RESUMO
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
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
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
Glaucoma is a multifactorial optic neuropathy characterized by retinal ganglion cell (RGC) death and axonal degeneration leading to irreversible blindness. Mutations in the myocilin (MYOC) gene are the most common genetic factors of primary open-angle glaucoma. To develop a genetic mouse model induced by the synergistic interaction of mutated myocilin and another significant risk factor, oxidative stress, we produced double-mutant mice (Tg-MYOC(Y437H/+)/Sod2(+/-)) bearing human MYOC with a Y437H point mutation and a heterozygous deletion of the gene for the primary antioxidant enzyme, superoxide dismutase 2 (SOD2). Sod2 is broadly expressed in most tissues including the trabecular meshwork (TM) and heterozygous Sod2 knockout mice exhibit the reduced SOD2 activity and oxidative stress in all studied tissues. Accumulation of Y437H myocilin in the TM induced endoplasmic reticulum stress and led to a 45% loss of smooth muscle alpha-actin positive cells in the eye drainage structure of 10- to 12-month-old Tg-MYOC(Y437H/+)/Sod2(+/-) mice as compared with wild-type littermates. Tg-MYOC(Y437H/+)/Sod2(+/-) mice had higher intraocular pressure, lost about 37% of RGCs in the peripheral retina, and exhibited axonal degeneration in the retina and optic nerve as compared with their wild-type littermates. Single-mutant littermates containing MYOC(Y437H/+) or Sod2(+/-) exhibited no significant pathological changes until 12 months of age. Additionally, we observed elevated expression of endothelial leukocyte adhesion molecule-1, a human glaucoma marker, in the TM of Tg-MYOC(Y437H/+)/Sod2(+/-) mice. This is the first reported animal glaucoma model that combines expression of a glaucoma-causing mutant gene and an additional mutation mimicking a deleterious environment factor that acts synergistically.
Assuntos
Proteínas do Citoesqueleto/genética , Epistasia Genética , Proteínas do Olho/genética , Glaucoma de Ângulo Aberto/genética , Glicoproteínas/genética , Heterozigoto , Mutação , Superóxido Dismutase/deficiência , Actinas/metabolismo , Animais , Apoptose/genética , Axônios/metabolismo , Axônios/patologia , Modelos Animais de Doenças , Selectina E/genética , Selectina E/metabolismo , Estresse do Retículo Endoplasmático/genética , Expressão Gênica , Glaucoma de Ângulo Aberto/diagnóstico , Haploinsuficiência , Pressão Intraocular , Camundongos , Camundongos Knockout , Doenças do Nervo Óptico/genética , Doenças do Nervo Óptico/patologia , Fenótipo , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Malha Trabecular/metabolismoRESUMO
Myocilin is a secreted glycoprotein that belongs to a family of olfactomedin domain-containing proteins. Although myocilin is detected in several ocular and nonocular tissues, the only reported human pathology related to mutations in the MYOCILIN gene is primary open-angle glaucoma. Functions of myocilin are poorly understood. Here we demonstrate that myocilin is a mediator of oligodendrocyte differentiation and is involved in the myelination of the optic nerve in mice. Myocilin is expressed and secreted by optic nerve astrocytes. Differentiation of optic nerve oligodendrocytes is delayed in Myocilin-null mice. Optic nerves of Myocilin-null mice contain reduced levels of several myelin-associated proteins including myelin basic protein, myelin proteolipid protein, and 2'3'-cyclic nucleotide 3'-phosphodiesterase compared with those of wild-type littermates. This leads to reduced myelin sheath thickness of optic nerve axons in Myocilin-null mice compared with wild-type littermates, and this difference is more pronounced at early postnatal stages compared with adult mice. Myocilin also affects differentiation of oligodendrocyte precursors in vitro. Its addition to primary cultures of differentiating oligodendrocyte precursors increases levels of tested markers of oligodendrocyte differentiation and stimulates elongation of oligodendrocyte processes. Myocilin stimulation of oligodendrocyte differentiation occurs through the NgR1/Lingo-1 receptor complex. Myocilin physically interacts with Lingo-1 and may be considered as a Lingo-1 ligand. Myocilin-induced elongation of oligodendrocyte processes may be mediated by activation of FYN and suppression of RhoA GTPase.
