance in vision and the myriad of blinding diseases characterized by their dysfunction and loss, but also as a model of other central nervous system diseases such as spinal cord injury and traumatic brain injury. This review summarizes current knowledge on the role of miRNA in retinal ganglion cell function, highlighting how perturbations can result in disease, and how modulating their abundance may provide a novel avenue of therapeutic research.
RESUMO
The purpose of this study was to characterize the miRNA profile of purified retinal ganglion cells (RGC) from healthy and diseased rat retina. Diseased retina includes those after a traumatic optic nerve crush (ONC), and after ocular hypertension/glaucoma. Rats were separated into four groups: healthy/intact, 7 days after laser-induced ocular hypertension, 2 days after traumatic ONC, and 7 days after ONC. RGC were purified from rat retina using microbeads conjugated to CD90.1/Thy1. RNA were sequenced using Next Generation Sequencing. Over 100 miRNA were identified that were significantly different in diseased retina compared to healthy retina. Considerable differences were seen in the miRNA expression of RGC 7 days after ONC, whereas after 2 days, few changes were seen. The miRNA profiles of RGC 7 days after ONC and 7 days after ocular hypertension were similar, but discrete miRNA differences were still seen. Candidate mRNA showing different levels of expression after retinal injury were manipulated in RGC cultures using mimics/AntagomiRs. Of the five candidate miRNA identified and subsequently tested for therapeutic efficacy, miR-194 inhibitor and miR-664-2 inhibitor elicited significant RGC neuroprotection, whereas miR-181a mimic and miR-181d-5p mimic elicited significant RGC neuritogenesis.
Assuntos
Glaucoma/genética , Glaucoma/patologia , MicroRNAs/metabolismo , Compressão Nervosa , Nervo Óptico/patologia , Células Ganglionares da Retina/metabolismo , Animais , Feminino , Regulação da Expressão Gênica , Pressão Intraocular/genética , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Neuritos/metabolismo , Neurogênese/genética , Neuroproteção/genética , Hipertensão Ocular/genética , Hipertensão Ocular/fisiopatologia , Ratos Sprague-DawleyRESUMO
Bone marrow mesenchymal stem cell (BMSC)-derived small extracellular vesicles (sEV) but not fibroblast sEV provide retinal ganglion cell (RGC) neuroprotection both in vitro and in vivo, with miRNAs playing an essential role. More than 40 miRNAs were more abundant in BMSC-sEV than in fibroblast-sEV. The purpose of this study was to test the in vitro and in vivo neuroprotective and axogenic properties of six candidate miRNAs (miR-26a, miR-17, miR-30c-2, miR-92a, miR-292, and miR-182) that were more abundant in BMSC-sEV than in fibroblast-sEV. Adeno-associated virus 2 (AAV2) expressing a combination of three of the above candidate miRNAs were added to heterogenous adult rat retinal cultures or intravitreally injected into rat eyes one week before optic nerve crush (ONC) injury. Survival and neuritogenesis of ßIII-tubulin+ RGCs was assessed in vitro, as well as the survival of RBPMS+ RGCs and regeneration of their axons in vivo. Retinal nerve fiber layer thickness (RNFL) was measured to assess axonal density whereas positive scotopic threshold response electroretinography amplitudes provided a readout of RGC function. Qualitative retinal expression of PTEN, a target of several of the above miRNAs, was used to confirm successful miRNA activity. AAV2 reliably transduced RGCs in vitro and in vivo. Viral delivery of miRNAs in vitro showed a trend towards neuroprotection but remained insignificant. Delivery of selected combinations of miRNAs (miR-17-5p, miR-30c-2 and miR-92a; miR-92a, miR-292 and miR-182) before ONC provided significant therapeutic benefits according to the above measurable endpoints. However, no single miRNA appeared to be responsible for the effects observed, whilst positive effects observed appeared to coincide with successful qualitative reduction in PTEN immunofluorescence in the retina. Viral delivery of miRNAs provides a possible neuroprotective strategy for injured RGCs that is conducive to therapeutic manipulation.
