RESUMO
Docosahexaenoic acid (DHA, C22:6 ω3) may be involved in various neuroprotective mechanisms that could prevent Alzheimer's disease (AD). Its influence has still been little explored regarding the dysfunction of the endolysosomal pathway, known as an early key event in the physiopathological continuum triggering AD. This dysfunction could result from the accumulation of degradation products of the precursor protein of AD, in particular the C99 fragment, capable of interacting with endosomal proteins and thus contributing to altering this pathway from the early stages of AD. This study aims to evaluate whether neuroprotection mediated by DHA can also preserve the endolysosomal function. AD-typical endolysosomal abnormalities were recorded in differentiated human SH-SY5Y neuroblastoma cells expressing the Swedish form of human amyloid precursor protein. This altered phenotype included endosome enlargement, the reduced secretion of exosomes, and a higher level of apoptosis, which confirmed the relevance of the cellular model chosen for studying the associated deleterious mechanisms. Second, neuroprotection mediated by DHA was associated with a reduced interaction of C99 with the Rab5 GTPase, lower endosome size, restored exosome production, and reduced neuronal apoptosis. Our data reveal that DHA may influence protein localization and interactions in the neuronal membrane environment, thereby correcting the dysfunction of endocytosis and vesicular trafficking associated with AD.
Assuntos
Doença de Alzheimer , Ácidos Docosa-Hexaenoicos , Endossomos , Lisossomos , Neurônios , Proteínas rab5 de Ligação ao GTP , Humanos , Ácidos Docosa-Hexaenoicos/farmacologia , Ácidos Docosa-Hexaenoicos/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Proteínas rab5 de Ligação ao GTP/metabolismo , Endossomos/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Neurônios/efeitos dos fármacos , Lisossomos/metabolismo , Linhagem Celular Tumoral , Precursor de Proteína beta-Amiloide/metabolismo , Apoptose , Fármacos Neuroprotetores/farmacologia , Sobrevivência Celular/efeitos dos fármacosRESUMO
Astroglia play an important role, providing de novo synthesized cholesterol to neurons in the form of ApoE-lipidated particles; disruption of this process can increase the risk of Alzheimer's disease. We recently reported that glia-specific suppression of the lipolysis-stimulated lipoprotein receptor (LSR) gene leads to Alzheimer's disease-like memory deficits. Since LSR is an Apo-E lipoprotein receptor, our objective of this study was to determine the effect of LSR expression modulation on cholesterol and ApoE output in mouse astrocytes expressing human ApoE3. qPCR analysis showed that siRNA-mediated lsr knockdown significantly increased expression of the genes involved in cholesterol synthesis, secretion, and metabolism. Analysis of media and lipoprotein fractions showed increased cholesterol and lipidated ApoE output in HDL-like particles. Further, lsr expression could be upregulated when astrocytes were incubated 5 days in media containing high levels (two-fold) of lipoprotein, or after 8 h treatment with 1 µM LXR agonist T0901317 in lipoprotein-deficient media. In both conditions of increased lsr expression, the ApoE output was repressed or unchanged despite increased abca1 mRNA levels and cholesterol production. We conclude that LSR acts as a sensor of lipoprotein content in the medium and repressor of ApoE release, while ABCA1 drives cholesterol efflux, thereby potentially affecting cholesterol load, ApoE lipidation, and limiting cholesterol trafficking towards the neuron.
Assuntos
Doença de Alzheimer , Receptores de Lipoproteínas , Doença de Alzheimer/metabolismo , Animais , Apolipoproteínas E/metabolismo , Astrócitos/metabolismo , Colesterol/metabolismo , Humanos , Lipólise , Camundongos , Receptores de Lipoproteínas/genética , Receptores de Lipoproteínas/metabolismoRESUMO
Perturbations of cholesterol metabolism have been linked to neurodegenerative diseases. Glia-neuron crosstalk is essential to achieve a tight regulation of brain cholesterol trafficking. Adequate cholesterol supply from glia via apolipoprotein E-containing lipoproteins ensures neuronal development and function. The lipolysis-stimulated lipoprotein receptor (LSR), plays an important role in brain cholesterol homeostasis. Aged heterozygote Lsr+/- mice show altered brain cholesterol distribution and increased susceptibility to amyloid stress. Since LSR expression is higher in astroglia as compared to neurons, we sought to determine if astroglial LSR deficiency could lead to cognitive defects similar to those of Alzheimer's disease (AD). Cre recombinase was activated in adult Glast-CreERT/lsrfl/fl mice by tamoxifen to induce astroglial Lsr deletion. Behavioral phenotyping of young and old astroglial Lsr KO animals revealed hyperactivity during the nocturnal period, deficits in olfactory function affecting social memory and causing possible apathy, as well as visual memory and short-term working memory problems, and deficits similar to those reported in neurodegenerative diseases, such as AD. Furthermore, GFAP staining revealed astroglial activation in the olfactory bulb. Therefore, astroglial LSR is important for working, spatial, and social memory related to sensory input, and represents a novel pathway for the study of brain aging and neurodegeneration.
