RESUMO
Biological membranes are functionalized by membrane-associated protein machinery. Membrane-associated transport processes, such as endocytosis, represent a fundamental and universal function mediated by membrane-deforming protein machines, by which small biomolecules and even micrometer-size substances can be transported via encapsulation into membrane vesicles. Although synthetic molecules that induce dynamic membrane deformation have been reported, a molecular approach enabling membrane transport in which membrane deformation is coupled with substance binding and transport remains critically lacking. Here, we developed an amphiphilic molecular machine containing a photoresponsive diazocine core (AzoMEx) that localizes in a phospholipid membrane. Upon photoirradiation, AzoMEx expands the liposomal membrane to bias vesicles toward outside-in fission in the membrane deformation process. Cargo components, including micrometer-size M13 bacteriophages that interact with AzoMEx, are efficiently incorporated into the vesicles through the outside-in fission. Encapsulated M13 bacteriophages are transiently protected from the external environment and therefore retain biological activity during distribution throughout the body via the blood following administration. This research developed a molecular approach using synthetic molecular machinery for membrane functionalization to transport micrometer-size substances and objects via vesicle encapsulation. The molecular design demonstrated in this study to expand the membrane for deformation and binding to a cargo component can lead to the development of drug delivery materials and chemical tools for controlling cellular activities.
Assuntos
Endocitose , Proteínas de Membrana , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Lipossomos/química , Transporte BiológicoRESUMO
The mammalian brain has very limited ability to regenerate lost neurons and recover function after injury. Promoting the migration of young neurons (neuroblasts) derived from endogenous neural stem cells using biomaterials is a new and promising approach to aid recovery of the brain after injury. However, the delivery of sufficient neuroblasts to distant injured sites is a major challenge because of the limited number of scaffold cells that are available to guide neuroblast migration. To address this issue, we have developed an amphiphilic peptide [(RADA)3-(RADG)] (mRADA)-tagged N-cadherin extracellular domain (Ncad-mRADA), which can remain in mRADA hydrogels and be injected into deep brain tissue to facilitate neuroblast migration. Migrating neuroblasts directly contacted the fiber-like Ncad-mRADA hydrogel and efficiently migrated toward an injured site in the striatum, a deep brain area. Furthermore, application of Ncad-mRADA to neonatal cortical brain injury efficiently promoted neuronal regeneration and functional recovery. These results demonstrate that self-assembling Ncad-mRADA peptides mimic both the function and structure of endogenous scaffold cells and provide a novel strategy for regenerative therapy.
Assuntos
Caderinas , Células-Tronco Neurais , Animais , Encéfalo , Neurônios , Peptídeos , MamíferosRESUMO
Ischemic stroke leads to acute neuron death and forms an injured core, triggering delayed cell death at the penumbra. The impaired brain functions after ischemic stroke are hardly recovered because of the limited regenerative properties. However, recent rodent intervention studies manipulating the extracellular environments at the subacute phase shed new light on the regenerative potency of the injured brain. This review introduces the rational design of artificial extracellular matrix (ECM) mimics using supramolecular peptidic scaffolds, which self-assemble via non-covalent bonds and form hydrogels. The facile customizability of the peptide structures allows tuning the hydrogels' physical and biochemical properties, such as charge states, hydrophobicity, cell adhesiveness, stiffness, and stimuli responses. Supramolecular peptidic materials can create safer and more economical drugs than polymer materials and cell transplantation. We also discuss the importance of activating developmental programs for the recovery at the subacute phase of ischemic stroke. Self-assembling molecular medicine mimicking the ECMs and activating developmental programs may stand as a new drug modality of regenerative medicine in various tissues.