Assuntos
Diferenciação Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Proteínas do Olho/metabolismo , Glicoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas da Mielina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Oligodendroglia/fisiologia , Nervo Óptico/citologia , Receptores de Superfície Celular/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Células Cultivadas , Chlorocebus aethiops , Proteínas do Citoesqueleto/genética , Proteínas do Olho/genética , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Gânglios Espinais/citologia , Regulação da Expressão Gênica/genética , Glicoproteínas/genética , Humanos , Técnicas In Vitro , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Mutação/genética , Proteínas da Mielina/genética , Proteínas do Tecido Nervoso/genética , Receptor Nogo 1 , Oligodendroglia/ultraestrutura , Receptores de Superfície Celular/genética , Células-Tronco/fisiologiaRESUMO
Myocilin, a causative gene for open angle glaucoma, encodes a secreted glycoprotein with poorly understood functions. To gain insight into its functions, we produced a stably transfected HEK293 cell line expressing myocilin under an inducible promoter and compared gene expression profiles between myocilin-expressing and vector control cell lines by a microarray analysis. A significant fraction of differentially expressed genes in myocilin-expressing cells was associated with cell growth and cell death, suggesting that myocilin may have a role in the regulation of cell growth and survival. Increased proliferation of myocilin-expressing cells was demonstrated by the WST-1 proliferation assay, direct cell counting, and immunostaining with antibodies against Ki-67, a cellular proliferation marker. Myocilin-containing conditioned medium also increased proliferation of unmodified HEK293 cells. Myocilin-expressing cells were more resistant to serum starvation-induced apoptosis than control cells. TUNEL-positive apoptotic cells were dramatically decreased, and two apoptotic marker proteins, cleaved caspase 7 and cleaved poly(ADP-ribose) polymerase, were significantly reduced in myocilin-expressing cells as compared with control cells under apoptotic conditions. In addition, myocilin-deficient mesenchymal stem cells exhibited reduced proliferation and enhanced susceptibility to serum starvation-induced apoptosis as compared with wild-type mesenchymal stem cells. Phosphorylation of ERK1/2 and its upstream kinases, c-Raf and MEK, was increased in myocilin-expressing cells compared with control cells. Elevated phosphorylation of ERK1/2 was also observed in the trabecular meshwork of transgenic mice expressing 6-fold higher levels of myocilin when compared with their wild-type littermates. These results suggest that myocilin promotes cell proliferation and resistance to apoptosis via the ERK1/2 MAPK signaling pathway.
Assuntos
Proliferação de Células , Proteínas do Citoesqueleto/metabolismo , Proteínas do Olho/metabolismo , Glicoproteínas/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Células-Tronco Mesenquimais/metabolismo , Animais , Apoptose/fisiologia , Caspase 7/genética , Caspase 7/metabolismo , Sobrevivência Celular/fisiologia , Proteínas do Citoesqueleto/genética , Proteínas do Olho/genética , Glicoproteínas/genética , Células HEK293 , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Transgênicos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/fisiologiaRESUMO
Adipose-derived mesenchymal stem cells (ASCs) hold promise for use in cell-based therapies. Their intrinsic anti-inflammatory properties are potentially useful for treatments of inflammatory conditions such as uveitis, while their ability to differentiate along multiple cell lineages suggests use in regenerating damaged or degenerated tissue. However, how ASCs will respond to the intraocular environment is poorly studied. We have recently reported that aqueous humor (AH), the fluid that nourishes the anterior segment of the eye, potently increases alkaline phosphatase (ALP) activity of ASCs, indicating osteogenic differentiation. Here, we expand on our previous findings to better define the nature of this response. To this end, we cultured ASCs in the presence of 0, 5, 10, and 20% AH and assayed them for ALP activity. We found ALP activity correlates with increasing AH concentrations from 5 to 20%, and that longer treatments result in increased ALP activity. By using serum free media and pretreating AH with dextran-coated charcoal, we found that serum and charcoal-adsorbable AH components augment but are not required for this response. Further, by heat-treating the AH, we established that thermally labile components are required for the osteogenic response. Finally, we showed myocilin, a protein present in AH, could induce ALP activity in ASCs. However, this was to a lesser extent than untreated 5% AH, and myocilin could only partially rescue the effect after heat treatment, documenting there were additional thermally labile constituents of AH involved in the osteogenic response. Our work adds to the understanding of the induction of ALP in ASCs following exposure to AH, providing important insight in how ASCs will be influenced by the ocular environment. In conclusion, increased osteogenic potential upon exposure to AH represents a potential challenge to developing ASC cell-based therapies directed at the eye.