Assuntos
Astrócitos/metabolismo , Transtornos da Memória/metabolismo , Memória de Curto Prazo , Receptores de Lipoproteínas/metabolismo , Olfato , Animais , Colesterol/metabolismo , Transtornos da Memória/genética , Camundongos , Receptores de Lipoproteínas/genéticaRESUMO
Perturbations of lipid homeostasis manifest as dyslipidemias and obesity, which are significant risk factors for atherosclerosis and diabetes. Lipoprotein receptors in the liver are key players in the regulation of lipid homeostasis, among which the hepatic lipolysis stimulated lipoprotein receptor, LSR, was recently shown to play an important role in the removal of lipoproteins from the circulation during the postprandial phase. Since heterozygous LSR+/- mice demonstrate moderate dyslipidemia and develop higher body weight gain in response to high-fat diet compared with littermate LSR+/+ controls, we questioned if LSR heterozygosity could affect genes related to hepatic lipid metabolism. A target-specific qPCR array for 84 genes related to lipid metabolism was performed on mRNA isolated from livers of 6 mo old female LSR+/- mice and LSR+/+ littermates following a 6 wk period on a standard (STD) or high-fat diet (60% kcal, HFD). Of the 84 genes studied, 32 were significantly downregulated in STD-LSR+/- mice compared with STD-LSR+/+, a majority of which were PPARα target genes involved in lipid metabolism and transport, and insulin and adipokine-signaling pathways. Of these 32 genes, 80% were also modified in HFD-LSR+/+, suggesting that STD-LSR+/- mice demonstrated a predisposition towards a "high-fat"-like profile, which could reflect dysregulation of liver lipid homeostasis. Since similar profiles of genes were affected by either LSR heterozygosity or by high-fat diet, this would suggest that LSR is a key receptor in regulating hepatic lipid homeostasis, and whose downregulation combined with a Western-type diet may increase predisposition to diet-induced obesity.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Homeostase/genética , Metabolismo dos Lipídeos/genética , Fígado/metabolismo , Receptores de Lipoproteínas/genética , Transcriptoma/genética , Animais , Regulação para Baixo/genética , Feminino , Heterozigoto , Insulina/genética , Lipídeos/genética , Camundongos , Obesidade/genética , Aumento de Peso/genéticaRESUMO
The death of retinal ganglion cells (RGC) leads to visual impairment and blindness in ocular neurodegenerative diseases, primarily in glaucoma and diabetic retinopathy; hence, mechanisms that contribute to protecting RGC from ischemia/hypoxia are of great interest. We here address the role of retinal glial (Müller) cells and of pigment-epithelium-derived factor (PEDF), one of the main neuroprotectants released from the glial cells. We show that the hypoxia-induced loss in the viability of cultured purified RGC is due to apoptosis, but that the number of viable RGC increases when co-cultured with Müller glial cells suggesting that glial soluble mediators attenuate the death of RGC. When PEDF was ablated from Müller cells a significantly lower number of RGC survived in RGC-Müller cell co-cultures indicating that PEDF is a major survival factor allowing RGC to escape cell death. We further found that RGC express a PEDF receptor known as patatin-like phospholipase domain-containing protein 2 (PNPLA2) and that PEDF exposure, as well as the presence of Müller cells, leads to an activation of nuclear factor (NF)-κB in RGC. Furthermore, adding an NF-κB inhibitor (SN50) to PEDF-treated RGC cultures reduced the survival of RGC. These findings strongly suggest that NF-κB activation in RGC is critically involved in the pro-survival action of Müller-cell derived PEDF and plays an important role in maintaining neuronal survival.