Assuntos
AVC Isquêmico , Engenharia Tecidual , Matriz Extracelular , Humanos , Hidrogéis/química , Medicina Molecular , Peptídeos/química , Medicina RegenerativaRESUMO
During injured tissue regeneration, the extracellular matrix plays a key role in controlling and coordinating various cellular events by binding and releasing secreted proteins in addition to promoting cell adhesion. Herein, we develop a cell-adhesive fiber-forming peptide that mimics the jigsaw-shaped hydrophobic surface in the dovetail-packing motif of glycophorin A as an artificial extracellular matrix for regenerative therapy. We show that the jigsaw-shaped self-assembling peptide forms several-micrometer-long supramolecular nanofibers through a helix-to-strand transition to afford a hydrogel under physiological conditions and disperses homogeneously in the hydrogel. The molecular- and macro-scale supramolecular properties of the jigsaw-shaped self-assembling peptide hydrogel allow efficient incorporation and sustained release of vascular endothelial growth factor, and demonstrate cell transplantation-free regenerative therapeutic effects in a subacute-chronic phase mouse stroke model. This research highlights a therapeutic strategy for injured tissue regeneration using the jigsaw-shaped self-assembling peptide supramolecular hydrogel.
Assuntos
Regeneração do Cérebro/fisiologia , Hidrogéis/química , Peptídeos/química , Proteínas/química , Adesivos , Animais , Engenharia Biomédica , Lesões Encefálicas/diagnóstico por imagem , Adesão Celular , Modelos Animais de Doenças , Feminino , Proteínas de Fluorescência Verde/química , Hidrogéis/uso terapêutico , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Camundongos Endogâmicos C57BL , Nanofibras , Sistema Nervoso , Peptídeos/uso terapêutico , Fator A de Crescimento do Endotélio VascularRESUMO
Invited for the cover of this issue is the group of Takahiro Muraoka at Tokyo University of Agriculture and Technology and collaborators. The image depicts nanofiber formation of an amphiphilic peptide with a central alkylene chain that shows non-cell adhesive properties. Read the full text of the article at 10.1002/chem.202100739.
Assuntos
Hidrogéis , Nanofibras , Adesivos , PeptídeosRESUMO
BACKGROUND AND AIMS: Synthetic cyclin-dependent kinase (CDK) 4/6 inhibitors exert antitumor effects by forcing RB1 in unphosphorylated status, causing not only cell cycle arrest but also cellular senescence, apoptosis, and increased immunogenicity. These agents currently have an indication in advanced breast cancers and are in clinical trials for many other solid tumors. HCC is one of promising targets of CDK4/6 inhibitors. RB family dysfunction is often associated with the initiation of HCC; however, this is revivable, as RB family members are not frequently mutated or deleted in this malignancy. APPROACH AND RESULTS: Loss of all Rb family members in transformation related protein 53 (Trp53)-/- mouse liver resulted in liver tumor reminiscent of human HCC, and re-expression of RB1 sensitized these tumors to a CDK4/6 inhibitor, palbociclib. Introduction of an unphosphorylatable form of RB1 (RB7LP) into multiple liver tumor cell lines induced effects similar to palbociclib. By screening for compounds that enhance the efficacy of RB7LP, we identified an I kappa B kinase (IKK)ß inhibitor Bay 11-7082. Consistently, RB7LP expression and treatment with palbociclib enhanced IKKα/ß phosphorylation and NF-κB activation. Combination therapy using palbociclib with Bay 11-7082 was significantly more effective in hepatoblastoma and HCC treatment than single administration. Moreover, blockade of IKK-NF-κB or AKT pathway enhanced effects of palbociclib on RB1-intact KRAS Kirsten rat sarcoma viral oncogene homolog mutated lung and colon cancers. CONCLUSIONS: In conclusion, CDK4/6 inhibitors have a potential to treat a wide variety of RB1-intact cancers including HCC when combined with an appropriate kinase inhibitor.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Aminopiridinas/farmacologia , Aminopiridinas/uso terapêutico , Animais , Benzimidazóis/farmacologia , Benzimidazóis/uso terapêutico , Carcinoma Hepatocelular/genética , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Células Hep G2 , Humanos , Técnicas In Vitro , Neoplasias Hepáticas/genética , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas Experimentais/genética , Camundongos , Transplante de Neoplasias , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Purinas/farmacologia , Purinas/uso terapêutico , Piridinas/uso terapêutico , Proteína do Retinoblastoma , Proteína Supressora de Tumor p53/genética , Proteínas de XenopusRESUMO