Assuntos
Tecido Adiposo/citologia , Fosfatase Alcalina/metabolismo , Humor Aquoso/fisiologia , Temperatura Alta , Células-Tronco Mesenquimais/enzimologia , Osteogênese/fisiologia , Análise de Variância , Humor Aquoso/química , Células Cultivadas , Meios de Cultura Livres de Soro/farmacologia , Proteínas do Citoesqueleto/farmacologia , Relação Dose-Resposta a Droga , Proteínas do Olho/farmacologia , Glicoproteínas/farmacologia , HumanosRESUMO
The development of neuroprotective strategies to attenuate retinal ganglion cell death could lead to novel therapies for chronic optic neuropathies such as glaucoma. Intravitreal transplantation of mesenchymal stem cells slows retinal ganglion cell death in models of optic nerve injury, but the mechanism of action remains unclear. Here we characterized the neuroprotective effects of mesenchymal stem cells and mesenchymal stem cell-derived factors in organotypic retinal explant culture and an in vivo model of ocular hypertensive glaucoma. Co-culture of rat and human bone marrow-derived mesenchymal stem cells with retinal explants increased retinal ganglion cell survival, after 7 days ex vivo, by â¼2-fold and was associated with reduced apoptosis and increased nerve fibre layer and inner plexiform layer thicknesses. These effects were not demonstrated by co-culture with human or mouse fibroblasts. Conditioned media from mesenchymal stem cells conferred neuroprotection, suggesting that the neuroprotection is mediated, at least partly, by secreted factors. We compared the concentrations of 29 factors in human mesenchymal stem cell and fibroblast conditioned media, and identified 11 enriched in the mesenchymal stem cell secretome. Treatment of retinal explants with a cocktail of these factors conferred retinal ganglion cell neuroprotection, with factors from the platelet-derived growth factor family being the most potent. Blockade of platelet-derived growth factor signalling with neutralizing antibody or with small molecule inhibitors of platelet-derived growth factor receptor kinase or downstream phosphatidylinositol 3 kinase eliminated retinal ganglion cell neuroprotection conferred by mesenchymal stem cell co-culture. Intravitreal injection of platelet-derived growth factor -AA or -AB led to profound optic nerve neuroprotection in vivo following experimental induction of elevated intraocular pressure. These data demonstrate that mesenchymal stem cells secrete a number of neuroprotective proteins and suggest that platelet-derived growth factor secretion in particular may play an important role in mesenchymal stem cell-mediated retinal ganglion cell neuroprotection. Furthermore, platelet-derived growth factor may represent an independent target for achieving retinal ganglion cell neuroprotection.