Assuntos
Células Ependimogliais/citologia , Proteínas do Olho/metabolismo , Fatores de Crescimento Neural/metabolismo , Células Ganglionares da Retina/citologia , Serpinas/metabolismo , Animais , Apoptose , Sobrevivência Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Células Ependimogliais/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Hipóxia/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Peptídeos/farmacologia , Interferência de RNA , Ratos , Ratos Long-Evans , Reação em Cadeia da Polimerase em Tempo Real , Células Ganglionares da Retina/metabolismoRESUMO
Netrin-4, a member of the netrin family, is a potent regulator of embryonic development. It promotes neurite extension and regulates pulmonary airway branching, vasculogenesis patterning, and endothelial proliferation in pathological angiogenesis. The initial characterization of netrin-4 expression was focused on epithelial-derived organs (kidney, lung and salivary gland) and the central nervous system. Ocular development is an ideal system to study netrin-4 expression and function, as it involves both ectodermal (cornea, lens and retina) and mesodermal (sclera and choroid) derivatives and has an extensive and well-characterized angiogenic process. Netrin-4 is expressed in all ocular tissues. It is a prominent component of the basement membranes of the lens and cornea, as well as all three basement membranes of the retina: the inner limiting membrane, vascular basement membranes, and Bruch's membrane. Netrin-4 is differentially deposited in vascular basement membranes, with more intense anti-netrin-4 reactivity on the arterial side. The retinal microcirculation also expresses netrin-4. In order to test the function of netrin-4 in vivo, we generated a conventional mouse lacking Ntn4 expression. Basement membrane formation in the cornea, lens and retina is undisrupted by netrin-4 deletion, demonstrating that netrin-4 is not a major structural component of these basement membranes. In the Ntn4 homozygous null (Ntn4-/-) cornea, the overall morphology of the cornea, as well as the epithelial, stromal and endothelial stratification are normal; however, epithelial cell proliferation is increased. In the Ntn4-/- retina, neurogenesis appears to proceed normally, as does retinal lamination. In the Ntn4-/- retina, retinal ganglion cell targeting is intact, although there are minor defects in axon fasciculation. In the retinal vasculature of the Ntn4-/- retina, the distribution patterns of astrocytes and the vasculature are largely normal, with the possible exception of increased branching in the deep capillary plexus, suggesting that netrin-4 may act as a negative regulator of angiogenesis. These data, taken together, suggest that netrin-4 is a negative regulator of corneal epithelial cell proliferation and retinal vascular branching in vivo, whereas netrin-4 may be redundant with other members of the netrin family in other ocular tissue development. Ntn4-/- mice may serve as a good model in which to study the role of netrins in vivo of the pathobiologic vascular remodeling in the retina and cornea.
Assuntos
Olho/metabolismo , Regulação da Expressão Gênica/fisiologia , Fatores de Crescimento Neural/genética , Animais , Axônios/fisiologia , Membrana Basal/metabolismo , Proliferação de Células , Epitélio Corneano/citologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfogênese/fisiologia , Neovascularização Fisiológica , Netrinas , Reação em Cadeia da Polimerase , Células Ganglionares da Retina/metabolismo , Vasos Retinianos/fisiologiaRESUMO
Survival of retinal ganglion cells (RGC) is compromised in several vision-threatening disorders such as ischemic and hypertensive retinopathies and glaucoma. Pigment epithelium-derived factor (PEDF) is a naturally occurring pleiotropic secreted factor in the retina. PEDF produced by retinal glial (Müller) cells is suspected to be an essential component of neuron-glial interactions especially for RGC, as it can protect this neuronal type from ischemia-induced cell death. Here we show that PEDF treatment can directly affect RGC survival in vitro. Using Müller cell-RGC-co-cultures we observed that activity of Müller-cell derived soluble mediators can attenuate hypoxia-induced damage and RGC loss. Finally, neutralizing the activity of PEDF in glia-conditioned media partially abolished the neuroprotective effect of glia, leading to an increased neuronal death in hypoxic condition. Altogether our results suggest that PEDF is crucially involved in the neuroprotective process of reactive Müller cells towards RGC.