Amphiphilic peptides bearing terminal alkyl tails form supramolecular nanofibers that are increasingly used as biomaterials with multiple functionalities. Insertion of alkylene chains in peptides can be designed as another type of amphiphilic peptide, yet the influence of the internal alkylene chains on self-assembly and biological properties remains poorly defined. Unlike the terminal alkyl tails, the internal alkylene chains can affect not only the hydrophobicity but also the flexibility and packing of the peptides. Herein, we demonstrate the supramolecular and biological effects of the central alkylene chain length inserted in a peptide. Insertion of the alkylene chain at the center of the peptide allowed for strengthened ß-sheet hydrogen bonds and modulation of the packing order, and consequently the amphiphilic peptide bearing C2 alkylene chain formed a hydrogel with the highest stiffness. Interestingly, the amphiphilic peptides bearing internal alkylene chains longer than C2 showed a diminished cell-adhesive property. This study offers a novel molecular design to tune mechanical and biological properties of peptide materials.
Assuntos
Hidrogéis , Nanofibras , Adesivos , Interações Hidrofóbicas e Hidrofílicas , PeptídeosRESUMO
Cell adhesion is a fundamental biological process involved in a wide range of cellular and biological activity. Integrin-ligand binding is largely responsible for cell adhesion with an extracellular matrix, and the RGD sequence is an epitope in ligand proteins such as fibronectin. The extracellular matrix consists of fibrous proteins with embedded ligands for integrins. Such a biological architecture has been reconstructed for biochemical, pharmaceutical, and biomaterial studies using artificial supramolecular systems to reproduce cell adhesion functionality, and fiber-forming self-assembling peptides containing RGD are one such promising material for this purpose. In this study, using RADA16 as a model fiber-forming peptide, a series of RGD-containing variants have been synthesized by the replacement of one alanine with glycine at different positions, in which all the variants consist of identical amino acid components. The position of the RGD unit influenced the supramolecular self-assembly of the amphiphilic peptide to inhibit ß-sheet formation (A6G) or twist the molecular alignment in ß-sheet-type assemblies (A10G and A14G). Furthermore, A10G and A14G formed assembled nanofibers, which afforded hydrogels with higher viscoelasticities than other RGD-containing variants. In contrast to A10G and A14G, which exhibit substantial cell adhesion functionality, the cell adhesion efficiencies of the other RGD-containing variants were significantly reduced. This suggests that the higher order structure could strongly influence the cell adhesion functionality of RGD-containing supramolecular nanofibers.
RESUMO
To facilitate efficient oxygen and nutrient delivery, blood vessels in the brain form three-dimensional patterns. However, little is known about how blood vessels develop stereographically in the neocortex and how they control the expansion and differentiation of neural progenitors during neocortical development. We show that highly vascularized and avascular regions are strictly controlled in a spatially and temporally restricted manner and are associated with distinct cell populations. Dividing basal progenitors and oligodendrocyte precursors preferentially contact honeycomb vessels, but dividing apical progenitors are localized in avascular regions without Flt1-positive endothelial cells but directly contact with sprouting neovascular tip cells. Therefore, not all blood vessels are associated equally with neural progenitors. Furthermore, a disruption of normal vascular patterning can induce abnormalities in neural development, whereas the impaired features of neural progenitors influenced angiogenesis patterning. These results indicate that close association between the nervous and vascular systems is essential for neocortex assembly.