Assuntos
Células-Tronco Mesenquimais/metabolismo , Fármacos Neuroprotetores/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Células Ganglionares da Retina/metabolismo , Animais , Axotomia/efeitos adversos , Técnicas de Cocultura/métodos , Humanos , Células-Tronco Mesenquimais/patologia , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Células Ganglionares da Retina/patologiaRESUMO
Myocilin is a secreted glycoprotein that is expressed in ocular and non-ocular tissues. Mutations in the MYOCILIN gene may lead to juvenile- and adult-onset primary open-angle glaucoma. Here we report that myocilin is expressed in bone marrow-derived mesenchymal stem cells (MSCs) and plays a role in their differentiation into osteoblasts in vitro and in osteogenesis in vivo. Expression of myocilin was detected in MSCs derived from mouse, rat, and human bone marrow, with human MSCs exhibiting the highest level of myocilin expression. Expression of myocilin rose during the course of human MSC differentiation into osteoblasts but not into adipocytes, and treatment with exogenous myocilin further enhanced osteogenesis. MSCs derived from Myoc-null mice had a reduced ability to differentiate into the osteoblastic lineage, which was partially rescued by exogenous extracellular myocilin treatment. Myocilin also stimulated osteogenic differentiation of wild-type MSCs, which was associated with activation of the p38, Erk1/2, and JNK MAP kinase signaling pathways as well as up-regulated expression of the osteogenic transcription factors Runx2 and Dlx5. Finally, cortical bone thickness and trabecular volume, as well as the expression level of osteopontin, a known factor of bone remodeling and osteoblast differentiation, were reduced dramatically in the femurs of Myoc-null mice compared with wild-type mice. These data suggest that myocilin should be considered as a target for improving the bone regenerative potential of MSCs and may identify a new role for myocilin in bone formation and/or maintenance in vivo.
Assuntos
Diferenciação Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Proteínas do Olho/metabolismo , Glicoproteínas/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Osteogênese/fisiologia , Animais , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/biossíntese , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Proteínas do Citoesqueleto/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas do Olho/genética , Glicoproteínas/genética , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Mutantes , Osteoblastos/citologia , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Regulação para Cima/fisiologiaRESUMO
The glaucoma-associated gene, myocilin, is expressed in ocular and non-ocular tissues including the peripheral nervous system, but its functions in these tissues remain poorly understood. We demonstrate that in sciatic nerve, myocilin is expressed in Schwann cells with high concentrations at the nodes of Ranvier. There, myocilin interacts with gliomedin, neurofascin, and NrCAM, which are essential for node formation and function. Treatment of isolated dorsal root ganglion cultures with myocilin stimulates clustering of the nodal proteins neurofascin and sodium channel Nav1.2. Sciatic nerves of myocilin null mice express reduced levels of several myelin-associated and basal membrane proteins compared with those of wild-type littermates. They also demonstrate reduced myelin sheath thickness and partial disorganization of the nodes. Myocilin signaling through ErbB2/3 receptors may contribute to these observed effects. Myocilin binds to ErbB2/ErbB3, activates these receptors, and affects the downstream PI3K-AKT signaling pathway. These data implicate a role for myocilin in the development and/or maintenance of myelination and nodes of Ranvier in sciatic nerve.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas do Olho/metabolismo , Glicoproteínas/metabolismo , Bainha de Mielina/metabolismo , Sistema Nervoso Periférico/metabolismo , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Animais , Proteínas do Citoesqueleto/genética , Proteínas do Olho/genética , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica , Glaucoma/metabolismo , Glicoproteínas/genética , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Mutação , Bainha de Mielina/genética , Fosforilação , Nós Neurofibrosos/metabolismo , Nervo Isquiático/metabolismo , Transdução de SinaisRESUMO
