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Multiple sclerosis (MS) is an idiopathic demyelinating disease in which meningeal inflammation correlates with accelerated disease progression. The study of meningeal inflammation in MS has been limited because of constrained access to MS brain/spinal cord specimens and the lack of experimental models recapitulating progressive MS. Unlike induced models, a spontaneously occurring model would offer a unique opportunity to understand MS immunopathogenesis and provide a compelling framework for translational research. We propose granulomatous meningoencephalomyelitis (GME) as a natural model to study neuropathological aspects of MS. GME is an idiopathic, progressive neuroinflammatory disease of young dogs with a female bias. In the GME cases examined in this study, the meninges displayed focal and disseminated leptomeningeal enhancement on magnetic resonance imaging, which correlated with heavy leptomeningeal lymphocytic infiltration. These leptomeningeal infiltrates resembled tertiary lymphoid organs containing large B cell clusters that included few proliferating Ki67+ cells, plasma cells, follicular dendritic/reticular cells, and germinal center B cell-like cells. These B cell collections were confined in a specialized network of collagen fibers associated with the expression of the lympho-organogenic chemokines CXCL13 and CCL21. Although neuroparenchymal perivascular infiltrates contained B cells, they lacked the immune signature of aggregates in the meningeal compartment. Finally, meningeal B cell accumulation correlated significantly with cortical demyelination reflecting neuropathological similarities to MS. Hence, during chronic neuroinflammation, the meningeal microenvironment sustains B cell accumulation that is accompanied by underlying neuroparenchymal injury, indicating GME as a novel, naturally occurring model to study compartmentalized neuroinflammation and the associated pathology thought to contribute to progressive MS.
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Linfocitos B/inmunología , Modelos Animales de Enfermedad , Meninges/inmunología , Esclerosis Múltiple Crónica Progresiva/inmunología , Animales , Linfocitos B/patología , Perros , Meninges/patología , Esclerosis Múltiple Crónica Progresiva/patologíaRESUMEN
The concerted actions of the CNS and the immune system are essential to coordinating the outcome of neuroinflammatory responses. Yet, the precise mechanisms involved in this crosstalk and their contribution to the pathophysiology of neuroinflammatory diseases largely elude us. Here, we show that the CNS-endogenous hedgehog pathway, a signal triggered as part of the host response during the inflammatory phase of multiple sclerosis and experimental autoimmune encephalomyelitis, attenuates the pathogenicity of human and mouse effector CD4 T cells by regulating their production of inflammatory cytokines. Using a murine genetic model, in which the hedgehog signalling is compromised in CD4 T cells, we show that the hedgehog pathway acts on CD4 T cells to suppress the pathogenic hallmarks of autoimmune neuroinflammation, including demyelination and axonal damage, and thus mitigates the development of experimental autoimmune encephalomyelitis. Impairment of hedgehog signalling in CD4 T cells exacerbates brain-brainstem-cerebellum inflammation and leads to the development of atypical disease. Moreover, we present evidence that hedgehog signalling regulates the pathogenic profile of CD4 T cells by limiting their production of the inflammatory cytokines granulocyte-macrophage colony-stimulating factor and interferon-γ and by antagonizing their inflammatory program at the transcriptome level. Likewise, hedgehog signalling attenuates the inflammatory phenotype of human CD4 memory T cells. From a therapeutic point of view, our study underlines the potential of harnessing the hedgehog pathway to counteract ongoing excessive CNS inflammation, as systemic administration of a hedgehog agonist after disease onset effectively halts disease progression and significantly reduces neuroinflammation and the underlying neuropathology. We thus unveil a previously unrecognized role for the hedgehog pathway in regulating pathogenic inflammation within the CNS and propose to exploit its ability to modulate this neuroimmune network as a strategy to limit the progression of ongoing neuroinflammation.