Assuntos
Proteínas do Olho/metabolismo , Fatores de Crescimento Neural/metabolismo , Neuroglia/metabolismo , Fármacos Neuroprotetores/metabolismo , Células Ganglionares da Retina/metabolismo , Serpinas/metabolismo , Animais , Sequência de Bases , Técnicas de Cocultura , Primers do DNA , Camundongos , Camundongos Endogâmicos BALB C , Neuroglia/patologia , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Células Ganglionares da Retina/citologiaRESUMO
The repair of central nerves remains a major challenge in regenerative neurobiology. Regenerative guides possessing critical features such as cell adhesion, physical guiding and topical stimulation are needed. To generate such a guide, silk protein materials are prepared using electrospinning. The silk is selected for this study due to its biocompatibility and ability to be electrospun for the formation of aligned biofunctional nanofibers. The addition of Brain Derived Neurotrophic Factor (BDNF), Ciliary Neurotrophic Factor (CNTF) or both to the electrospun fibers enable enhanced function without impact to the structure or the surface morphology. Only a small fraction of the loaded growth factors is released over time allowing the fibers to continue to provide these factors to the cells for extended periods of time. The entrapped factors remain active and available to the cells as rat retinal ganglion cells (RGCs) exhibit longer axonal growth when in contact with the biofunctionalized fibers. Compare to non-functionalized fibers, the growth of neurites increased 2 fold on fibers containing BDNF, 2.5 fold with fibers containing CNTF and by almost 3-fold on fibers containing both factors. The results demonstrate the potential of aligned and functionalized electrospun silk fibers to promote nerve growth in the central nervous system, underlying the great potential of complex biomaterials in neuroregenerative strategies following axotomy and nerve crush traumas.
RESUMO
The dystrophin-associated-protein complex (DAPC) has been extensively characterized in the central nervous system where it is localized both in neuronal and glial cells. Few studies have characterized this complex in the neurohypophysis. To further study this complex in pituicytes, the resident astroglia of the neurophypophysis, we used adult pituicyte cultures and determined the expression and localization of dystrophins/utrophins and the DAPC by RT-PCR, western blotting and immunofluorescence. Our data show that the pituicytes express dystrophins, utrophins and several members of the DAPC including dystroglycans, δ-, γ-sarcoglycans, α-dystrobrevin-1 and α1-syntrophin. Double immunofluorescence analysis shows that laminin colocalizes with dystroglycan, suggesting that similarly to muscle and astrocytes, the DAPC interacts with the extracellular matrix in pituicytes. Collectively these findings show that dystrophins/utrophins and members of the DAPC are expressed in pituicytes where they may form multiprotein complexes and play a role in the retraction-reinsertion of pituicyte endfeet during specific physiological conditions.
Assuntos
Complexo de Proteínas Associadas Distrofina/metabolismo , Distrofina/metabolismo , Neuro-Hipófise/citologia , Isoformas de Proteínas/metabolismo , Utrofina/metabolismo , Animais , Células Cultivadas , Distrofina/genética , Complexo de Proteínas Associadas Distrofina/química , Complexo de Proteínas Associadas Distrofina/genética , Proteínas Associadas à Distrofina/genética , Proteínas Associadas à Distrofina/metabolismo , Perfilação da Expressão Gênica , Humanos , Laminina/genética , Laminina/metabolismo , Masculino , Neuro-Hipófise/química , Neuro-Hipófise/metabolismo , Isoformas de Proteínas/genética , Ratos , Ratos Wistar , Utrofina/genéticaRESUMO
Niemann-Pick type C (NPC) disease is an inherited lysosomal storage disease and caused by mutations in Npc1 or Npc2, which mediate cooperatively the egress of cholesterol from lysosomes. The disease entails progressive neurodegeneration, whose cause is poorly understood. Here, we report that Npc1 is distributed in distinct layers of the mouse retina and that its deficiency causes striking retinal degeneration in 2-month-old mice with signs of age-related maculopathies. This includes impaired visual function, accumulation of lipofuscin in the retinal pigment epithelium layer, degeneration of photoreceptor outer segments, disruption of synaptic layers and an increase in autophagy markers in the ganglion cell layer. Moreover, the lack of Npc1 results in the upregulation of proteins that mediate cellular cholesterol release in the retina. Our findings suggest that Npc1 is required for normal retinal function and that its absence may serve as model to study age-related degeneration of the retina.
