Name: exogenous administration of microsomes-associated alpha-synuclein aggregates to primary neurons as a powerful cell model of fibrils formation
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这项工作得到意大利大学和研究部 (MIUR) 的支持, 通过职业重新融合补助金计划 (青年研究员 RLM 项目) 和 Scuola 师范学校 Superiore。我们感谢美国明尼苏达州大学的迈克尔. 李教授提供的 Prp 人 A53T αS Tg 小鼠, 其中聚合体是孤立的。

通过国家和国际实验动物福利和试验法 (欧洲经济共同体理事会指令 86/609, 1987年12月12日和指令 2010/63/欧盟, 2010年9月22日) 批准并充分遵守了对野生动植物和 Tg 动物的使用。本文所描述的所有协议都遵循了我们机构的动物保育指南。
1. A53T αS Tg 小鼠微粒相关αS 团聚体的分离
<ol><li>制备由250毫米蔗糖、20毫米 HEPES、10毫米氯化钾、1.5 毫米氯化镁2、2毫米 EDTA 和1x 磷酸酶/蛋白酶抑制剂组成的均匀化缓冲液。把缓冲器放在冰上。</li>
	<li>用聚四氟乙烯杵均质器, 用 10-15 冲程, 融汇 1:10 (瓦特/v) 冰冷均匀缓冲液中的新鲜或冰冻组织。</li>
	<li>使用冷冻离心机将初始匀浆 (1-2 毫升) 转移到离心管和离心机, 在4°c 时, 用一个冷藏的分离器除去细胞核和未间断的细胞 (P1)。丢弃 P1。</li>
	<li>使用冷冻离心机将上清 (S1) 到干净的离心管和离心机 S1 1万 x 克20分钟, 以获得第二上清 (S10) 和颗粒 (P10)。 注: P10 是一种含有线粒体和突触体的粗膜颗粒。丢弃 P10。</li>
	<li>将上清 (S10) 到聚碳酸酯瓶 (> 1 毫升) 和离心机 S10 在 10万 x g, 1 小时4摄氏度使用 ultracentrifuge 和固定角度转子 (90 Ti)。 注: 上清是纯细胞质分数, 而颗粒, P100, 含有微粒相关αS 骨料。</li>
	<li>并用重悬 P100 颗粒与均匀缓冲的500µL。将 P100 转移到清洁的离心管和离心机在 1万 x g 20 分钟在4°c 在一个冷藏离心机。</li>
	<li>用100µL 均匀化缓冲器丢弃上清和并用重悬 P100。 注意: 这个分数是微粒相关的αS 聚合体。</li>
	<li>油脂实验 2 s 的样品 [设定输出功率1瓦特 (RMS)]。将样品存放在摄氏-80 摄氏度。</li>
	<li>后天, 用分析法确定蛋白质量。</li>
</ol>2. 西方污点
注: 微粒相关αS 集料的生化特性由西方印迹评价。
<ol><li>在垂直电泳仪上铸造梯度 4-20% 三甘氨酸聚丙烯酰胺凝胶。</li>
	<li>负载1µg 微粒相关的αS 分数, 溶解在变性样品缓冲。</li>
	<li>在不同的井中, 加载5µL 蛋白质标准标记。</li>
	<li>在三/甘氨酸/SDS 运行缓冲液中 100 V 的凝胶, 直到蛋白质标记达到凝胶的末端。</li>
	<li>使用碱性碳酸盐缓冲器 (10 毫米 NaCO3, 3 毫米 NaHCO3, 20% 甲醇) 将蛋白质转移到硝化膜上, 4 摄氏度, 200 毫安, 恒定。</li>
	<li>用 pbs-非离子表面活性剂 0.05% (pbs t) 与5% 不含脂肪的干牛奶在一个轨道振动筛上用30分钟的 RT 阻挡膜。</li>
	<li>简要地, 用 PBS 冲洗膜。</li>
	<li>用 Syn-1 (1:5, 000) 或 pSer129-αS 抗体 (1:5, 000) 在的非脂肪干牛奶中孵育膜, 在一个轨道振动筛上的2.5% °c。</li>
	<li>在轨道振动筛上用 PBS 清洗膜10分钟。</li>
	<li>用抗鼠或抗兔酶共轭二级抗体 (1:3, 000) 在 RT 中的2.5% 个非脂肪干牛奶中孵育膜。</li>
	<li>在轨道振动筛上用 PBS 清洗膜10分钟。重复3x。</li>
	<li>通过常规化学发光试剂盒获得信号。</li>
</ol>3. 主要神经元培养
注: 主要神经元培养是从 P0 鼠海马或皮质 (线 C57BL/6) 中制备的。整个过程, 减去离心步骤, 是在细胞培养罩下, 在无菌条件下进行的。