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Linfocitos T CD4-Positivos/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Proteínas Hedgehog/inmunología , Inflamación/inmunología , Animales , Encéfalo/inmunología , Encéfalo/patología , Linfocitos T CD4-Positivos/patología , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Proteínas Hedgehog/metabolismo , Humanos , Inflamación/metabolismo , Ratones , Médula Espinal/inmunología , Médula Espinal/patologíaRESUMEN
Inter-alpha inhibitor proteins (IAIPs) are naturally occurring immunomodulatory molecules found in most tissues. We have reported ontogenic changes in the expression of IAIPs in brain during development in sheep and abundant expression of IAIPs in fetal and neonatal rodent brain in a variety of cellular types and brain regions. Although a few studies identified bikunin, light chain of IAIPs, in adult human brain, the presence of the complete endogenous IAIP protein complex has not been reported in human brain. In this study, we examined the immunohistochemical expression of endogenous IAIPs in human cerebral cortex from early in development through the neonatal period and in adults using well-preserved postmortem brains. We examined total, nuclear, and cytoplasmic staining of endogenous IAIPs and their expression in neurofilament light polypeptide-positive neurons and glial fibrillary acidic protein (GFAP)-positive astrocytes. IAIPs were ubiquitously detected for the first time in cerebral cortical cells at 24-26, 27-28, 29-36, and 37-40 weeks of gestation and in adults. Quantitative analyses revealed that IAIPs were predominately localized in the nucleus in all age groups, but cytoplasmic IAIP expression was more abundant in adult than in the younger ages. Immunoreactivity of IAIPs was expressed in neurons and astrocytes in all age groups. In addition, IAIP co-localization with GFAP-positive astrocytes was more abundant in adults than in the developing brain. We conclude that IAIPs exhibit ubiquitous expression, and co-localize with neurons and astrocytes in the developing and adult human brain suggesting a potential role for IAIPs in development and endogenous neuroprotection.
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alfa-Globulinas/metabolismo , Encéfalo/metabolismo , Neuronas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento/metabolismo , Astrocitos/metabolismo , Femenino , Feto/metabolismo , Edad Gestacional , Humanos , Lactante , Masculino , Persona de Mediana EdadRESUMEN
Microvascular accumulation and neuronal overproduction of amyloid-beta peptide (Abeta) are pathologic features of Alzheimer's disease (AD). In this study, we examined the receptor for advanced glycation endproducts (RAGE), a multi-ligand receptor found in both neurons and cerebral microvascular endothelia that binds Abeta. RAGE expression was assessed in aged controls (n = 6), patients with early AD-like pathology (n = 6), and severe, Braak V-VI AD (n = 6). Human hippocampi were stained with a specific polyclonal antibody directed against RAGE (Research Diagnostics, Flanders, NJ). Immunoreactivity was localized in both neurons and cerebral endothelial cells. Quantitative image-analyses were performed on grayscale images to assess the total surface area of endothelial RAGE immunoreaction product in cross sections of cerebral microvessels (5-20 microm). Confocal images were acquired for confirmation of RAGE immunoreactivity in both microvessels and neurons by coupling RAGE with CD-31 and neurofilament, respectively. A significant increase in endothelial RAGE immunoreactivity was found in severe Braak V-VI AD patients when compared to aged controls (p < 0.001), and when compared to patients with early AD pathology (p = 0.0125). In addition, a significant increase in endothelial RAGE immunoreactivity was witnessed when comparing aged controls having no reported AD pathology with patients having early AD-like pathology (p = 0.038). Our data suggest that microvascular RAGE levels increase in conjunction with the onset of AD, and continue to increase linearly as a function of AD pathologic severity (p < 0.0001).
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Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Hipocampo/metabolismo , Hipocampo/patología , Receptores Inmunológicos/metabolismo , Anciano , Anciano de 80 o más Años , Amiloide/metabolismo , Progresión de la Enfermedad , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Masculino , Microcirculación/fisiología , Persona de Mediana Edad , Receptor para Productos Finales de Glicación AvanzadaRESUMEN
BACKGROUND: In Alzheimer's disease, there are striking changes in CSF composition that relate to altered choroid plexus (CP) function. Studying CP tissue gene expression at the blood-cerebrospinal fluid barrier could provide further insight into the epithelial and stromal responses to neurodegenerative disease states. METHODS: Transcriptome-wide Affymetrix microarrays were used to determine disease-related changes in gene expression in human CP. RNA from post-mortem samples of the entire lateral ventricular choroid plexus was extracted from 6 healthy controls (Ctrl), 7 patients with advanced (Braak and Braak stage III-VI) Alzheimer's disease (AD), 4 with frontotemporal dementia (FTD) and 3 with Huntington's disease (HuD). Statistics and agglomerative clustering were accomplished with MathWorks, MatLab; and gene set annotations by comparing input sets to GeneGo ( http://www.genego.com ) and Ingenuity ( http://www.ingenuity.com ) pathway sets. Bonferroni-corrected hypergeometric p-values of < 0.1 were considered a significant overlap between sets. RESULTS: Pronounced differences in gene expression occurred in CP of advanced AD patients vs. Ctrls. Metabolic and immune-related pathways including acute phase response, cytokine, cell adhesion, interferons, and JAK-STAT as well as mTOR were significantly enriched among the genes upregulated. Methionine degradation, claudin-5 and protein translation genes were downregulated. Many gene expression changes in AD patients were observed in FTD and HuD (e.g., claudin-5, tight junction downregulation), but there were significant differences between the disease groups. In AD and HuD (but not FTD), several neuroimmune-modulating interferons were significantly enriched (e.g., in AD: IFI-TM1, IFN-AR1, IFN-AR2, and IFN-GR2). AD-associated expression changes, but not those in HuD and FTD, were enriched for upregulation of VEGF signaling and immune response proteins, e.g., interleukins. HuD and FTD patients distinctively displayed upregulated cadherin-mediated adhesion. CONCLUSIONS: Our transcript data for human CP tissue provides genomic and mechanistic insight for differential expression in AD vs. FTD vs. HuD for stromal as well as epithelial components. These choroidal transcriptome characterizations elucidate immune activation, tissue functional resiliency, and CSF metabolic homeostasis. The BCSFB undergoes harmful, but also important functional and adaptive changes in neurodegenerative diseases; accordingly, the enriched JAK-STAT and mTOR pathways, respectively, likely help the CP in adaptive transcription and epithelial repair and/or replacement when harmed by neurodegeneration pathophysiology. We anticipate that these precise CP translational data will facilitate pharmacologic/transgenic therapies to alleviate dementia.