Assuntos
Neocórtex/citologia , Neocórtex/embriologia , Neovascularização Fisiológica , Células-Tronco Neurais/citologia , Animais , Diferenciação Celular , Hipóxia Celular , Polaridade Celular , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Feminino , Humanos , Cadeias beta de Integrinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Neocórtex/irrigação sanguínea , Neocórtex/ultraestrutura , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Pseudópodes/metabolismo , Nicho de Células-Tronco , Fatores de TempoRESUMO
Photon upconversion (UC) from near-infrared (NIR) light to visible light has enabled optogenetic manipulations in deep tissues. However, materials for NIR optogenetics have been limited to inorganic UC nanoparticles. Herein, NIR-light-triggered optogenetics using biocompatible, organic TTA-UC hydrogels is reported. To achieve triplet sensitization even in highly viscous hydrogel matrices, a NIR-absorbing complex is covalently linked with energy-pooling acceptor chromophores, which significantly elongates the donor triplet lifetime. The donor and acceptor are solubilized in hydrogels formed from biocompatible Pluronic F127 micelles, and heat treatment endows the excited triplets in the hydrogel with remarkable oxygen tolerance. Combined with photoactivatable Cre recombinase technology, NIR-light stimulation successfully performs genome engineering resulting in the formation of dendritic-spine-like structures of hippocampal neurons.
Assuntos
Complexos de Coordenação/química , Corantes Fluorescentes/química , Hidrogéis/química , Osmio/química , Perileno/química , Genoma , Raios Infravermelhos , Cinética , Micelas , Estrutura Molecular , Optogenética/métodos , Oxigênio/química , Fótons , Poloxâmero/química , Relação Estrutura-Atividade , TermodinâmicaRESUMO
Self-assembling peptides that are capable of adopting ß-sheet structures can generate nanofibers that lead to hydrogel formation. Herein, to tune the supramolecular morphologies, mechanical properties, and stimuli responses of the hydrogels, we investigated glycine substitution in a ß-sheet-forming amphiphilic peptide. Glycine substitution generally enhances conformational flexibility. Indeed, glycine substitution in an amphiphilic peptide weakened the hydrogels or even inhibited the gelation. However, unexpectedly, glycine substitution at the center of the peptide molecule significantly enhanced the hydrogel stiffness. The central glycine substitution affected the molecular packing and led to twisted ß-sheet structures and to nanofiber bundling, which likely led to the stiffened hydrogel. Importantly, the supramolecular structures were accurately predicted by molecular dynamics simulations, demonstrating the helpfulness of these techniques for the identification of self-assembling peptides. The hydrogel formed by the amphiphilic peptide with the central glycine substitution had cell adhesive function, and showed a reversible thermal gel-to-sol transition. Thus, glycine substitution is effective in modulating self-assembling structures, rheological properties, and dynamics of biofunctional self-assembling peptides.
Assuntos
Adesivos/química , Glicinérgicos/metabolismo , Glicina/química , Peptídeos/química , Glicinérgicos/química , Hidrogéis/química , Simulação de Dinâmica Molecular , Nanofibras/química , ReologiaRESUMO
Radial glia (RG) are embryonic neural stem cells (NSCs) that produce neuroblasts and provide fibers that act as a scaffold for neuroblast migration during embryonic development. Although they normally disappear soon after birth, here we found that RG fibers can persist in injured neonatal mouse brains and act as a scaffold for postnatal ventricular-subventricular zone (V-SVZ)-derived neuroblasts that migrate to the lesion site. This injury-induced maintenance of RG fibers has a limited time window during post-natal development and promotes directional saltatory movement of neuroblasts via N-cadherin-mediated cell-cell contacts that promote RhoA activation. Transplanting an N-cadherin-containing scaffold into injured neonatal brains likewise promotes migration and maturation of V-SVZ-derived neuroblasts, leading to functional improvements in impaired gait behaviors. Together these results suggest that RG fibers enable postnatal V-SVZ-derived neuroblasts to migrate toward sites of injury, thereby enhancing neuronal regeneration and functional recovery from neonatal brain injuries.