Assuntos
MicroRNAs/genética , Regeneração Nervosa , Traumatismos do Nervo Óptico/patologia , Células Ganglionares da Retina/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Modelos Animais de Doenças , Eletrorretinografia , Feminino , MicroRNAs/metabolismo , Nervo Óptico/metabolismo , Nervo Óptico/patologia , Traumatismos do Nervo Óptico/metabolismo , Ratos , Ratos Sprague-Dawley , Células Ganglionares da Retina/patologiaRESUMO
Extracellular vesicles (EV), which include exosomes and microvesicles, are secreted from virtually every cell. EV contain mRNA, miRNA, lipids and proteins and can deliver this expansive cargo into nearby cells as well as over long distances via the blood stream. Great interest has been given to them for their role in cell to cell communication, disease progression, or as biomarkers, and more recent studies have interrogated their potential as a therapeutic that may replace paracrine-acting cell therapies. The retina is a conveniently accessible component of the central nervous system and the proposed paradigm for the testing of many cell therapies. Recently, several studies have been published demonstrating that the delivery of EV/exosomes into the eye can elicit significant therapeutic effects in several models of retinal disease. We summarize results from currently available studies, demonstrating their efficacy in multiple eye disease models as well as highlighting where future research efforts should be directed.
Assuntos
Gerenciamento Clínico , Vesículas Extracelulares/metabolismo , Doenças Retinianas/terapia , Transporte Biológico , Biomarcadores/metabolismo , Exossomos , Humanos , Doenças Retinianas/metabolismoRESUMO
Purpose: To investigate the possible role of activating transcription factor 3 (ATF3) in retinal ganglion cell (RGC) neuroprotection and optic nerve regeneration after optic nerve crush (ONC). Methods: Overexpression of proteins of interest (ATF3, phosphatase and tensin homolog [PTEN], placental alkaline phosphatase, green fluorescent protein) in the retina was achieved by intravitreal injections of recombinant adenovirus-associated viruses (rAAVs) expressing corresponding proteins. The number of RGCs and αRGCs was evaluated by immunostaining retinal sections and whole-mount retinas with antibodies against RNA binding protein with multiple splicing (RBPMS) and osteopontin, respectively. Axonal regeneration was assessed via fluorophore-coupled cholera toxin subunit B labeling. RGC function was evaluated by recording positive scotopic threshold response. Results: The level of ATF3 is preferentially elevated in osteopontin+/RBPMS+ αRGCs following ONC. Overexpression of ATF3 by intravitreal injection of rAAV 2 weeks before ONC promoted RBPMS+ RGC survival and preserved RGC function as assessed by positive scotopic threshold response recordings 2 weeks after ONC. However, overexpression of ATF3 and simultaneous downregulation of PTEN, a negative regulator of the mTOR pathway, combined with ONC, only moderately promoted short distance RGC axon regeneration (200 µm from the lesion site) but did not provide additional RGC neuroprotection compared with PTEN downregulation alone. Conclusions: These results reveal a neuroprotective effect of ATF3 in the retina following injury and identify ATF3 as a promising agent for potential treatments of optic neuropathies.