Genetic studies have linked myocilin to open angle glaucoma, but the functions of the protein in the eye and other tissues have remained elusive. The purpose of this investigation was to elucidate myocilin function(s). We identified α1-syntrophin, a component of the dystrophin-associated protein complex (DAPC), as a myocilin-binding candidate. Myocilin interacted with α1-syntrophin via its N-terminal domain and co-immunoprecipitated with α1-syntrophin from C2C12 myotubes and mouse skeletal muscle. Expression of 15-fold higher levels of myocilin in the muscles of transgenic mice led to the elevated association of α1-syntrophin, neuronal nitric-oxide synthase, and α-dystroglycan with DAPC, which increased the binding of laminin to α-dystroglycan and Akt signaling. Phosphorylation of Akt and Forkhead box O-class 3, key regulators of muscle size, was increased more than 3-fold, whereas the expression of muscle-specific RING finger protein-1 and atrogin-1, muscle atrophy markers, was decreased by 79 and 88%, respectively, in the muscles of transgenic mice. Consequently, the average size of muscle fibers of the transgenic mice was increased by 36% relative to controls. We suggest that intracellular myocilin plays a role as a regulator of muscle hypertrophy pathways, acting through the components of DAPC.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas do Citoesqueleto/metabolismo , Distrofina/metabolismo , Proteínas do Olho/metabolismo , Glicoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Animais , Diferenciação Celular/fisiologia , Citoplasma/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Olho/genética , Expressão Gênica/fisiologia , Glicoproteínas/genética , Hipertrofia/metabolismo , Camundongos , Camundongos Transgênicos , Complexos Multiproteicos/metabolismo , Músculo Esquelético/citologia , Atrofia Muscular/metabolismo , Mioblastos/citologia , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologiaRESUMO
Olfm1, a secreted highly conserved glycoprotein, is detected in peripheral and central nervous tissues and participates in neural progenitor maintenance, cell death in brain, and optic nerve arborization. In this study, we identified Olfm1 as a molecule promoting axon growth through interaction with the Nogo A receptor (NgR1) complex. Olfm1 is coexpressed with NgR1 in dorsal root ganglia and retinal ganglion cells in embryonic and postnatal mice. Olfm1 specifically binds to NgR1, as judged by alkaline phosphatase assay and coimmunoprecipitation. The addition of Olfm1 inhibited the growth cone collapse of dorsal root ganglia neurons induced by myelin-associated inhibitors, indicating that Olfm1 attenuates the NgR1 receptor functions. Olfm1 caused the inhibition of NgR1 signaling by interfering with interaction between NgR1 and its coreceptors p75NTR or LINGO-1. In zebrafish, inhibition of optic nerve extension by olfm1 morpholino oligonucleotides was partially rescued by dominant negative ngr1 or lingo-1. These data introduce Olfm1 as a novel NgR1 ligand that may modulate the functions of the NgR1 complex in axonal growth.
Assuntos
Axônios/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas da Matriz Extracelular/fisiologia , Glicoproteínas/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Células COS , Chlorocebus aethiops , Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Proteínas de Fluorescência Verde/biossíntese , Cones de Crescimento/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas da Mielina/fisiologia , Proteínas Nogo , Nervo Óptico/citologia , Nervo Óptico/embriologia , Especificidade de Órgãos , Células PC12 , Ligação Proteica , Ratos , Receptor de Fator de Crescimento Neural/metabolismo , Peixe-Zebra , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Retinitis pigmentosa (RP) is a phenotypically and genetically heterogeneous group of inherited retinal degenerations characterized clinically by night blindness, progressive constriction of the visual fields, and loss of vision, and pathologically by progressive loss of rod and then cone photoreceptors. Autosomal-recessive RP (arRP) in a consanguineous Pakistani family previously linked to chromosome 2p22.3-p24.1 is shown to result from a homozygous missense mutation (c.1015T>C [p.C339R]) in ZNF513, encoding a presumptive transcription factor. znf513 is expressed in the retina, especially in the outer nuclear layer, inner nuclear layer, and photoreceptors. Knockdown of znf513 in zebrafish reduces eye size, retinal thickness, and expression of rod and cone opsins and causes specific loss of photoreceptors. These effects are rescued by coinjection with wild-type (WT) but not p.C339R-znf513 mRNA. Both normal and p.C339R mutant ZNF513 proteins expressed in COS-7 cells localize to the nucleus. ChIP analysis shows that only the wild-type but not the mutant ZNF513 binds to the Pax6, Sp4, Arr3, Irbp, and photoreceptor opsin promoters. These results suggest that the ZNF513 p.C339R mutation is responsible for RP in this family and that ZNF513 plays a key role in the regulation of photoreceptor-specific genes in retinal development and photoreceptor maintenance.