<ol><li>治疗盖玻片 (直径18毫米), 65% 硝酸溶液至少12小时的 RT。</li>
	<li>除去硝酸。使用 PBS 10x 和蒸馏水冲洗两次盖玻片。</li>
	<li>在24井菜肴中插入盖玻片, 并将其涂上聚 d-赖氨酸 (0.1 毫克/毫升, 不育蒸馏水或 PBS 1x), 1 小时37摄氏度。</li>
	<li>去除聚 d-赖氨酸, 用蒸馏水冲洗盖玻片三次。储存在4摄氏度, 直到需要。</li>
	<li>用斩首弄死老鼠幼崽, 并将头部与尸体分开。把头放在盘子上, 轻轻地解剖皮肤。</li>
	<li>通过使用细剪刀, 通过在大脑的底座上做切口来打开头骨。把头骨的两半分开, 小心地取出。</li>
	<li>通过使用镊子, 从基地中掐掉大脑, 隔离大脑皮层和海马体。将它们转移到两个单独的菜肴中, 其中包括汉克的平衡盐溶液 (HBSS) 培养基, 含有1% 青霉素/链霉素。</li>
	<li>对每只动物重复步骤3.6 和3.7。注意, 整个过程不应超过 30-45 分钟。</li>
	<li>将所收集的组织剁碎, 并将其转化为两个50毫升圆锥管 (一个用于海马体和一个皮质), 10 毫升的 HBSS 培养基含有胰蛋白酶0.1%。在水浴中孵育7分钟, 在37摄氏度。 注: 不需要搅拌。</li>
	<li>添加1毫升的 DMEM 含有10% 胎牛血清 (血清) 和10µg/毫升 DNase 匀浆, 以阻止胰蛋白酶活性。</li>
	<li>离心机将所产生的分离组织在 200 x g 上, 在5分钟内在离心后的 RT 上移除上清。 注: 颗粒代表从组织中解剖的细胞。</li>
	<li>并用重悬在电镀培养基中的细胞, 含有 2% B27, 2 毫米谷氨酰胺, 6 毫克/毫升葡萄糖, 10% 血清, 12.5 µM 谷氨酸和10µg/毫升庆大霉素。</li>
	<li>聚 d-赖氨酸盖玻片上的板块离解神经元根据比例: 1 cortex/12 井和 1 hippocampus/6 井, 每井产生大约 15万/20万个细胞。保持孵化器的细胞在37摄氏度。</li>
	<li>后天 (天体外, DIV 1), 取代电镀培养基与含有 2% B27, 10 µg/毫升庆大霉素和2毫米谷氨酰胺培养基。</li>
	<li>在 DIV 2, 去除1/3 的培养基和添加1/3 的新鲜培养基含有2.5 µM 胞嘧啶苷 48 h, 以减少胶质污染。</li>
	<li>维持37摄氏度的神经元, 每三天更换一半的培养基。</li>
</ol>4. 神经元治疗
注: 治疗已在 7 DIV 进行。所有步骤都是在无菌条件下的细胞培养罩下进行的。图 1显示了 7 DIV 皮层神经元培养密度的例子。
<ol><li>从三种不同患病的 Tg 小鼠的脊髓中获得的微粒相关的αS 聚集物, 以便有一个1的µg/µL 溶液, 使用原来的均匀化缓冲稀释。</li>
	<li>取出1/3 的培养基, 用新鲜培养基 (含 2% B27、1x 庆大霉素和2毫米谷氨酰胺) 轻轻替换。</li>
	<li>将1µg 池微粒相关的αS 聚合体添加到细胞培养基中。在37摄氏度的孵化器中返回神经元。</li>
	<li>每3天1周, 加1/3 的新鲜培养基。请勿更换介质。只是添加它。</li>
	<li>经过1周的治疗后, 取出1/3 的培养基, 用新鲜培养基 (含 2% B27、10µg/毫升庆大霉素和2毫米谷氨酰胺) 轻轻替换。每3天重复一次。</li>
	<li>修复2周后的神经元 (DIV 21)。</li>
</ol>5. 免疫荧光
<ol><li>固定的神经元与2% 多聚甲醛在 PBS 1x 和5% 蔗糖溶液15分钟在 RT 不震动, 在一个化学油烟机。</li>
	<li>卸下固定解决方案。简单地, 用 PBS 1x 清洗, 3 次。 注意: 轻轻地执行所有的洗涤步骤, 因为主神经元不坚定地坚持聚赖氨酸盖玻片。</li>
	<li>Permeabilize 神经元0.3% 的非离子表面活性剂在 PBS 1x 5 分钟的 RT。</li>
	<li>简单地, 用 PBS 1x 清洗, 3 次。</li>
	<li>在 PBS 1x 上孵化3% 只血清的神经元, 用于在轨道振动筛上阻断不特异的结合部位。</li>
	<li>孵化神经元与适当的原发性抗体溶于3% 血清在 PBS 1x, O/N 在4°c 在一个轨道振动筛。 注: syn303 (1:1, 000), 小鼠αS (1:200), pser129-αS (1:1, 000) 和头 (1:10,000) 抗体使用。</li>