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Enfermedad de Alzheimer/metabolismo , Plexo Coroideo/metabolismo , Demencia Frontotemporal/metabolismo , Enfermedad de Huntington/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Expresión Génica , Homeostasis/fisiología , Humanos , Masculino , Análisis por Micromatrices , Persona de Mediana Edad , TranscriptomaRESUMEN
Decreased clearance of potentially toxic metabolites, due to aging changes, likely plays a significant role in the accumulation of amyloid-beta (Aß) peptides and other macromolecules in the brain of the elderly and in the patients with Alzheimer's disease (AD). Aging is the single most important risk factor for AD development. Aß transport receptor proteins expressed at the blood-brain barrier are significantly altered with age: the efflux transporters lipoprotein receptor-related protein 1 and P-glycoprotein are reduced, whereas the influx transporter receptor for advanced glycation end products is increased. These receptors play an important role in maintaining brain biochemical homeostasis. We now report that, in a rat model of aging, gene transcription is altered in aging, as measured by Aß receptor gene messenger RNA (mRNA) at 3, 6, 9, 12, 15, 20, 30, and 36 months. Gene mRNA expression from isolated cerebral microvessels was measured by quantitative polymerase chain reaction. Lipoprotein receptor-related protein 1 and P-glycoprotein mRNA were significantly reduced in aging, and receptor for advanced glycation end products was increased, in parallel with the changes seen in receptor protein expression. Transcriptional changes appear to play a role in aging alterations in blood-brain barrier receptor expression and Aß accumulation.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Envejecimiento/genética , Envejecimiento/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Barrera Hematoencefálica/metabolismo , Expresión Génica/genética , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Transporte de Proteínas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor para Productos Finales de Glicación Avanzada/genética , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Animales , Masculino , Ratas Endogámicas F344 , Transcripción GenéticaRESUMEN
Expression of the orphan C2orf40 gene is associated with the aggregation of the neurofibrillary tangle-protein tau in transgenic mice, tumor suppression, the induction of senescence in CNS, and the activation of microglia and peripheral mononuclear leukocytes. This gene also encodes several secreted pro- and anti-inflammatory neuropeptide-like cytokines, suggesting they might be implicated in the inflammatory component(s) of Alzheimer's disease (AD). Accordingly, we evaluated human AD and control brains for expression changes by RT-qPCR, Western blot, and histological changes by immunolabeling. RT-qPCR demonstrated increased cortical gene expression in AD. The molecular form of Ecrg4 detected in cortex was 8-10 kDa, which was shown previously to interact with the innate immunity receptor complex. Immunocytochemical studies showed intensely stained microglia and intravascular blood-borne monocytes within cerebral cortical white matter of AD patients. Staining was diminished within cortical neurons, except for prominent staining in neurofibrillary tangles. Choroid plexuses showed a decreasing trend. These findings support our hypothesis that c2orf40 participates in the neuroimmune response in AD.