Assuntos
Degeneração Macular/patologia , Proteínas/metabolismo , Retina/patologia , Animais , Autofagia , Colesterol/metabolismo , Eletrorretinografia , Peptídeos e Proteínas de Sinalização Intracelular , Degeneração Macular/genética , Degeneração Macular/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Proteína C1 de Niemann-Pick , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/metabolismo , Doença de Niemann-Pick Tipo C/patologia , Fagossomos/metabolismo , Fagossomos/ultraestrutura , Proteínas/genética , Retina/citologia , Retina/metabolismoRESUMO
Previous studies suggest that glial cells contribute to synaptogenesis in specific neurons from the postnatal CNS. Here, we studied whether this is true for Purkinje cells (PCs), which represent a unique neuronal cell type due to their large size, massive synaptic input, and high vulnerability. Using new glia-free cultures enriched in PCs from postnatal mice we show that these neurons survived and grew, but displayed only low levels of excitatory and inhibitory synaptic activity. Coculture with glial cells strongly enhanced the frequency and size of spontaneous and miniature excitatory synaptic currents as well as neurite growth and branching. Immunocytochemical staining for microtubule-associated protein 2- (MAP2-) positive neurites revealed impaired dendrite formation in PCs under glia-free conditions, which can explain the absence of synaptic activity. Glial signals strongly enhanced dendritogenesis in PCs and thus their ability to receive excitatory synaptic input from granule cells (GCs). The enhancement of dendrite formation was mimicked by glia-conditioned medium (GCM), whereas the increase in synaptic activity required physical presence of glia. This indicated that dendrite development is necessary but not sufficient for PCs to receive excitatory synaptic input and that synaptogenesis requires additional signals. The level of inhibitory synaptic activity was low even in cocultures due to a low incidence of inhibitory interneurons. Taken together, our results reinforce the idea that glial cells promote synaptogenesis in specific neuronal cell types.
Assuntos
Dendritos/fisiologia , Neuroglia/fisiologia , Células de Purkinje/citologia , Sinapses/fisiologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Cerebelo/citologia , Técnicas de Cocultura/métodos , Meios de Cultivo Condicionados/farmacologia , Dendritos/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Glutamato Descarboxilase/metabolismo , Complexo Antígeno L1 Leucocitário/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Associadas aos Microtúbulos/metabolismo , Neuroglia/química , Técnicas de Patch-Clamp/métodos , Sinapses/efeitos dos fármacos , Antígenos Thy-1/metabolismoRESUMO
The retina expresses several laminins in the outer plexiform layer (OPL), where they may provide an extracellular scaffold for synapse stabilization. Mice with a targeted deletion of the laminin ß2 gene (Lamb2) exhibit retinal disruptions: photoreceptor synapses in the OPL are disorganized and the retinal physiological response is attenuated. We hypothesize that laminins are required for proper trans-synaptic alignment. To test this, we compared the distribution, expression, association and modification of several pre- and post-synaptic elements in wild-type and Lamb2-null retinae. A potential laminin receptor, integrin α3, is at the presynaptic side of the wild-type OPL. Another potential laminin receptor, dystroglycan, is at the post-synaptic side of the wild-type OPL. Integrin α3 and dystroglycan can be co-immunoprecipitated with the laminin ß2 chain, demonstrating that they may bind laminins. In the absence of the laminin ß2 chain, the expression of many pre-synaptic components (bassoon, kinesin, among others) is relatively undisturbed although their spatial organization and anchoring to the membrane is disrupted. In contrast, in the Lamb2-null, ß-dystroglycan (ß-DG) expression is altered, co-localization of ß-DG with dystrophin and the glutamate receptor mGluR6 is disrupted, and the post-synaptic bipolar cell components mGluR6 and GPR179 become dissociated, suggesting that laminins mediate scaffolding of post-synaptic components. In addition, although pikachurin remains associated with ß-DG, pikachurin is no longer closely associated with mGluR6 or α-DG in the Lamb2-null. These data suggest that laminins act as links among pre- and post-synaptic laminin receptors and α-DG and pikachurin in the synaptic space to maintain proper trans-synaptic alignment.
