RESUMEN
The present study was designed to characterize transduction of non-human primate brain and spinal cord with a modified adeno-associated virus serotype 2, incapable of binding to the heparan sulfate proteoglycan receptor, referred to as AAV2-HBKO. AAV2-HBKO was infused into the thalamus, intracerebroventricularly or via a combination of both intracerebroventricular and thalamic delivery. Thalamic injection of this modified vector encoding GFP resulted in widespread CNS transduction that included neurons in deep cortical layers, deep cerebellar nuclei, several subcortical regions, and motor neuron transduction in the spinal cord indicative of robust bidirectional axonal transport. Intracerebroventricular delivery similarly resulted in widespread cortical transduction, with one striking distinction that oligodendrocytes within superficial layers of the cortex were the primary cell type transduced. Robust motor neuron transduction was also observed in all levels of the spinal cord. The combination of thalamic and intracerebroventricular delivery resulted in transduction of oligodendrocytes in superficial cortical layers and neurons in deeper cortical layers. Several subcortical regions were also transduced. Our data demonstrate that AAV2-HBKO is a powerful vector for the potential treatment of a wide number of neurological disorders, and highlight that delivery route can significantly impact cellular tropism and pattern of CNS transduction.
Asunto(s)
Terapia Genética , Vectores Genéticos/efectos adversos , Neuronas/efectos de los fármacos , Parvovirinae/genética , Médula Espinal/efectos de los fármacos , Animales , Transporte Axonal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/patología , Proteínas de la Cápside/administración & dosificación , Proteínas de la Cápside/genética , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/patología , Dependovirus , Modelos Animales de Enfermedad , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Proteoglicanos de Heparán Sulfato/administración & dosificación , Proteoglicanos de Heparán Sulfato/genética , Humanos , Infusiones Intraventriculares , Neuronas Motoras/efectos de los fármacos , Neuronas/patología , Primates , Médula Espinal/patología , Tálamo/efectos de los fármacosRESUMEN
Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder in which the aggregation and deposition of amyloid-ß (Aß) peptides in the brain are central to its pathogenesis. In healthy brains, Aß is effectively metabolized with little accumulation. Cellular uptake and subsequent degradation of Aß is one of the major pathways for its clearance in the brain. Increasing evidence has demonstrated significant roles for the low-density lipoprotein receptor-related protein 1 (LRP1) in the metabolism of Aß in neurons, glia cells, and along the brain vasculatures. Heparan sulfate proteoglycan (HSPG) has also been implicated in several pathogenic features of AD, including its colocalization with amyloid plaques. Here, we demonstrate that HSPG and LRP1 cooperatively mediate cellular Aß uptake. Fluorescence-activated cell sorter and confocal microscopy revealed that knockdown of LRP1 suppresses Aß uptake, whereas overexpression of LRP1 enhances this process in neuronal cells. Heparin, which antagonizes HSPG, significantly inhibited cellular Aß uptake. Importantly, treatment with heparin or heparinase blocked LRP1-mediated cellular uptake of Aß. We further showed that HSPG is more important for the binding of Aß to the cell surface than LRP1. The critical roles of HSPG in cellular Aß binding and uptake were confirmed in Chinese hamster ovary cells genetically deficient in HSPG. We also showed that heparin and a neutralizing antibody to LRP1 suppressed Aß uptake in primary neurons. Our findings demonstrate that LRP1 and HSPG function in a cooperative manner to mediate cellular Aß uptake and define a major pathway through which Aß gains entry to neuronal cells.