Assuntos
Fator 3 Ativador da Transcrição/fisiologia , Neuroproteção/fisiologia , Traumatismos do Nervo Óptico/fisiopatologia , Células Ganglionares da Retina/fisiologia , Fator 3 Ativador da Transcrição/metabolismo , Animais , Axônios/patologia , Camundongos , Camundongos Endogâmicos C57BL , Compressão Nervosa , Regeneração Nervosa/fisiologia , Traumatismos do Nervo Óptico/metabolismo , Traumatismos do Nervo Óptico/patologia , Células Ganglionares da Retina/patologiaRESUMO
Purpose: To determine whether priming of bone marrow mesenchymal stem cells (MSCs) by signals from injured retina, particularly tumor necrosis factor α (TNFα), increase their exosomes' neuroprotective efficacy on retinal ganglion cells (RGCs). Methods: MSCs were primed with retinal cell culture conditioned medium, with or without the TNFα blocker etanercept or TNFα prior to isolation of exosomes. MSC conditioned medium or exosomes were added to rat retinal cultures or human stem cell-derived retinal ganglion cell (hRGC) cultures, and RGC neuroprotective effects were quantified. Luminex assays were used to compare primed versus unprimed exosomes. Results: MSC conditioned medium and exosomes exerted a significant neuroprotective effect on injured rat and hRGC. This effect was significantly increased after MSCs were primed with retinal conditioned medium or TNFα. Blocking of TNFα signaling with etanercept prevented priming-induced RGC neuroprotective efficacy. Priming increased PEDF and VEGF-AA exosomal abundance. Conclusions: MSC exosomes promote RGC survival not just in rodent retinal cultures but also with hRGC. Their efficacy can be further enhanced through TNFα priming with the mechanism of action potentially mediated, at least in part, through increased levels of PEDF and VEGF-AA.
Assuntos
Células-Tronco Mesenquimais/fisiologia , Fármacos Neuroprotetores/farmacologia , Células Ganglionares da Retina/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Animais , Células Cultivadas , Etanercepte/farmacologia , Exossomos , Proteínas do Olho/metabolismo , Feminino , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Fatores de Crescimento Neural/metabolismo , Neuroproteção/efeitos dos fármacos , Ratos Sprague-Dawley , Serpinas/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Purpose: To determine if bone marrow-derived stem cell (BMSC) small extracellular vesicles (sEV) promote retinal ganglion cell (RGC) neuroprotection in the genetic DBA/2J mouse model of glaucoma for 12 months. Methods: BMSC sEV and control fibroblast-derived sEV were intravitreally injected into 3-month-old DBA/2J mice once a month for 9 months. IOP and positive scotopic threshold responses were measured from 3 months: IOP was measured monthly and positive scotopic threshold responses were measured every 3 months. RGC neuroprotection was determined in wholemounts stained with RNA binding protein with multiple splicing (RBPMS), whereas axonal damage was assessed using paraphenylenediamine staining. Results: As expected, DBA/2J mice developed chronic ocular hypertension beginning at 6 months. The delivery of BMSC sEV, but not fibroblast sEV, provided significant neuroprotective effects for RBPMS+ RGC while significantly reducing the number of degenerating axons seen in the optic nerve. BMSC sEV significantly preserved RGC function in 6-month-old mice, but provided no benefit at 9 and 12 months. Conclusions: BMSC sEV are an effective neuroprotective treatment in a chronic model of ocular hypertension for 1 year, preserving RGC numbers and protecting against axonal degeneration.
Assuntos
Modelos Animais de Doenças , Vesículas Extracelulares/fisiologia , Glaucoma/prevenção & controle , Células-Tronco Mesenquimais/fisiologia , Doenças do Nervo Óptico/prevenção & controle , Animais , Western Blotting , Eletrorretinografia , Exossomos/genética , Feminino , Glaucoma/genética , Imuno-Histoquímica , Pressão Intraocular/fisiologia , Camundongos , Camundongos Endogâmicos DBA , Fibras Nervosas/patologia , Neuroproteção , Visão Noturna/fisiologia , Hipertensão Ocular/genética , Hipertensão Ocular/prevenção & controle , Doenças do Nervo Óptico/genética , Células Ganglionares da Retina/patologiaRESUMO
Purpose: The compound heterozygous mutations in the ß subunit of chaperonin containing TCP-1 (CCT), encoded by CCT2, lead to the Leber congenital amaurosis (LCA). In this study, a cct2 mutant line of zebrafish was established to investigate the role of CCT2 mutations in LCA in vertebrates. Methods: A cct2 mutant zebrafish line was produced using the CRISPR-Cas9 system. Changes in the eyes of developing wild-type and mutant larvae were monitored using microscopy, immunostaining, TUNEL, and EdU assays. Phenotypic rescue of mutant phenotype was investigated by injection of CCT2 RNA into zebrafish embryos. Results: The cct2 mutation (L394H-7del) led to the synthesis of a mutated cctß protein with the L394H replacement and deletion of 7 amino acid residues (positions 395-401). The homozygous cct2-L394H-7del mutant exhibited a small eye phenotype at 2 days post fertilization (dpf) and was embryonically lethal after 5 dpf. In homozygous cct2-L394H-7del mutants, the retinal ganglion cell differentiation was attenuated, retinal cell cycle was affected, and the neural retinal cell death was significantly increased at 2 dpf compared with wild-type. Injection of RNA encoding wild-type human CCTß rescued the small eye phenotype, reduced retinal cell death, and restored the levels of CCTß protein and the major client protein Gß1 that were significantly reduced in the homozygous cct2-L394H-7del mutant compared with wild-type. These results indicate that cct2 plays an essential role in retinal development by regulating the cell cycle. Conclusions: The retinal pathology observed in the homozygous cct2-L394H-7del mutants resembles the retinal pathology of human LCA patients.