Assuntos
Proteínas do Olho/genética , Genes Recessivos/genética , Mutação/genética , Células Fotorreceptoras de Vertebrados/metabolismo , Retinose Pigmentar/genética , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Cromatina/metabolismo , Mapeamento Cromossômico , Análise Mutacional de DNA , Proteínas do Olho/química , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Marcadores Genéticos , Humanos , Escore Lod , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Linhagem , Células Fotorreceptoras de Vertebrados/patologia , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Fatores de Transcrição/química , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/químicaRESUMO
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
The MYOCILIN gene encodes a secreted glycoprotein which is highly expressed in eye drainage structures. Mutations in this gene may lead to juvenile open-angle glaucoma and adult onset primary open-angle glaucoma, one of the leading causes of irreversible blindness in the world. Functions of wild-type myocilin are still unclear. We have recently demonstrated that myocilin is a modulator of Wnt signaling and may affect actin cytoskeleton organization. Here we report that myocilin and its naturally occurring proteolytic fragments, similar to Wnt3a, are able to stimulate trabecular meshwork, NIH3T3, and FHL124 cell migration with the N-terminal proteolytic fragment of myocilin lacking the olfactomedin domain producing the highest stimulatory effect. Stimulation of cell migration occurs through activation of the integrin-focal adhesion kinase (FAK)-serine/threonine kinase (AKT) signaling pathway. Inhibition of FAK by siRNA reduced the stimulatory action of myocilin by threefold. Activation of several components of this signaling pathway was also demonstrated in the eyes of transgenic mice expressing elevated levels of myocilin in the eye drainage structures. These data extend the similarities between actions of myocilin and Wnt proteins acting through a ß-catenin-independent mechanism. The modification of the migratory ability of cells by myocilin may play a role in normal functioning of the eye anterior segment and its pathology including glaucoma.
Assuntos
Movimento Celular , Proteínas do Citoesqueleto/metabolismo , Proteínas do Olho/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Glicoproteínas/metabolismo , Integrinas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Malha Trabecular/enzimologia , Animais , Movimento Celular/efeitos dos fármacos , Proteínas do Citoesqueleto/genética , Ativação Enzimática , Proteínas do Olho/genética , Quinase 1 de Adesão Focal/genética , Glicoproteínas/genética , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células NIH 3T3 , Fragmentos de Peptídeos/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Malha Trabecular/efeitos dos fármacos , Transfecção , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteína Wnt3 , Proteína Wnt3ARESUMO
Mutations in the myocilin gene are associated with juvenile and adult-onset primary open-angle glaucoma. However, the pathogenic mechanisms of myocilin-induced glaucoma are still largely unknown. To investigate these mechanisms, we developed stably transfected HEK293 cell lines expressing wild-type or mutant (Y437H and I477N) myocilins under an inducible promoter. Expression of two mutant myocilins led to different levels of endoplasmic reticulum stress and increased apoptosis after treatment of cells with hydrogen peroxide. The Y437H mutant myocilin cell line showed the highest sensitivity to the oxidant treatment. Several antioxidant genes were down-regulated in the Y437H mutant myocilin cell line, but not in other cell lines. The Y437H mutant myocilin cell line also produced more reactive oxygen species than other cell lines examined. Consistent with the data obtained in cultured cells, the endoplasmic reticulum stress marker, 78 kDa glucose-regulated protein, was up-regulated, whereas antioxidant proteins, paraoxonase 2 and glutathione peroxidase 3, were down-regulated in the eye angle tissue of 18-month-old transgenic mice expressing Y437H myocilin mutant. In addition, a pro-apoptotic factor, CCAAT/enhancer-binding protein-homologous protein, was up-regulated in the aged transgenic mouse angle tissue. Our results suggest that expression of mutated myocilins may have a sensitization effect, which can lead to a severe phenotype in combination with oxidative stress. Mutant myocilins may confer different sensitivity to oxidative stress depending on the mutation.