Assuntos
Laminina/metabolismo , Células Fotorreceptoras/metabolismo , Receptores de Laminina/metabolismo , Sinapses/metabolismo , Animais , Camundongos , Camundongos KnockoutRESUMO
The regulation of cholesterol, an essential brain lipid, ensures proper neuronal development and function, as demonstrated by links between perturbations of cholesterol metabolism and neurodegenerative diseases, including Alzheimer's disease. The central nervous system (CNS) acquires cholesterol via de novo synthesis, where glial cells provide cholesterol to neurons. Both lipoproteins and lipoprotein receptors are key elements in this intercellular transport, where the latter recognize, bind and endocytose cholesterol containing glia-produced lipoproteins. CNS lipoprotein receptors are like those in the periphery, among which include the ApoB, E binding lipolysis stimulated lipoprotein receptor (LSR). LSR is a multimeric protein complex that has multiple isoforms including α and α', which are seen as a doublet at 68 kDa, and ß at 56 kDa. While complete inactivation of murine lsr gene is embryonic lethal, studies on lsr +/- mice revealed altered brain cholesterol distribution and cognitive functions. In the present study, LSR profiling in different CNS regions revealed regiospecific expression of LSR at both RNA and protein levels. At the RNA level, the hippocampus, hypothalamus, cerebellum, and olfactory bulb, all showed high levels of total lsr compared to whole brain tissues, whereas at the protein level, only the hypothalamus, olfactory bulb, and retina showed the highest levels of total LSR. Interestingly, major regional changes in LSR expression were observed in aged mice which suggests changes in cholesterol homeostasis in specific structures in the aging brain. Immunocytostaining of primary cultures of mature murine neurons and glial cells isolated from different CNS regions showed that LSR is expressed in both neurons and glial cells. However, lsr RNA expression in the cerebellum was predominantly higher in glial cells, which was confirmed by the immunocytostaining profile of cerebellar neurons and glia. Based on this observation, we would propose that LSR in glial cells may play a key role in glia-neuron cross talk, particularly in the feedback control of cholesterol synthesis to avoid cholesterol overload in neurons and to maintain proper functioning of the brain throughout life.
Assuntos
Envelhecimento/metabolismo , Encéfalo/metabolismo , Receptores de Lipoproteínas/metabolismo , Envelhecimento/genética , Animais , Encéfalo/anatomia & histologia , Colesterol/metabolismo , Homeostase , Humanos , Lipólise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuroglia/metabolismo , Neurônios/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Lipoproteínas/deficiência , Receptores de Lipoproteínas/genética , Distribuição Tecidual , TranscriptomaRESUMO
The implication of the cyanotoxin ß-N-methylamino-l-alanine (BMAA) in long-lasting neurodegenerative disorders is still a matter of controversy. It has been alleged that chronic ingestion of BMAA through the food chain could be a causative agent of amyotrophic lateral sclerosis (ALS) and several related pathologies including Parkinson syndrome. Both in vitro and in vivo studies of the BMAA mode of action have focused on different molecular targets, demonstrating its toxicity to neuronal cells, especially motoneurons, and linking it to human neurodegenerative diseases. Historically, the hypothesis of BMAA-induced excitotoxicity following the stimulation of glutamate receptors has been established. However, in this paradigm, most studies have shown acute, rather than chronic effects of BMAA. More recently, the interaction of this toxin with neuromelanin, a pigment present in the nervous system, has opened a new research perspective. The issues raised by this toxin are related to its kinetics of action, and its possible incorporation into cellular proteins. It appears that BMAA neurotoxic activity involves different targets through several mechanisms known to favour the development of neurodegenerative processes.
Assuntos
Diamino Aminoácidos/metabolismo , Doenças Neurodegenerativas/metabolismo , Animais , Toxinas de Cianobactérias , Humanos , Melaninas/metabolismo , Receptores de Glutamato/metabolismoRESUMO
Although a major risk factor for Alzheimer's disease (AD), the "aging" parameter is not systematically considered in preclinical validation of anti-AD drugs. To explore how aging affects neuronal reactivity to anti-AD agents, the ciliary neurotrophic factor (CNTF)-associated pathway was chosen as a model. Comparison of the neuroprotective properties of CNTF in 6- and 18-month old mice revealed that CNTF resistance in the older animals is associated with the exclusion of the CNTF-receptor subunits from rafts and their subsequent dispersion to non-raft cortical membrane domains. This age-dependent membrane remodeling prevented both the formation of active CNTF-receptor complexes and the activation of prosurvival STAT3 and ERK1/2 pathways, demonstrating that age-altered membranes impaired the reactivity of potential therapeutic targets. CNTF-receptor distribution and CNTF signaling responses were improved in older mice receiving dietary docosahexaenoic acid, with CNTF-receptor functionality being similar to those of younger mice, pointing toward dietary intervention as a promising adjuvant strategy to maintain functional neuronal membranes, thus allowing the associated receptors to respond appropriately to anti-AD agents.