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P-glycoprotein (P-gp), part of the blood-brain barrier, limits drug access to the brain and is the target for therapies designed to improve drug penetration. P-gp also extrudes brain amyloid-beta (Aß). Accumulation of Aß is a hallmark of Alzheimer's disease (AD). Aß accumulates in normal aging and in AD primarily due to decreased Aß clearance. This is a preliminary report on the relative protein and messenger RNA expression of P-gp in human brains, ages 20-100 years, including AD subjects. In these preliminary studies, cortical endothelial P-gp expression decreased in AD compared with controls (p < 0.001). Trends in P-gp expression in human aging are similar to aging rats. Microvessel P-gp messenger RNA remained unchanged with aging and AD. Aß plaques were found in 42.8% of normal subjects (54.5% of those older than 50 years). A qualitative analysis showed that P-gp expression is lower than the group mean in subjects older than 75 years but increased if younger. Decreased P-gp expression may be related to Aß plaques in aging and AD. Downregulating P-gp to allow pharmaceuticals into the central nervous system may increase Aß accumulation.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Envejecimiento/patología , Enfermedad de Alzheimer/patología , Amiloide/metabolismo , Encéfalo/metabolismo , Regulación de la Expresión Génica/fisiología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Péptidos beta-Amiloides/metabolismo , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Encéfalo/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fragmentos de Péptidos/metabolismo , Escalas de Valoración Psiquiátrica , ARN Mensajero/metabolismo , Adulto JovenRESUMEN
BACKGROUND: Normal pressure hydrocephalus (NPH) is most common in the elderly and has a high co-morbidity with Alzheimer's disease (AD) and cerebrovascular disease (CVD). To understand the relationship between NPH, AD and CVD, we investigated how chronic hydrocephalus impacts brain amyloid-beta peptide (Aß) accumulation and vascular pathology in an AD transgenic rodent model. Previously we showed that the altered CSF physiology produced by kaolin-hydrocephalus in older wild-type Sprague-Dawley rats increased Aß and hyperphosphorylated Tau (Silverberg et. al. Brain Res. 2010, 1317:286-296). We postulated that hydrocephalus would similarly affect an AD rat model. METHODS: Thirty-five transgenic rats (tgAPP21) that express high levels of human APP and naturally overproduce Aß40 were used. Six- (n = 7) and twelve-month-old (n = 9) rats had hydrocephalus induced by cisternal kaolin injection. We analyzed Aß burden (Aß40, Aß42 and oligomeric Aß) and vascular integrity (Masson trichrome and Verhoeff-Van Gieson) by immunohistochemistry and chemical staining at 10 weeks (n = 8) and 6 months (n = 5) post hydrocephalus induction. We also analyzed whether the vascular pathology seen in tgAPP21 rats, which develop amyloid angiopathy, was accelerated by hydrocephalus. Age-matched naïve and sham-operated tgAPP21 rats served as controls (n = 19). RESULTS: In hydrocephalic tgAPP21 rats, compared to naïve and sham-operated controls, there was increased Aß 40 and oligomeric Aß in hippocampal and cortical neurons at 10 weeks and 6 months post-hydrocephalus induction. No dense-core amyloid plaques were seen, but diffuse Aß immunoreactivity was evident in neurons. Vascular pathology was accelerated by the induction of hydrocephalus compared to controls. In the six-month-old rats, subtle degenerative changes were noted in vessel walls at 10 weeks post-kaolin, whereas at six months post-kaolin and in the 12-month-old hydrocephalic rats more pronounced amyloid angiopathic changes were seen, with frequent large areas of infarction noted. CONCLUSIONS: Kaolin-hydrocephalus can accelerate intraneuronal Aß40 accumulation and vascular pathology in tgAPP21 rats. In addition, disrupted CSF production and reduced CSF turnover results in impaired Aß clearance and accelerated vascular pathology in chronic hydrocephalus. The high co-morbidity seen in NPH, AD and CVD is likely not to be an age-related coincidence, but rather a convergence of pathologies related to diminished CSF clearance.
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The goals of this research were to describe age-related changes in brain biochemistry and behavior, and the relationships between them. The chronological ages of greatest change are particularly important for targeting interventions. In this experiment, 36 Fischer 344/Brown-Norway rats (3, 12, 20, and 30 months old) were trained in lever boxes on temporal discrimination tasks. The greatest response rate decrease and response pattern change occurred between 12 and 20 months. The biochemical results showed that amyloid-beta peptides (Aß40 and Aß42) increased with age. The endothelial expression of the Aß influx transporter (RAGE) also increased, and the expression of Aß efflux transporter (LPR-1) decreased, with age. The greatest change in the biochemical measures also were between 12 and 20 months. Twenty additional rats were analyzed for stem cell proliferation, and neurogenesis decreased with age, particularly between about 12 and 20 months. These early changes in brain, biochemistry, and behavior provide opportunity for new therapies or prophylaxis.