Assuntos
Lesões Encefálicas/patologia , Lesões Encefálicas/fisiopatologia , Movimento Celular , Neuroglia/patologia , Neurônios/patologia , Recuperação de Função Fisiológica , Animais , Animais Recém-Nascidos , Caderinas/metabolismo , Ventrículos Laterais/patologia , Neuroglia/metabolismo , Neuroglia/ultraestrutura , Neurônios/metabolismo , Neurônios/ultraestrutura , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Neuronal differentiation and cell-cycle exit are tightly coordinated, even in pathological situations. When pathological neurons re-enter the cell cycle and progress through the S phase, they undergo cell death instead of division. However, the mechanisms underlying mitotic resistance are mostly unknown. Here, we have found that acute inactivation of retinoblastoma (Rb) family proteins (Rb, p107 and p130) in mouse postmitotic neurons leads to cell death after S-phase progression. Checkpoint kinase 1 (Chk1) pathway activation during the S phase prevented the cell death, and allowed the division of cortical neurons that had undergone acute Rb family inactivation, oxygen-glucose deprivation (OGD) or in vivo hypoxia-ischemia. During neurogenesis, cortical neurons became protected from S-phase Chk1 pathway activation by the DNA methyltransferase Dnmt1, and underwent cell death after S-phase progression. Our results indicate that Chk1 pathway activation overrides mitotic safeguards and uncouples neuronal differentiation from mitotic resistance.
Assuntos
Divisão Celular , Quinase 1 do Ponto de Checagem/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Neurônios/citologia , Neurônios/enzimologia , Animais , Morte Celular , Hipóxia Celular , Sobrevivência Celular , DNA (Citosina-5-)-Metiltransferase 1 , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Glucose/deficiência , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Neurogênese , Oxigênio , Proteína do Retinoblastoma/metabolismo , Fase S , Transdução de Sinais , Acidente Vascular Cerebral/patologiaRESUMO
Ischemic brain stroke is caused by blood flow interruption, leading to focal ischemia, neuron death, and motor, sensory, and/or cognitive dysfunctions. Angiogenesis, neovascularization from existing blood vessel, is essential for tissue growth and repair. Proangiogenic therapy for stroke is promising for preventing excess neuron death and improving functional recovery. Vascular endothelial growth factor (VEGF) is a critical factor for angiogenesis by promoting the proliferation, the survival, and the migration of endothelial cells. Here, angiogenic biomaterials to support injured brain regeneration are developed. Porous laminin (LN)-rich sponge (LN-sponge), on which histidine-tagged VEGF (VEGF-Histag) is immobilized via affinity interaction is developed. In an in vivo mouse stroke model, transplanting VEGF-Histag-LN-sponge produces remarkably stronger angiogenic activity than transplanting LN-sponge with soluble VEGF. The findings indicate that using affinity interactions to immobilize VEGF is a practical approach for developing angiogenic biomaterials for regenerating the injured brain.
Assuntos
Isquemia Encefálica , Proteínas Imobilizadas , Laminina , Neovascularização Fisiológica/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular , Animais , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Modelos Animais de Doenças , Implantes de Medicamento/química , Implantes de Medicamento/farmacologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/farmacologia , Laminina/química , Laminina/farmacologia , Camundongos , Porosidade , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Cerebral ischemic stroke is a main cause of chronic disability. However, there is currently no effective treatment to promote recovery from stroke-induced neurological symptoms. Recent studies suggest that after stroke, immature neurons, referred to as neuroblasts, generated in a neurogenic niche, the ventricular-subventricular zone, migrate toward the injured area, where they differentiate into mature neurons. Interventions that increase the number of neuroblasts distributed at and around the lesion facilitate neuronal repair in rodent models for ischemic stroke, suggesting that promoting neuroblast migration in the post-stroke brain could improve efficient neuronal regeneration. To move toward the lesion, neuroblasts form chain-like aggregates and migrate along blood vessels, which are thought to increase their migration efficiency. However, the molecular mechanisms regulating these migration processes are largely unknown. Here we studied the role of ß1-class integrins, transmembrane receptors for extracellular matrix proteins, in these migrating neuroblasts. We found that the neuroblast chain formation and blood vessel-guided migration critically depend on ß1 integrin signaling. ß1 integrin facilitated the adhesion of neuroblasts to laminin and the efficient translocation of their soma during migration. Moreover, artificial laminin-containing scaffolds promoted neuroblast chain formation and migration toward the injured area. These data suggest that laminin signaling via ß1 integrin supports vasculature-guided neuronal migration to efficiently supply neuroblasts to injured areas. This study also highlights the importance of vascular scaffolds for cell migration in development and regeneration.