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Fibroblastos/metabolismo , Proteoglicanos de Heparán Sulfato/metabolismo , Neuronas/metabolismo , Fragmentos de Péptidos/metabolismo , Receptores de LDL/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Transporte Biológico , Western Blotting , Células CHO , Línea Celular , Células Cultivadas , Cricetinae , Cricetulus , Embrión de Mamíferos , Endocitosis/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Femenino , Fibroblastos/efectos de los fármacos , Citometría de Flujo , Técnicas de Silenciamiento del Gen , Proteoglicanos de Heparán Sulfato/antagonistas & inhibidores , Proteoglicanos de Heparán Sulfato/deficiencia , Proteoglicanos de Heparán Sulfato/genética , Heparina/farmacología , Hipotálamo/citología , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Ratones , Microscopía Confocal , Neuronas/efectos de los fármacos , Embarazo , ARN Interferente Pequeño , Receptores de LDL/genética , Transfección , Proteínas Supresoras de Tumor/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
OBJECTIVE: To investigate the expression of HSPG in glomerular base membrane of adriamycin-induced nephropathy (AN) rats, and the effect of Qufengtongluo recipe on HSPG mRNA expression and proteinuria in AN rats. METHODS: One hundred forty rats were used in this study, including 32 rats in normal control group. AN was induced in the left rats by a single tail intravenous injection of adriamycin. Three weeks later, 90 AN rats were randomly divided into five groups; the nephropathy group (B, n=18), the Qufeng group (C, n=18), Qufeng and prednisone group (D, n=18), prednisone group(E,n=18) and benazepri group (F, n= 18). The rats in these five groups were treated with different combination of Qufeng recipe and prednisone. In each group, renal tissue samples were collected at week 3 and 7. The distribution, expression of HSPG was examined by indirect immunofluorescence, and semi-quantity RT-PCR, respectively. RESULTS: (1) In AN rats, the diffuse fusion and effacement of foot processes were observed when model established. (2) Compared with nephropathy group, the average fluorescence intensity of HSPG dramatically increased in Qufeng group and prednisone group (P < 0.01), similarly, it also increased in D and F groups (P < 0.01). (3) Compared with nephropathy group, the expression of HSPG mRNA was significantly up-regulated in other groups. (P < 0.01), especially in C and F groups. There was significant negative correlation between the expression of HSPG and quantity of 24-hour proteinuria. CONCLUSION: The abnormal expression of HSPG and their altered distributions may be an important molecular mechanism that leads to the occurrence and development of proteinuria in AN rats. The effect of Qufengtongluo recipe on nephrotic syndrome might be related to the alteration of HSPG expression and distribution in glomerulus.
Asunto(s)
Doxorrubicina , Medicamentos Herbarios Chinos/farmacología , Membrana Basal Glomerular/metabolismo , Proteoglicanos de Heparán Sulfato/metabolismo , Enfermedades Renales/inducido químicamente , Animales , Medicamentos Herbarios Chinos/uso terapéutico , Proteoglicanos de Heparán Sulfato/genética , Enfermedades Renales/metabolismo , Masculino , Fitoterapia , Prednisona/uso terapéutico , Proteinuria/inducido químicamente , Proteinuria/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Schwartz-Jampel syndrome (SJS) is an autosomal-recessive condition characterized by muscle stiffness and chondrodysplasia. It is due to loss-of-function hypomorphic mutations in the HSPG2 gene that encodes for perlecan, a proteoglycan secreted into the basement membrane. The origin of muscle stiffness in SJS is debated. To resolve this issue, we performed an electrophysiological investigation of an SJS mouse model with a missense mutation in the HSPG2 gene. Compound muscle action potential amplitudes, distal motor latencies, repetitive nerve stimulation tests, and sensory nerve conduction velocities of SJS mice were normal. On electromyography (EMG), neuromyotonic discharges, that is, bursts of motor unit action potentials firing at high rates (120-300 HZ), were constantly observed in SJS mice in all muscles, except in the diaphragm. Neuromyotonic discharges were not influenced by general anesthesia and disappeared with curare administration. They persisted after complete motor nerve section, terminating only with Wallerian degeneration. These results demonstrate that perlecan deficiency in SJS provokes a neuromyotonic syndrome. The findings further suggest a distal axonal localization of the generator of neuromyotonic discharges. SJS should now be considered as an inherited disorder with peripheral nerve hyperexcitability.