Assuntos
Apoptose/genética , Ciclo Celular/genética , Chaperonina com TCP-1/genética , Amaurose Congênita de Leber/genética , Retina/patologia , Deleção de Sequência , Proteínas de Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Western Blotting , Sistemas CRISPR-Cas , Modelos Animais de Doenças , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Microscopia , Dados de Sequência Molecular , Fenótipo , RNA/genética , Peixe-ZebraRESUMO
Purpose: To investigate the benefit of bone marrow mesenchymal stem cell (BMSC)-derived small extracellular vesicles (sEV) as an intravitreal (ivit) therapy in two rat models of glaucoma and to determine and identify candidate miRNA involved in the mechanism. Methods: sEV were isolated from human BMSC and fibroblasts and ivit injected into adult rats after induction of elevated IOP. IOP was elevated using either intracameral injection of microbeads or laser photocoagulation of circumferential limbal vessels and the trabecular meshwork. Retinal nerve fiber layer (RNFL) thickness was measured using optical coherence tomography, positive scotopic threshold response (pSTR) recorded using ERG, and RNA binding protein with multiple splicing (RBPMS+) retinal ganglion cell (RGC) counted using retinal wholemounts. sEV miRNA were sequenced using RNAseq. Results: sEV isolated from BMSC promoted significant neuroprotection of RGC while preventing RNFL degenerative thinning and loss of pSTR. sEV proved therapeutically efficacious when ivit injected every week or every month, but ineffective with longer delays between treatments. Knockdown of Argonaute2 (AGO2), a protein critical for miRNA function and packing into sEV prior to sEV isolation, significantly attenuated the above effects. Addition of BMSC sEV (but not fibroblast sEV) reduced death of cultured purified RGC. RNAseq identified 43 miRNA upregulated in BMSC sEV in comparison to fibroblast sEV, which yielded no neuroprotective effects. Conclusions: Injection of BMSC-derived sEV into the vitreous provided significant therapeutic benefit to glaucomatous eyes. The neuroprotective effect of sEV, at least partially, may be explained by direct action on RGC through miRNA-dependent mechanisms.