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Envejecimiento/metabolismo , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Actividad Motora/fisiología , Neurogénesis , Fragmentos de Péptidos/metabolismo , Factores de Edad , Animales , Encéfalo/fisiología , Condicionamiento Operante , Discriminación en Psicología/fisiología , Masculino , Proteínas de Transporte de Membrana/metabolismo , Ratas , Ratas Endogámicas F344RESUMEN
BACKGROUND: Amyloid accumulation in the brain parenchyma is a hallmark of Alzheimer's disease (AD) and is seen in normal aging. Alterations in cerebrospinal fluid (CSF) dynamics are also associated with normal aging and AD. This study analyzed CSF volume, production and turnover rate in relation to amyloid-beta peptide (Aß) accumulation in the aging rat brain. METHODS: Aging Fischer 344/Brown-Norway hybrid rats at 3, 12, 20, and 30 months were studied. CSF production was measured by ventriculo-cisternal perfusion with blue dextran in artificial CSF; CSF volume by MRI; and CSF turnover rate by dividing the CSF production rate by the volume of the CSF space. Aß40 and Aß42 concentrations in the cortex and hippocampus were measured by ELISA. RESULTS: There was a significant linear increase in total cranial CSF volume with age: 3-20 months (p < 0.01); 3-30 months (p < 0.001). CSF production rate increased from 3-12 months (p < 0.01) and decreased from 12-30 months (p < 0.05). CSF turnover showed an initial increase from 3 months (9.40 day-1) to 12 months (11.30 day-1) and then a decrease to 20 months (10.23 day-1) and 30 months (6.62 day-1). Aß40 and Aß42 concentrations in brain increased from 3-30 months (p < 0.001). Both Aß42 and Aß40 concentrations approached a steady state level by 30 months. CONCLUSIONS: In young rats there is no correlation between CSF turnover and Aß brain concentrations. After 12 months, CSF turnover decreases as brain Aß continues to accumulate. This decrease in CSF turnover rate may be one of several clearance pathway alterations that influence age-related accumulation of brain amyloid.
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Encapsulated human mesenchymal stem cells(MSC) are studied in a double transgenic mouse model of Alzheimer's disease (AD) after intraventricular implantation at 3 months of age. Abeta 40/42 deposition, and glial (GFAP) and microglial (CD11b) immunoreactivity were investigated 2 months after transplantation of either native MSC or MSC transfected with glucagon-like peptide-1 (GLP-1). CD11b immunostaining in the frontal lobes was significantly decreased in the GLP-1 MSC group compared to the untreated controls. Also, the plaque associated GFAP immunoreactivity was only observed in one of four animals in the GLP-1 MSC group. Abeta 40 whole brain ELISA was decreased in the MSC group: 86.06±5.2 pg/ml (untreated control) vs. 78.67±11.2 pg/ml (GLP-1 MSC group) vs.70.9±11.1 pg/ml (MSC group, p<0.05). Intraventricular transplantation of native and GLP-1 transfected MSC has been shown effective. Decreased amyloid deposition or suppression of glial and microglial responses were observed. However, encapsulation of MSC may alter their biological activity.
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Enfermedad de Alzheimer/terapia , Terapia Genética/métodos , Péptido 1 Similar al Glucagón/genética , Trasplante de Células Madre Mesenquimatosas/métodos , Alginatos/farmacología , Péptidos beta-Amiloides/análisis , Péptidos beta-Amiloides/metabolismo , Animales , Materiales Biocompatibles/farmacología , Cápsulas , Línea Celular , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Ácido Glucurónico/farmacología , Ácidos Hexurónicos/farmacología , Humanos , Masculino , Células Madre Mesenquimatosas , Ratones , Ratones Transgénicos , TransfecciónRESUMEN
BACKGROUND: Neuropeptides may have considerable potential in the treatment of acute and chronic neurological diseases. Encapsulated genetically engineered cells have been suggested as a means for sustained local delivery of such peptides to the brain. In our experiments, we studied human mesenchymal stem cells which were transfected to produce glucagon-like peptide-1 (GLP-1). METHODS: Cells were packed in a water-permeable mesh bag containing 400 polymeric microcapsules, each containing 3000 cells. The mesh bags were either transplanted into the subdural space, into the brain parenchyma or into the cerebral ventricles of the cat brain. Mesh bags were explanted after two weeks, and cell viability, as well as GLP-1 concentration in the cerebrospinal fluid (CSF), was measured. RESULTS: Viability of cells did not significantly differ between the three implantation sites. However, CSF concentration of GLP-1 was significantly elevated only after ventricular transplantation with a maximum concentration of 73 pM (binding constant = 70 pM). CONCLUSIONS: This study showed that ventricular cell-based delivery of soluble factors has the capability to achieve concentrations in the CSF which may become pharmacologically active. Despite the controversy about the pharmacokinetic limitations of ventricular drug delivery, there might be a niche in this for encapsulated cell biodelivery of soluble, highly biologically-effective neuropeptides of low molecular weight like GLP-1.