Assuntos
Apoptose/fisiologia , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Mutação , Estresse Oxidativo/fisiologia , Adolescente , Adulto , Animais , Linhagem Celular/efeitos dos fármacos , Criança , Pré-Escolar , Glaucoma de Ângulo Aberto/genética , Glaucoma de Ângulo Aberto/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Camundongos , Camundongos Transgênicos , Análise em Microsséries , Dados de Sequência Molecular , Oxidantes/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Malha Trabecular/citologia , Malha Trabecular/metabolismo , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Adulto JovemRESUMO
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
Olfactomedin 1 (Olfm1) is a secreted glycoprotein belonging to a family of olfactomedin domain-containing proteins. It is involved in the regulation of neural crest production in chicken and promotes neuronal differentiation in Xenopus. Here, we investigate the functions of Olfm1 in zebrafish eye development. Overexpression of full-length Olfm1, and especially its BMY form lacking the olfactomedin domain, increased the thickness of the optic nerve and produced a more extended projection field in the optic tectum compared with control embryos. In contrast, injection of olfm1-morpholino oligonucleotide (Olfm1-MO) reduced the eye size, inhibited optic nerve extension, and increased the number of apoptotic cells in the retinal ganglion cell and inner nuclear layers. Overexpression of full-length Olfm1 increased the lateral separation of the expression domains of eye-field markers, rx3 and six3. The Olfm1-MO had the opposite effect. These data suggest that zebrafish Olfm1 may play roles in the early eye determination, differentiation, optic nerve extension, and branching of the retinal ganglion cell axon terminals, with the N-terminal region of Olfm1 being critical for these effects. Injection of RNA encoding WIF-1, a secreted inhibitor of Wnt signaling, caused changes in the expression pattern of rx3 similar to those observed after Olfm1-MO injection. Simultaneous overexpression of WIF-1 and Olfm1 abolished the WIF-1 effect. Physical interaction of WIF-1 and Olfm1 was demonstrated by coimmunoprecipitation experiments. We concluded that Olfm1 serves as a modulator of Wnt signaling.
Assuntos
Axônios/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Glicoproteínas/fisiologia , Retina/crescimento & desenvolvimento , Transdução de Sinais/fisiologia , Proteínas Wnt/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Proteínas da Matriz Extracelular/biossíntese , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/genética , Glicoproteínas/biossíntese , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Camundongos , Camundongos Knockout , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Retina/embriologia , Proteínas Wnt/antagonistas & inibidores , Proteínas Wnt/metabolismo , Peixe-ZebraRESUMO
A family of olfactomedin domain-containing proteins consists of at least 13 members in mammals. Although the first protein belonging to this family, olfactomedin, was isolated and partially characterized from frog olfactory neuroepithelim almost 20 years ago, the functions of many family members remain elusive. Most of the olfactomedin domain-containing proteins, similar to frog olfactomedin, are secreted glycoproteins that demonstrate specific expression patterns. Other family members are membrane-bound proteins that may serve as receptors. More than half of the olfactomedin domain-containing genes are expressed in neural tissues. Data obtained over the last several years demonstrate that olfactomedin domain-containing proteins play important roles in neurogenesis, neural crest formation, dorsal ventral patterning, cell-cell adhesion, cell cycle regulation, and tumorigenesis and may serve as modulators of critical signaling pathways (Wnt, bone morphogenic protein). Mutations in two genes encoding myocilin and olfactomedin 2 were implicated in glaucoma, and a growing number of evidence indicate that other genes belonging to the family of olfactomedin domain-containing proteins may contribute to different human disorders including psychiatric disorders. In this review, we summarize recent advances in understanding the possible roles of these proteins with special emphasis on the proteins that are preferentially expressed and function in neural tissues.