Assuntos
Modelos Animais de Doenças , Vesículas Extracelulares/metabolismo , Glaucoma/prevenção & controle , Células-Tronco Mesenquimais/fisiologia , Neuroproteção/fisiologia , Doenças do Nervo Óptico/prevenção & controle , Animais , Proteínas Argonautas/genética , Eletrorretinografia , Feminino , Técnicas de Silenciamento de Genes , Glaucoma/metabolismo , Glaucoma/patologia , Pressão Intraocular/fisiologia , Injeções Intravítreas , MicroRNAs/genética , Fibras Nervosas/patologia , Doenças do Nervo Óptico/metabolismo , Doenças do Nervo Óptico/patologia , Ratos , Ratos Sprague-Dawley , Células Ganglionares da Retina/patologia , Tomografia de Coerência ÓpticaRESUMO
The olfm1a and olfm1b genes in zebrafish encode conserved secreted glycoproteins. These genes are preferentially expressed in the brain and retina starting from 16 h post-fertilization until adulthood. Functions of the Olfm1 gene is still unclear. Here, we produced and analyzed a null zebrafish mutant of both olfm1a and olfm1b genes (olfm1 null). olfm1 null fish were born at a normal Mendelian ratio and showed normal body shape and fertility as well as no visible defects from larval stages to adult. Olfm1 proteins were preferentially localized in the synaptosomes of the adult brain. Olfm1 co-immunoprecipitated with GluR2 and soluble NSF attachment protein receptor complexes indicating participation of Olfm1 in both pre- and post-synaptic events. Phosphorylation of GluR2 was not changed while palmitoylation of GluR2 was decreased in the brain synaptosomal membrane fraction of olfm1 null compared with wt fish. The levels of GluR2, SNAP25, flotillin1, and VAMP2 were markedly reduced in the synaptic microdomain of olfm1 null brain compared with wt. The internalization of GluR2 in retinal cells and the localization of VAMP2 in brain synaptosome were modified by olfm1 null mutation. This indicates that Olfm1 may regulate receptor trafficking from the intracellular compartments to the synaptic membrane microdomain, partly through the alteration of post-translational GluR2 modifications such as palmitoylation. Olfm1 may be considered a novel regulator of the composition and function of the α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor complex.
Assuntos
Proteínas da Matriz Extracelular/genética , Glicoproteínas/genética , Transporte Proteico/genética , Receptores de AMPA/metabolismo , Animais , Encéfalo/metabolismo , Técnicas de Inativação de Genes , Peixe-ZebraRESUMO
Purpose: To elucidate functions of wild-type myocilin, a secreted glycoprotein associated with glaucoma. Methods: Lysates of mouse eyes were used for immunoprecipitation with affinity-purified antibodies against mouse myocilin. Shotgun proteomic analysis was used for the identification of proteins interacting with myocilin. Colocalization of myocilin and tissue inhibitor of metalloproteinases 3 (TIMP3) in different eye structures was investigated by a multiplex fluorescent in situ hybridization and immunofluorescent labeling with subsequent confocal microscopy. Matrix metalloproteinase 2 (MMP2) activity assay was used to test effects of myocilin on TIMP3 inhibitory action. Results: TIMP3 was identified by a shotgun proteomic analysis as a protein that was coimmunoprecipitated with myocilin from eye lysates of wild-type and transgenic mice expressing elevated levels of mouse myocilin but not from lysates of transgenic mice expressing mutated mouse myocilin. Interaction of myocilin and TIMP3 was confirmed by coimmunoprecipitation of myocilin and TIMP3 from HEK293 cells transiently transfected with cDNAs encoding these proteins. The olfactomedin domain of myocilin is essential for interaction with TIMP3. In the eye, the main sites of myocilin and TIMP3 colocalization are the trabecular meshwork, sclera, and choroid. Using purified proteins, it has been shown that myocilin markedly enhanced the inhibitory activity of TIMP3 toward MMP2. Conclusions: Myocilin may serve as a modulator of TIMP3 activity via interactions with the myocilin olfactomedin domain. Our data imply that in the case of MYOCILIN null or some glaucoma-causing mutations, inhibitory activity of TIMP3 toward MMP2 might be reduced, mimicking deleterious mutations in the TIMP3 gene.