Assuntos
Envelhecimento/genética , Envelhecimento/fisiologia , Encéfalo/citologia , Membrana Celular/fisiologia , Neurônios/citologia , Nootrópicos/uso terapêutico , Animais , Fator Neurotrófico Ciliar/fisiologia , Gorduras Insaturadas na Dieta , Ácidos Docosa-Hexaenoicos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Microdomínios da Membrana , Camundongos Endogâmicos C57BL , Receptor do Fator Neutrófico Ciliar/fisiologia , Fator de Transcrição STAT3/metabolismo , Transdução de SinaisRESUMO
Oligomeric amyloid-ß (Aß) peptide contributes to impaired synaptic connections and neurodegenerative processes, and as such, represents a primary therapeutic target for Alzheimer's disease (AD)-modifying approaches. However, the lack of efficacy of drugs that inhibit production of Aß demonstrates the need for a better characterization of its toxic effects, both on synaptic and neuronal function. Here, we used conditioned medium obtained from recombinant HEK-AßPP cells expressing the human amyloid-ß protein precursor (Aß-CM), to investigate Aß-induced neurotoxic and synaptotoxic effects. Characterization of Aß-CM revealed that it contained picomolar amounts of cell-secreted Aß in its soluble form. Incubation of primary cortical neurons with Aß-CM led to significant decreases in synaptic protein levels as compared to controls. This effect was no longer observed in neurons incubated with conditioned medium obtained from HEK-AßPP cells grown in presence of the γ-secretase inhibitor, Semagacestat or LY450139 (LY-CM). However, neurotoxic and pro-apoptotic effects of Aß-CM were only partially prevented using LY-CM, which could be explained by other deleterious compounds related to chronic oxidative stress that were released by HEK-AßPP cells. Indeed, full neuroprotection was observed in cells exposed to LY-CM by additional treatment with the antioxidant resveratrol, or with the pluripotent n-3 polyunsaturated fatty acid docosahexaenoic acid. Inhibition of Aß production appeared necessary but insufficient to prevent neurodegenerative effects associated with AD due to other neurotoxic compounds that could exert additional deleterious effects on neuronal function and survival. Therefore, association of various types of protective agents needs to be considered when developing strategies for AD treatment.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Análise de Variância , Animais , Azepinas/farmacologia , Caspase 3/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Meios de Cultivo Condicionados/farmacologia , Embrião de Mamíferos , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Fosfopiruvato Hidratase/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , TransfecçãoRESUMO
Lipids are the fundamental structural components of biological membranes. For a long time considered as simple barriers segregating aqueous compartments, membranes are now viewed as dynamic interfaces providing a molecular environment favorable to the activity of membrane-associated proteins. Interestingly, variations in membrane lipid composition, whether quantitative or qualitative, play a crucial role in regulation of membrane protein functionalities. Indeed, a variety of alterations in brain lipid composition have been associated with the processes of normal and pathological aging. Although not establishing a direct cause-and-effect relationship between these complex modifications in cerebral membranes and the process of cognitive decline, evidence shows that alterations in membrane lipid composition affect important physicochemical properties notably impacting the lateral organization of membranes, and thus microdomains. It has been suggested that preservation of microdomain functionality may represent an effective strategy for preventing or decelerating neuronal dysfunction and cerebral vulnerability, processes that are both aggravated by aging. The working hypothesis developed in this review proposes that preservation of membrane organization, for example, through nutritional supplementation of docosahexaenoic acid, could prevent disturbances in and preserve effective cerebral function.