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BACKGROUND: Age is the major risk factor for many neurodegenerative diseases, including Alzheimer's disease (AD). There is an accumulation of amyloid-beta peptides (Aß) in both the AD brain and the normal aging brain. Clearance of Aß from the brain occurs via active transport at the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB). With increasing age, the expression of the Aß efflux transporters is decreased and the Aß influx transporter expression is increased at the BBB, adding to the amyloid burden in the brain. Expression of the Aß transporters at the choroid plexus (CP) epithelium as a function of aging was the subject of this study. METHODS: This project investigated the changes in expression of the Aß transporters, the low density lipoprotein receptor-related protein-1 (LRP-1), P-glycoprotein (P-gp), LRP-2 (megalin) and the receptor for advanced glycation end-products (RAGE) at the BCSFB in Brown-Norway/Fischer rats at ages 3, 6, 9, 12, 20, 30 and 36 months, using real time RT-PCR to measure transporter mRNA expression, and immunohistochemistry (IHC) to measure transporter protein in isolated rat CP. RESULTS: There was an increase in the transcription of the Aß efflux transporters, LRP-1 and P-gp, no change in RAGE expression and a decrease in LRP-2, the CP epithelium influx transporter, at the BCSFB with aging. Decreased Aß42 concentration in the CP, as measured by quantitative IHC, was associated with these Aß transporter alterations. CONCLUSIONS: Age-dependent alterations in the CP Aß transporters are associated with a decrease in Aß42 accumulation in the CP, and are reciprocal to the changes seen in these transporters at the BBB, suggesting a possible compensatory role for the BCSFB in Aß clearance in aging.
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Conditions that compromise the blood-brain barrier (BBB) have been increasingly implicated in the pathogenesis of Alzheimer disease (AD). AGRIN is a heparan sulfate proteoglycan found abundantly in basement membranes of the cerebral vasculature, where it has been proposed to serve a functional role in the BBB. Furthermore, AGRIN is the major heparan sulfate proteoglycan associated with amyloid plaques in AD brains. To examine the relationship of AGRIN, the BBB, and AD-related pathologies, we generated mice in which the Agrn gene was deleted from either endothelial cells or neurons using gene targeting or was overexpressed using a genomic transgene construct. These mice were combined with a transgenic model of AD that over expresses disease-associated forms of amyloid precursor protein and presenilin 1. In mice lacking endothelial cell expression of Agrn, the BBB remained intact but aquaporin 4 levels were reduced, indicating that the loss of AGRIN affects BBB-associated components. This change in Agrn resulted in an increase in ß-amyloid (Aß) in the brain. Conversely, overexpression of Agrn decreased Aß deposition, whereas elimination of Agrn from neurons did not change Aß levels. These results indicate that AGRIN is important for maintaining BBB composition and that changes in Agrn expression (particularly vessel-associated AGRIN) influence Aß homeostasis in mouse models of AD.
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Agrina/metabolismo , Péptidos beta-Amiloides/metabolismo , Acuaporina 4/metabolismo , Encéfalo/metabolismo , Agrina/genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Encéfalo/patología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Ratones , Ratones Transgénicos , Neuronas/metabolismo , Neuronas/patología , Placa Amiloide/metabolismo , Placa Amiloide/patología , Receptores Colinérgicos/metabolismoRESUMEN
By virtue of its ability to regulate the composition of cerebrospinal fluid (CSF), the choroid plexus (CP) is ideally suited to instigate a rapid response to traumatic brain injury (TBI) by producing growth regulatory proteins. For example, Esophageal Cancer Related Gene-4 (Ecrg4) is a tumor suppressor gene that encodes a hormone-like peptide called augurin that is present in large concentrations in CP epithelia (CPe). Because augurin is thought to regulate senescence, neuroprogenitor cell growth and differentiation in the CNS, we evaluated the kinetics of Ecrg4 expression and augurin immunoreactivity in CPe after CNS injury. Adult rats were injured with a penetrating cortical lesion and alterations in augurin immunoreactivity were examined by immunohistochemistry. Ecrg4 gene expression was characterized by in situ hybridization. Cell surface augurin was identified histologically by confocal microscopy and biochemically by sub-cellular fractionation. Both Ecrg4 gene expression and augurin protein levels were decreased 24-72 hrs post-injury but restored to uninjured levels by day 7 post-injury. Protein staining in the supraoptic nucleus of the hypothalamus, used as a control brain region, did not show a decrease of auguin immunoreactivity. Ecrg4 gene expression localized to CPe cells, and augurin protein to the CPe ventricular face. Extracellular cell surface tethering of 14 kDa augurin was confirmed by cell surface fractionation of primary human CPe cells in vitro while a 6-8 kDa fragment of augurin was detected in conditioned media, indicating release from the cell surface by proteolytic processing. In rat CSF however, 14 kDa augurin was detected. We hypothesize the initial release and proteolytic processing of augurin participates in the activation phase of injury while sustained Ecrg4 down-regulation is dysinhibitory during the proliferative phase. Accordingly, augurin would play a constitutive inhibitory function in normal CNS while down regulation of Ecrg4 gene expression in injury, like in cancer, dysinhibits proliferation.