Assuntos
Proteínas do Citoesqueleto/fisiologia , Proteínas do Olho/fisiologia , Glicoproteínas/fisiologia , Metaloproteinase 2 da Matriz/metabolismo , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Animais , Western Blotting , Corpo Ciliar/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Expressão Gênica , Células HEK293 , Células Hep G2 , Humanos , Imunoprecipitação , Hibridização in Situ Fluorescente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Proteômica , RNA Mensageiro/genética , Inibidor Tecidual de Metaloproteinase-3/genética , Malha Trabecular/metabolismo , TransfecçãoRESUMO
Purpose: Our previous experiments demonstrated that intravitreal injection of platelet-derived growth factor-AA (PDGF-AA) provides retinal ganglion cell (RGC) neuroprotection in a rodent model of glaucoma. Here we used PDGFRα-enhanced green fluorescent protein (EGFP) mice to identify retinal cells that may be essential for RGC protection by PDGF-AA. Methods: PDGFRα-EGFP mice expressing nuclear-targeted EGFP under the control of the PDGFRα promoter were used. Localization of PDGFRα in the neural retina was investigated by confocal imaging of EGFP fluorescence and immunofluorescent labeling with a panel of antibodies recognizing different retinal cell types. Primary cultures of mouse RGCs were produced by immunopanning. Neurobiotin injection of amacrine cells in a flat-mounted retina was used for the identification of EGFP-positive amacrine cells in the inner nuclear layer. Results: In the mouse neural retina, PDGFRα was preferentially localized in the ganglion cell and inner nuclear layers. Immunostaining of the retina demonstrated that astrocytes in the ganglion cell layer and a subpopulation of amacrine cells in the inner nuclear layer express PDGFRα, whereas RGCs (in vivo or in vitro) did not. PDGFRα-positive amacrine cells are likely to be Type 45 gamma-aminobutyric acidergic (GABAergic) wide-field amacrine cells. Conclusions: These data indicate that the neuroprotective effect of PDGF-AA in a rodent model of glaucoma could be mediated by astrocytes and/or a subpopulation of amacrine cells. We suggest that after intravitreal injection of PDGF-AA, these cells secrete factors protecting RGCs.
Assuntos
Células Amácrinas/metabolismo , Astrócitos/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Células Ganglionares da Retina/efeitos dos fármacos , Animais , Biotina/análogos & derivados , Biotina/farmacologia , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Colina O-Acetiltransferase/metabolismo , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Microscopia de Fluorescência , Neuroproteção , Fármacos Neuroprotetores , Células Ganglionares da Retina/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
The loss of retinal ganglion cells (RGC) and their axons is one of the leading causes of blindness and includes traumatic (optic neuropathy) and degenerative (glaucoma) eye diseases. Although no clinical therapies are in use, mesenchymal stem cells (MSC) have demonstrated significant neuroprotective and axogenic effects on RGC in both of the aforementioned models. Recent evidence has shown that MSC secrete exosomes, membrane enclosed vesicles (30-100 nm) containing proteins, mRNA and miRNA which can be delivered to nearby cells. The present study aimed to isolate exosomes from bone marrow-derived MSC (BMSC) and test them in a rat optic nerve crush (ONC) model. Treatment of primary retinal cultures with BMSC-exosomes demonstrated significant neuroprotective and neuritogenic effects. Twenty-one days after ONC and weekly intravitreal exosome injections; optical coherence tomography, electroretinography, and immunohistochemistry was performed. BMSC-derived exosomes promoted statistically significant survival of RGC and regeneration of their axons while partially preventing RGC axonal loss and RGC dysfunction. Exosomes successfully delivered their cargo into inner retinal layers and the effects were reliant on miRNA, demonstrated by the diminished therapeutic effects of exosomes derived from BMSC after knockdown of Argonaute-2, a key miRNA effector molecule. This study supports the use of BMSC-derived exosomes as a cell-free therapy for traumatic and degenerative ocular disease. Stem Cells Translational Medicine 2017;6:1273-1285.