Assuntos
Encéfalo/metabolismo , Movimento Celular , Integrina beta1/metabolismo , Neurônios/metabolismo , Transdução de Sinais , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Vasos Sanguíneos/metabolismo , Encéfalo/irrigação sanguínea , Encéfalo/ultraestrutura , Células Cultivadas , Técnicas de Cocultura , Feminino , Integrina beta1/genética , Laminina/metabolismo , Masculino , Camundongos Endogâmicos ICR , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Microscopia Eletrônica , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Acidente Vascular Cerebral/metabolismo , Alicerces TeciduaisRESUMO
Point mutations in the vacuolar protein sorting 35 gene (VPS35) have been associated with an autosomal dominant form of late-onset Parkinson disease (PARK17), but there has been considerable debate over whether it is caused by a loss- or gain-of-function mechanism and over the intracellular target site of neurotoxicity. To investigate the pathogenesis of PARK17 in vivo, we generated Vps35 D620N knock-in (KI) mice, expressing the homologous mutant protein with endogenous patterns of expression, simultaneously with Vps35 deletion 1 (Del1) mice, which carry 1bp deletion in the exon15 of Vps35, by CRISPR/Cas9-mediated genome engineering. Neither homozygous nor heterozygous Vps35 D620N KI mice suffered from premature death or developed clear neurodegeneration up to 70 weeks of age. Vps35 Del1 allele appeared to be a null or at least severely hypomorphic allele and homozygous Vps35 Del1 showed early embryonic lethality. Heterozygous crossings between Del1 and D620N knock-in mice revealed that the D620N/Del1 compound heterozygous mice, but not heterozygous Del1 mice, suffered from survival disadvantage. In vivo microdialysis showed that DA release evoked by 120 mM potassium chloride was significantly reduced in the caudate putamen of adult homozygous Vps35 D620N KI mice. Taken together, these results suggest that Vps35 D620N allele is a partial-loss-of-function allele and that such a genetic predisposition and age-related alterations in the nigrostriatal dopamine system cooperatively influence the pathogenesis of PARK17.
Assuntos
Modelos Animais de Doenças , Dopamina/metabolismo , Mutação , Doença de Parkinson/metabolismo , Proteínas de Transporte Vesicular/genética , Animais , Técnicas de Introdução de Genes , Homozigoto , Camundongos , Neostriado/metabolismo , Neostriado/fisiopatologia , Doença de Parkinson/genética , Doença de Parkinson/fisiopatologiaRESUMO
The cerebral cortex is responsible for higher functions of the central nervous system (CNS), such as movement, sensation, and cognition. When the cerebral cortex is severely injured, these functions are irreversibly impaired. Although recent neurobiological studies reveal that the cortex has the potential for regeneration, therapies for functional recovery face some technological obstacles. Biomaterials have been used to evoke regenerative potential and promote regeneration in several tissues, including the CNS. This review presents a brief overview of new therapeutic strategies for cortical regeneration from the perspectives of neurobiology and biomaterial engineering, and discusses a promising technology for evoking the regenerative potential of the cerebral cortex.