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Lesiones Encefálicas/metabolismo , Plexo Coroideo/metabolismo , Proteínas de Neoplasias/metabolismo , Animales , Lesiones Encefálicas/genética , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Hibridación in Situ , Masculino , Microscopía Confocal , Proteínas de Neoplasias/genética , Ratas , Ratas Sprague-Dawley , Proteínas Supresoras de TumorRESUMEN
BACKGROUND: The content and composition of cerebrospinal fluid (CSF) is determined in large part by the choroid plexus (CP) and specifically, a specialized epithelial cell (CPe) layer that responds to, synthesizes, and transports peptide hormones into and out of CSF. Together with ventricular ependymal cells, these CPe relay homeostatic signals throughout the central nervous system (CNS) and regulate CSF hydrodynamics. One new candidate signal is augurin, a newly recognized 14 kDa protein that is encoded by esophageal cancer related gene-4 (Ecrg4), a putative tumor suppressor gene whose presence and function in normal tissues remains unexplored and enigmatic. The aim of this study was to explore whether Ecrg4 and its product augurin, can be implicated in CNS development and the response to CNS injury. METHODS: Ecrg4 gene expression in CNS and peripheral tissues was studied by in situ hybridization and quantitative RT-PCR. Augurin, the protein encoded by Ecrg4, was detected by immunoblotting, immunohistochemistry and ELISA. The biological consequence of augurin over-expression was studied in a cortical stab model of rat CNS injury by intra-cerebro-ventricular injection of an adenovirus vector containing the Ecrg4 cDNA. The biological consequences of reduced augurin expression were evaluated by characterizing the CNS phenotype caused by Ecrg4 gene knockdown in developing zebrafish embryos. RESULTS: Gene expression and immunohistochemical analyses revealed that, the CP is a major source of Ecrg4 in the CNS and that Ecrg4 mRNA is predominantly localized to choroid plexus epithelial (CPe), ventricular and central canal cells of the spinal cord. After a stab injury into the brain however, both augurin staining and Ecrg4 gene expression decreased precipitously. If the loss of augurin was circumvented by over-expressing Ecrg4 in vivo, BrdU incorporation by cells in the subependymal zone decreased. Inversely, gene knockdown of Ecrg4 in developing zebrafish embryos caused increased proliferation of GFAP-positive cells and induced a dose-dependent hydrocephalus-like phenotype that could be rescued by co-injection of antisense morpholinos with Ecrg4 mRNA. CONCLUSION: An unusually elevated expression of the Ecrg4 gene in the CP implies that its product, augurin, plays a role in CP-CSF-CNS function. The results are all consistent with a model whereby an injury-induced decrease in augurin dysinhibits target cells at the ependymal-subependymal interface. We speculate that the ability of CP and ependymal epithelium to alter the progenitor cell response to CNS injury may be mediated, in part by Ecrg4. If so, the canonic control of its promoter by DNA methylation may implicate epigenetic mechanisms in neuroprogenitor fate and function in the CNS.
RESUMEN
Aging is the most important single risk factor for developing Alzheimer disease. We measured amyloid-beta peptide (Abeta) levels in rat cerebral cortex and hippocampus during normal aging of Brown-Norway/Fischer rats. Amyloid-beta accumulation was associated with expression of the Abeta influx transporter, the receptor for advanced glycation end-products (RAGEs) at the blood-brain barrier. Rats at selected ages from 3 to 36 months were analyzed by 1) immunohistochemistry for amyloid deposition and quantitative microvessel surface area RAGE expression, 2) ELISA for cortical Abeta40 and Abeta42 concentrations, and 3) Western blotting of microvessel proteins for RAGE expression. Immunohistochemistry showed increasing accumulation of brain Abeta with aging. By ELISA analysis, both Abeta40 and Abeta42 concentrations in cortical homogenates rose sharply from 9 to 12 months. The Abeta42 continued to rise up to age 30 months, whereas Abeta40 stabilized after 12 months. The expression of RAGE initially decreased between 3 and 12 months but then increased between 12 and 34 months by immunohistochemistry. On immunoblotting, RAGE decreased up to 9 months and then progressively increased up to 36 months. These data indicate an association between amyloid and microvessel RAGE during aging. An increase in capillary RAGE expression seems to play a role in the later Abeta accumulation but not in the initial increase.