Assuntos
Células da Medula Óssea/citologia , Exossomos/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/metabolismo , Animais , Células Cultivadas , Eletrorretinografia , Feminino , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: The objective of this study is to investigate the role and underlying mechanism of Olfactomedin 2 (Olfm2) in smooth muscle cell (SMC) phenotypic modulation and vascular remodeling. APPROACH AND RESULTS: Platelet-derived growth factor-BB induces Olfm2 expression in primary SMCs while modulating SMC phenotype as shown by the downregulation of SMC marker proteins. Knockdown of Olfm2 blocks platelet-derived growth factor-BB-induced SMC phenotypic modulation, proliferation, and migration. Conversely, Olfm2 overexpression inhibits SMC marker expression. Mechanistically, Olfm2 promotes the interaction of serum response factor with the runt-related transcription factor 2 that is induced by platelet-derived growth factor-BB, leading to a decreased interaction between serum response factor and myocardin, causing a repression of SMC marker gene transcription and consequently SMC phenotypic modulation. Animal studies show that Olfm2 is upregulated in balloon-injured rat carotid arteries. Knockdown of Olfm2 effectively inhibits balloon injury-induced neointima formation. Importantly, knockout of Olfm2 in mice profoundly suppresses wire injury-induced neointimal hyperplasia while restoring SMC contractile protein expression, suggesting that Olfm2 plays a critical role in SMC phenotypic modulation in vivo. CONCLUSIONS: Olfm2 is a novel factor mediating SMC phenotypic modulation. Thus, Olfm2 may be a potential target for treating injury-induced proliferative vascular diseases.
Assuntos
Lesões das Artérias Carótidas/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fator de Resposta Sérica/metabolismo , Fatores de Transcrição/metabolismo , Remodelação Vascular , Animais , Aorta Torácica/metabolismo , Becaplermina , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/patologia , Células Cultivadas , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/genética , Masculino , Camundongos , Camundongos Knockout , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Neointima , Proteínas Nucleares/metabolismo , Fenótipo , Ligação Proteica , Proteínas Proto-Oncogênicas c-sis/farmacologia , Interferência de RNA , Ratos Sprague-Dawley , Transdução de Sinais , Transativadores/metabolismo , Transfecção , Remodelação Vascular/efeitos dos fármacosRESUMO
Retinal ganglion cells (RGC) bear the sole responsibility of propagating visual stimuli to the brain. Their axons, which make up the optic nerve, project from the retina to the brain through the lamina cribrosa and in rodents, decussate almost entirely at the optic chiasm before synapsing at the superior colliculus. For many traumatic and degenerative ocular conditions, the dysfunction and/or loss of RGC is the primary determinant of visual loss and are the measurable endpoints in current research into experimental therapies. To actually measure these endpoints in rodent models, techniques must ascertain both the quantity of surviving RGC and their functional capacity. Quantification techniques include phenotypic markers of RGC, retrogradely transported fluorophores and morphological measurements of retinal thickness whereas functional assessments include electroretinography (flash and pattern) and visual evoked potential. The importance of the accuracy and reliability of these techniques cannot be understated, nor can the relationship between RGC death and dysfunction. The existence of up to 30 types of RGC complicates the measuring process, particularly as these may respond differently to disease and treatment. Since the above techniques may selectively identify and ignore particular subpopulations, their appropriateness as measures of RGC survival and function may be further limited. This review discusses the above techniques in the context of their subtype specificity.
Assuntos
Glaucoma/diagnóstico , Fibras Nervosas/patologia , Células Ganglionares da Retina/patologia , Vias Visuais/fisiopatologia , Animais , Eletrorretinografia , Potenciais Evocados Visuais , Glaucoma/fisiopatologia , Humanos , Pressão Intraocular , Tomografia de Coerência ÓpticaRESUMO
To safeguard proteomic integrity, cells rely on the proteasome to degrade aberrant polypeptides, but it is unclear how cells remove defective proteins that have escaped degradation owing to proteasome insufficiency or dysfunction. Here we report a pathway termed misfolding-associated protein secretion, which uses the endoplasmic reticulum (ER)-associated deubiquitylase USP19 to preferentially export aberrant cytosolic proteins. Intriguingly, the catalytic domain of USP19 possesses an unprecedented chaperone activity, allowing recruitment of misfolded proteins to the ER surface for deubiquitylation. Deubiquitylated cargos are encapsulated into ER-associated late endosomes and secreted to the cell exterior. USP19-deficient cells cannot efficiently secrete unwanted proteins, and grow more slowly than wild-type cells following exposure to a proteasome inhibitor. Together, our findings delineate a protein quality control (PQC) pathway that, unlike degradation-based PQC mechanisms, promotes protein homeostasis by exporting misfolded proteins through an unconventional protein secretion process.