Assuntos
Envelhecimento , Encéfalo/metabolismo , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/prevenção & controle , Encéfalo/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/administração & dosagem , Ácidos Docosa-Hexaenoicos/metabolismo , Humanos , Microdomínios da Membrana/química , Microdomínios da Membrana/efeitos dos fármacos , Modelos Biológicos , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/metabolismoRESUMO
The ectoderm gives rise not only to the skin but also to the entire CNS. This common embryonic lineage suggests that some molecular isoforms might serve analogous functions in both tissues. Indeed, not only are laminins important components of dermal adhesion mechanisms, but they also regulate some aspects of synaptic development in both the CNS and the PNS. In the skin, laminins are part of a hemidesmosome complex essential for basal keratinocyte adhesion that includes collagen XVII (BP180) and BPAG1 (dystonin/BP230). Here, we show that CNS neurons also express collagen XVII and BPAG1 and that these molecules are expressed in the adult and developing retina. In the retina, isoforms of collagen XVII and BPAG1 are colocalized with laminins at photoreceptor synapses and around photoreceptor outer segments; both molecules are expressed by rods, whereas cones express collagen XVII but not BPAG1. Moreover, biochemical data demonstrate that collagen XVII complexes with retinal laminins. We propose that collagen XVII and BPAG1 isoforms may help to anchor elements of the rod photoreceptor cytomatrix to the extracellular matrix.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Autoantígenos/metabolismo , Sistema Nervoso Central/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Colágenos não Fibrilares/metabolismo , Proteínas da Gravidez/metabolismo , Retina/metabolismo , Animais , Animais Recém-Nascidos , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/genética , Autoantígenos/química , Autoantígenos/genética , Northern Blotting/métodos , Western Blotting/métodos , Proteínas de Ligação ao Cálcio/metabolismo , Bovinos , Linhagem Celular , Colagenases/farmacologia , Imuno-Histoquímica/métodos , Imunoprecipitação/métodos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Cinesinas , Laminina/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Modelos Moleculares , Proteínas Musculares/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Colágenos não Fibrilares/química , Colágenos não Fibrilares/genética , Aglutinina de Amendoim/metabolismo , Proteínas da Gravidez/química , Proteínas da Gravidez/genética , RNA Mensageiro/metabolismo , Ratos , Retina/citologia , Retina/efeitos dos fármacos , Retina/crescimento & desenvolvimento , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo , Vimentina/metabolismo , Colágeno Tipo XVIIRESUMO
Cholesterol is a multifacetted molecule, which serves as essential membrane component, as cofactor for signaling molecules and as precursor for steroid hormones. Despite intense research on the diverse aspects of cholesterol, the role of cholesterol in the nervous system is still little understood. Our recent studies on primary cultures of highly purified neurons from the rodent central nervous system (CNS) suggest that during development, neurons reduce or even abandon cholesterol synthesis and import cholesterol from astrocytes via lipoproteins. Neurons use the glia-derived cholesterol to form numerous and efficient synapses. This provokes new ideas about the role of astrocytes as cholesterol producers and about the function of cholesterol in the CNS and its involvement in neurodegenerative diseases.
Assuntos
Sistema Nervoso Central/metabolismo , Colesterol/fisiologia , Lipídeos de Membrana/fisiologia , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Comunicação Celular , Colesterol/biossíntese , Humanos , Lipoproteínas/metabolismo , Camundongos , Doenças Neurodegenerativas/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Ratos , Transdução de SinaisRESUMO
Photodynamic therapy (PDT) has shown to be a promising technique to treat various forms of malignant neoplasia. The photodynamic eradication of the tumor cells is achieved by applying a photosensitizer either locally or systemically and following local activation through irradiation of the tumor mass with light of a specific wavelength after a certain time of incubation. Due to preferential accumulation of the photosensitizer in tumor cells, this procedure allows a selective inactivation of the malignant tumor while sparing the surrounding tissue to the greatest extent. These features and requirements make the PDT an attractive therapeutic option for the treatment of retinoblastoma, especially when surgical enucleation is a curative option. This extreme solution is still in use in case of tumours that are resistant to conventional chemotherapy or handled too late due to poor access to medical care in less advanced country. In this study we initially conducted in-vitro investigations of the new cationic water-soluble photo sensitizer tetrahydroporphyrin-tetratosylat (THPTS) regarding its photodynamic effect on human Rb-1 and Y79 retinoblastoma cells. We were able to show, that neither the incubation with THPTS without following illumination, nor the sole illumination showed a considerable effect on the proliferation of the retinoblastoma cells, whereas the incubation with THPTS combined with following illumination led to a maximal cytotoxic effect on the tumor cells. Moreover the phototoxicity was lower in normal primary cells from retinal pigmented epithelium demonstrating a higher phototoxic effect of THPTS in cancer cells than in this normal retinal cell type. The results at hand form an encouraging foundation for further in-vivo studies on the therapeutic potential of this promising photosensitizer for the eyeball and vision preserving as well as potentially curative therapy of retinoblastoma.