Asunto(s)
Envejecimiento/fisiología , Péptidos beta-Amiloides/metabolismo , Amiloide/metabolismo , Barrera Hematoencefálica/metabolismo , Regulación de la Expresión Génica/fisiología , Receptores Inmunológicos/metabolismo , Factores de Edad , Animales , Encéfalo/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Masculino , Proteínas del Tejido Nervioso/metabolismo , Fragmentos de Péptidos/metabolismo , Ratas , Ratas Endogámicas F344 , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/genéticaRESUMEN
AD pathology is often seen in cortical biopsies of NPH patients. It remains unclear whether these findings are coincidental or causally related. In an aged animal model of NPH, we quantify Abeta and pTau accumulation and describe its temporal and spatial distribution. One-year-old male Sprague-Dawley rats had hydrocephalus induced by cisternal kaolin injection. Immunohistochemistry (IMHC) for AbetaPP, Abeta40, Abeta42 and pTau (epitope pT231) and ELISA for Abeta40, Abeta42 and pT231 were performed on controls and after 2, 6 and 10 weeks of hydrocephalus. Rats had double-label fluorescence IMHC for localization of Abeta42 and pT231. IMHC showed no change in neuronal AbetaPP expression following hydrocephalus. Abeta42 appeared earliest in CSF clearance pathways, p<0.05, and also showed significant rises in perivascular spaces and in cortical parenchyma. Mean ELISA values for Abeta40 and Abeta42 increased three- to four-fold in hydrocephalic rats at 6 and 10 weeks. Abeta40 increased between 2 and 6 weeks (p=0.0001), and remained stable at 10 (p=0.0002); whereas Abeta42 was elevated at 2 weeks (p<0.04) and remained at 6 (p=0.015). PTau at 6 and 10 weeks showed AD-like increased neuronal somatic staining and loss of dendritic staining. ELISA demonstrated increased pT231 in hydrocephalic rats at 10 weeks (p<0.0002). Double-label fluorescence for Abeta42 and pT231 revealed intraneuronal co-localization. Hydrocephalus in the elderly rat, therefore, can induce both Abeta and pTau accumulation. As distinct from brain injury models, no increase in AbetaPP expression was demonstrated. Rather, altered CSF dynamics appears to impair Abeta clearance in this NPH model.
Asunto(s)
Envejecimiento/metabolismo , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Hidrocéfalo Normotenso/metabolismo , Proteínas tau/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Corteza Cerebral/metabolismo , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Hidrocéfalo Normotenso/inducido químicamente , Caolín , Masculino , Neuronas/metabolismo , Fragmentos de Péptidos/metabolismo , Fosforilación , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Reduced clearance of amyloid ß peptides (Aß) across the blood-brain barrier contributes to amyloid accumulation in Alzheimer disease. Amyloid ß efflux transport is via the endothelial low-density lipoprotein receptor-related protein 1 (LRP-1) and P-glycoprotein (P-gp), whereas Aß influx transport is via the receptor for advanced glycation end products. Because age is the major risk factor for developing Alzheimer disease, we measured LRP-1 and P-gp expression and associated transporter expression with Aß accumulation in aging rats. Quantitative LRP-1 and P-gp microvessel expression was measured by immunohistochemistry (IHC); LRP-1 and P-gp expression were assessed in microvessel isolates by Western blotting. There was an age-dependent loss of capillary LRP-1 across all ages (3-36 months) by IHC (linear trend p = 0.0004) and between 3 and 20 months by Western blotting (linear trend p < 0.0001). There was a late (30-36 months) P-gp expression loss by IHC (p < 0.05) and Western blotting (p = 0.0112). Loss of LRP-1 correlated with Aß42 accumulation (p = 0.0121) and very nearly with Aß40 (p = 0.0599) across all ages. Expression of LRP-1 correlated negatively with the expression of receptor for advanced glycation end products (p < 0.0004). These data indicate that alterations in LRP-1 and P-gp expression seem to contribute progressively to Aß accumulation in aging.