Asunto(s)
Fibras Musculares Esqueléticas/fisiología , Osteocondrodisplasias/patología , Osteocondrodisplasias/fisiopatología , Nervios Periféricos/fisiopatología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Fenómenos Biofísicos , Curare/farmacología , Modelos Animales de Enfermedad , Estimulación Eléctrica/métodos , Electromiografía/métodos , Proteoglicanos de Heparán Sulfato/deficiencia , Proteoglicanos de Heparán Sulfato/genética , Ratones , Ratones Transgénicos , Mutación Missense/genética , Conducción Nerviosa/efectos de los fármacos , Conducción Nerviosa/genética , Conducción Nerviosa/fisiología , Fármacos Neuromusculares no Despolarizantes/farmacología , Osteocondrodisplasias/genética , Tiempo de Reacción/efectos de los fármacos , Tiempo de Reacción/fisiologíaRESUMEN
Recent studies have shown that proteoglycans play an important role in the development of vascular disease and renal failure. In this study, the effects of angiotensin II (AngII) type 1 (AT1) and type 2 (AT2) receptor stimulation on glycosaminoglycan and proteoglycan core protein synthesis in vascular smooth muscle cells (VSMC) were examined. Treatment of AT1 receptor-expressing VSMC with AngII resulted in a dose-dependent and time-dependent increase (2- to 4-fold) in (3)H-glucosamine/(35)S-sulfate incorporation, which was abolished by pretreatment with the AT1 receptor antagonist, losartan. The effects of AngII were inhibited by the epidermal growth factor receptor inhibitor, AG1478, and the mitagen-activated protein kinase kinase inhibitor, PD98059, but not the protein kinase C inhibitors, chelerythrine and staurosporine. AngII treatment also resulted in significant increases in the mRNA of the core proteins, versican, biglycan, and perlecan. The effects of AT2 receptor stimulation were examined by retroviral transfection of VSMC with the AT2 receptor. Stimulation of the AT2 receptor in these VSMC-AT2 cells resulted in a significant (1.3-fold) increase in proteoglycan synthesis, which was abolished by the AT2 receptor antagonist, PD123319, and attenuated by pretreatment with pertussis toxin. These results implicate both AT1 and AT2 receptors in the regulation of proteoglycan synthesis and suggest the involvement of epidermal growth factor receptor-dependent tyrosine kinase pathways and G alpha i/o-mediated mechanisms in the effects of the two receptors.
Asunto(s)
Proteínas de la Matriz Extracelular , Músculo Liso Vascular/metabolismo , Proteoglicanos/biosíntesis , Receptores de Angiotensina/fisiología , Agrecanos , Angiotensina II/farmacología , Antagonistas de Receptores de Angiotensina , Animales , Células Cultivadas , ADN Complementario/genética , Glicoproteínas/genética , Proteoglicanos de Heparán Sulfato/genética , Imidazoles/farmacología , Lectinas Tipo C , Losartán/farmacología , Masculino , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Piridinas/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2RESUMEN
Heparan sulfate (HS) chains accumulate in both the medium and the cell layer of mesangial cell cultures. When given in fresh medium to quiescent cultures at naturally occurring concentrations, they suppress entry into the cell cycle and progression to DNA synthesis. We have attempted to identify the proteoglycan (PG) source of the antimitogenic HS chains from mesangial cell layers (HS(c)) and medium (HS(c)). When cells were labeled for 16 hours with [35S]sulfate, 25% of the label was found in intracellular HS chains and 5% in extracellular HSPGs. Cell-surface HSPGs accounted for the remaining 70% of the label associated with cell-layer HS and were released by either trypsin or 2% Triton X-100. About 20% of this cell-surface fraction was released by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC), and probably represents glypican-like PG; glypican mRNA was present in the cells. The remainder of this fraction could be incorporated into liposomes, indicating the presence of hydrophobic transmembrane regions suggestive of syndecans. Upon purification and deglycosylation, an antiserum to rat liver HSPGs that reacts primarily with syndecan-2 showed a strong signal corresponding to this protein and three weaker bands that may represent additional syndecans. mRNAs for syndecan-1, -2, and -4 were present in the cultures. Syndecan-1 and -2 mRNAs were increased 30 minutes after stimulation of quiescent rat mesangial cells (RMCs) with serum. Heparin, HS(c), and HS(m) all prevented this increase. Syndecan-4 mRNA was not affected by serum, heparin, or HS. In pulse-chase experiments, the amount of 35S appearing in the cellular protein-free HS fraction was accounted for almost entirely by cell-surface PGs, as matrix-associated label was a minor contribution at the end of the pulse-labeling. The appearance of [35S]HS in cell extracts was unaffected by phospholipase C treatment, indicating that turnover of the newly labeled syndecan fraction is the source of the antimitogenic HS chains.