Previous studies have shown that sirtuin 1(SIRT1) reduces the production of neuronal amyloid beta(Aβ) and inhibits the inflammatory response of glial cells, thereby generating a neuroprotective effect against Aβ...Previous studies have shown that sirtuin 1(SIRT1) reduces the production of neuronal amyloid beta(Aβ) and inhibits the inflammatory response of glial cells, thereby generating a neuroprotective effect against Aβ neurotoxicity in animal models of Alzheimer's disease. However, the protective effect of SIRT1 on astrocytes is still under investigation. This study established a time point model for the clearance of Aβ in primary astrocytes. Results showed that 12 hours of culture was sufficient for endocytosis of oligomeric Aβ, and 36 hours sufficient for effective degradation. Immunofluorescence demonstrated that Aβ degradation in primary astrocytes relies on lysosome function. Enzymatic agonists or SIRT1 inhibitors were used to stimulate cells over a concentration gradient. Aβ was co-cultured for 36 hours in medium. Western blot assay results under different conditions revealed that SIRT1 relies on its deacetylase activity to promote intracellular Aβ degradation. The experiment further screened SIRT1 using quantitative proteomics to investigate downstream, differentially expressed proteins in the Aβ degradation pathway and selected the ones related to enzyme activity of SIRT1. Most of the differentially expressed proteins detected are close to the primary astrocyte lysosomal pathway. Immunofluorescence staining demonstrated that SIRT1 relies on its deacetylase activity to upregulate lysosome number in primary astrocytes. Taken together, these findings confirm that SIRT1 relies on its deacetylase activity to upregulate lysosome number, thereby facilitating oligomeric Aβ degradation in primary astrocytes.展开更多
BACKGROUND: Astrocytes are considered to provide nutritional support in the central nervous system. However, recent studies have confirmed that astrocytes also play an important role in chronic pain. OBJECTIVE: To i...BACKGROUND: Astrocytes are considered to provide nutritional support in the central nervous system. However, recent studies have confirmed that astrocytes also play an important role in chronic pain. OBJECTIVE: To investigate the effects of intrathecal injection of fluorocitrate, minocycline or both on astrocyte activation and proliferation in the spinal dorsal horn of compressed dorsal root ganglion in rats. DESIGN, TIME AND SETTING: The neurology randomized controlled animal study was performed at the Jiangsu Institute of Anesthesia Medicine, from September 2006 to April 2007. MATERIALS: A total of 96 male Sprague Dawley rats, aged 6-8 weeks, were selected for this study. Following intrathecal catheterization, 80 rats underwent steel bar insertion into the L4-5 intervertebral foramina to make a stable compression on the L4-5 posterior root ganglion. Thus rat models of ganglion compression were established. Minocycline and fluorocitrate were purchased from Sigma, USA. METHODS: A total of 96 rats were randomly and equally divided into six groups. Rat L4, L5 transverse process and intervertebral foramina were exposed in the sham operation group, but without steel bar insertion. The model group did not receive any manipulations. Rats in the phosphate buffered saline (PBS) group were intrathecally injected with 0.01 mmol/L PBS (20 μL). Rats in the fluorocitrate group were subjected to 1 μmol/L fluorocitrate (20 μL). Rats in the minocycline group were intrathecally injected with 5 g/L minocycline (20 μL). Rats in the minocycline and fluorocitrate group received a mixture (20 μL) of 5 g/L minocycline and 1 μmol/L fluorocitrate. Following model establishment, drugs were administered once a day. MAIN OUTCOME MEASURES: At 7 and 14 days following model induction, glial fibrillary acidic protein expression in the spinal dorsal horn was measured by immunofluorescence microscopy. Six sections with significant glial fibrillary acidic protein -positive expression were obtained to count astrocytes under an inverted microscope. RESULTS: No significant differences in astrocyte count were detected between the fluorocitrate and model groups. Cell bodies were small with a few processes in the fluorocitrate group, compared with the model group. The astrocyte count decreased significantly in the minocycline group and the minocycline and fluorocitrate group compared with the sham operation, model, PBS and fluorocitrate groups (P 〈 0.01). The decrease in astrocyte count was mainly found in layers Ⅲ–Ⅳ of the spinal dorsal horn. Cell body volume was smaller and process numbers were fewer in the minocycline group and the minocycline and fluorocitrate group, compared with the model and PBS groups. CONCLUSION: Fluorocitrate can inhibit astrocyte activation, but does not affect astrocyte proliferation. However, minocycline can inhibit the activation and proliferation of astrocytes.展开更多