RESUMO
Retinal development requires precise temporal and spatial coordination of cell cycle exit, cell fate specification, cell migration and differentiation. When this process is disrupted, retinoblastoma, a developmental tumor of the retina, can form. Epigenetic modulators are central to precisely coordinating developmental events, and many epigenetic processes have been implicated in cancer. Studying epigenetic mechanisms in development is challenging because they often regulate multiple cellular processes; therefore, elucidating the primary molecular mechanisms involved can be difficult. Here we explore the role of Brg1 (Smarca4) in retinal development and retinoblastoma in mice using molecular and cellular approaches. Brg1 was found to regulate retinal size by controlling cell cycle length, cell cycle exit and cell survival during development. Brg1 was not required for cell fate specification but was required for photoreceptor differentiation and cell adhesion/polarity programs that contribute to proper retinal lamination during development. The combination of defective cell differentiation and lamination led to retinal degeneration in Brg1-deficient retinae. Despite the hypocellularity, premature cell cycle exit, increased cell death and extended cell cycle length, retinal progenitor cells persisted in Brg1-deficient retinae, making them more susceptible to retinoblastoma. ChIP-Seq analysis suggests that Brg1 might regulate gene expression through multiple mechanisms.
Assuntos
DNA Helicases/genética , DNA Helicases/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Retina/metabolismo , Retinoblastoma/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Animais , Apoptose , Padronização Corporal , Adesão Celular , Ciclo Celular , Diferenciação Celular , Linhagem da Célula , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Epigênese Genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Camundongos , Microftalmia/genética , Retina/patologia , Fatores de Tempo , TransgenesRESUMO
Oxidative stress has a ubiquitous role in neurodegenerative diseases and oxidative damage in specific regions of the brain is associated with selective neurodegeneration. We previously reported that Alzheimer disease (AD) model mice showed decreased insulin-degrading enzyme (IDE) levels in the cerebrum and accelerated phenotypic features of AD when crossbred with alpha-tocopherol transfer protein knockout (Ttpa-/-) mice. To further investigate the role of chronic oxidative stress in AD pathophysiology, we performed DNA microarray analysis using young and aged wild-type mice and aged Ttpa-/- mice. Among the genes whose expression changed dramatically was Phospholipase A2 group 3 (Pla2g3); Pla2g3 was identified because of its expression profile of cerebral specific up-regulation by chronic oxidative stress in silico and in aged Ttpa-/- mice. Immunohistochemical studies also demonstrated that human astrocytic Pla2g3 expression was significantly increased in human AD brains compared with control brains. Moreover, transfection of HEK293 cells with human Pla2g3 decreased endogenous IDE expression in a dose-dependent manner. Our findings show a key role of Pla2g3 on the reduction of IDE, and suggest that cerebrum specific increase of Pla2g3 is involved in the initiation and/or progression of AD.
Assuntos
Envelhecimento/genética , Doença de Alzheimer/genética , Cérebro/metabolismo , Fosfolipases A2 do Grupo III/genética , Insulisina/genética , Doença de Alzheimer/metabolismo , Animais , Proteínas de Transporte/genética , Células Cultivadas , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Fosfolipases A2 do Grupo III/metabolismo , Células HEK293 , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos , Estresse Oxidativo , Regulação para CimaRESUMO
After brain injury, neuroblasts generated from endogenous neural stem cells migrate toward the injured site using blood vessels as a scaffold, raising the possibility of reconstructing blood vessel network scaffolds as a strategy for promoting endogenous neuronal regeneration. In this study, we designed biomaterials based on the components and morphology of blood vessel scaffolds, and examined their ability to guide the migration of neuroblasts into a brain lesion site in mice. Transplanted porous sponge containing components of the basement membrane (BM) matrix enhanced neuroblast migration into the lesion, and detailed morphological examination suggested that the infiltrating cells used the BM sponge as a migration scaffold. Laminin (LN)-rich porous sponge also enhanced the migration of neuroblasts into the lesion, whereas BM gel and gelatin porous sponge did not. We conclude that the transplantation of LN-rich porous sponge promotes neuroblast migration into cortical lesions. This study highlights the possibility of using artificial blood vessel scaffolds to promote the regeneration of injured cerebral cortex.