基金supported by the National Natural Science Foundation of China,No.31670832,31470807,31270872a grant from the National Key Research and Development Program of China,No.2016YFA0500301a grant from the State Key Laboratory of Protein and Plant Gene Research,College of Life Sciences,Peking University,China
文摘Previous studies have shown that sirtuin 1(SIRT1) reduces the production of neuronal amyloid beta(Aβ) and inhibits the inflammatory response of glial cells, thereby generating a neuroprotective effect against Aβ neurotoxicity in animal models of Alzheimer's disease. However, the protective effect of SIRT1 on astrocytes is still under investigation. This study established a time point model for the clearance of Aβ in primary astrocytes. Results showed that 12 hours of culture was sufficient for endocytosis of oligomeric Aβ, and 36 hours sufficient for effective degradation. Immunofluorescence demonstrated that Aβ degradation in primary astrocytes relies on lysosome function. Enzymatic agonists or SIRT1 inhibitors were used to stimulate cells over a concentration gradient. Aβ was co-cultured for 36 hours in medium. Western blot assay results under different conditions revealed that SIRT1 relies on its deacetylase activity to promote intracellular Aβ degradation. The experiment further screened SIRT1 using quantitative proteomics to investigate downstream, differentially expressed proteins in the Aβ degradation pathway and selected the ones related to enzyme activity of SIRT1. Most of the differentially expressed proteins detected are close to the primary astrocyte lysosomal pathway. Immunofluorescence staining demonstrated that SIRT1 relies on its deacetylase activity to upregulate lysosome number in primary astrocytes. Taken together, these findings confirm that SIRT1 relies on its deacetylase activity to upregulate lysosome number, thereby facilitating oligomeric Aβ degradation in primary astrocytes.
基金the Social Development Science and Technology Plan Program of Jiangsu Province, No. B2004515
文摘BACKGROUND: Astrocytes are considered to provide nutritional support in the central nervous system. However, recent studies have confirmed that astrocytes also play an important role in chronic pain. OBJECTIVE: To investigate the effects of intrathecal injection of fluorocitrate, minocycline or both on astrocyte activation and proliferation in the spinal dorsal horn of compressed dorsal root ganglion in rats. DESIGN, TIME AND SETTING: The neurology randomized controlled animal study was performed at the Jiangsu Institute of Anesthesia Medicine, from September 2006 to April 2007. MATERIALS: A total of 96 male Sprague Dawley rats, aged 6-8 weeks, were selected for this study. Following intrathecal catheterization, 80 rats underwent steel bar insertion into the L4-5 intervertebral foramina to make a stable compression on the L4-5 posterior root ganglion. Thus rat models of ganglion compression were established. Minocycline and fluorocitrate were purchased from Sigma, USA. METHODS: A total of 96 rats were randomly and equally divided into six groups. Rat L4, L5 transverse process and intervertebral foramina were exposed in the sham operation group, but without steel bar insertion. The model group did not receive any manipulations. Rats in the phosphate buffered saline (PBS) group were intrathecally injected with 0.01 mmol/L PBS (20 μL). Rats in the fluorocitrate group were subjected to 1 μmol/L fluorocitrate (20 μL). Rats in the minocycline group were intrathecally injected with 5 g/L minocycline (20 μL). Rats in the minocycline and fluorocitrate group received a mixture (20 μL) of 5 g/L minocycline and 1 μmol/L fluorocitrate. Following model establishment, drugs were administered once a day. MAIN OUTCOME MEASURES: At 7 and 14 days following model induction, glial fibrillary acidic protein expression in the spinal dorsal horn was measured by immunofluorescence microscopy. Six sections with significant glial fibrillary acidic protein -positive expression were obtained to count astrocytes under an inverted microscope. RESULTS: No significant differences in astrocyte count were detected between the fluorocitrate and model groups. Cell bodies were small with a few processes in the fluorocitrate group, compared with the model group. The astrocyte count decreased significantly in the minocycline group and the minocycline and fluorocitrate group compared with the sham operation, model, PBS and fluorocitrate groups (P 〈 0.01). The decrease in astrocyte count was mainly found in layers Ⅲ–Ⅳ of the spinal dorsal horn. Cell body volume was smaller and process numbers were fewer in the minocycline group and the minocycline and fluorocitrate group, compared with the model and PBS groups. CONCLUSION: Fluorocitrate can inhibit astrocyte activation, but does not affect astrocyte proliferation. However, minocycline can inhibit the activation and proliferation of astrocytes.