	<li>取出抗体溶液, 简单地用 PBS 1x, 3 次清洗。</li>
	<li>孵化神经元与适当的荧光二级抗体溶解在 PBS 3% 的血清, 1 小时的 RT 在黑暗中的一个轨道振动筛。</li>
	<li>取出抗体溶液, 简单地用 PBS 1x, 3 次清洗。</li>
	<li>染色神经元与 DAPI 溶液 (0.1 µg/毫升在 PBS 1x) 15 分钟的 RT 在黑暗的轨道振动筛。</li>
	<li>取出抗体溶液, 简单地用 PBS 1x, 3 次清洗。</li>
	<li>使用 antifade 安装介质在幻灯片上安装盖玻片。</li>
</ol>

<ol>
	<li>Goedert, M., Spillantini, M. G., Del Tredici, K., Braak, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=100+years+of+Lewy+pathology.">100 years of Lewy pathology.</a> Nat. Rev. Neurol. 9, 13-24 (2012).</li><li>Visanji, N. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#945;-Synuclein-Based+Animal+Models+of+Parkinson's+Disease:+Challenges+and+Opportunities+in+a+New+Era.">&#945;-Synuclein-Based Animal Models of Parkinson's Disease: Challenges and Opportunities in a New Era.</a> Trends Neurosci. 39, 750-762 (2016).</li><li>Luk, K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathological+&#945;-Synuclein+Transmission+Initiates+Parkinson-like+Neurodegeneration+in+Nontransgenic+Mice.">Pathological &#945;-Synuclein Transmission Initiates Parkinson-like Neurodegeneration in Nontransgenic Mice.</a> Science. 338, 949-953 (2012).</li><li>Rey, N. L., Petit, G. H., Bousset, L., Melki, R., Brundin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+human+&#945;-synuclein+from+the+olfactory+bulb+to+interconnected+brain+regions+in+mice.">Transfer of human &#945;-synuclein from the olfactory bulb to interconnected brain regions in mice.</a> Acta Neuropathol. (Berl). 126, 555-573 (2013).</li><li>Guo, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+&#945;-Synuclein+Strains+Differentially+Promote+Tau+Inclusions+in+Neurons.">Distinct &#945;-Synuclein Strains Differentially Promote Tau Inclusions in Neurons.</a> Cell. 154, 103-117 (2013).</li><li>Masuda-Suzukake, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion-like+spreading+of+pathological+&#945;-synuclein+in+brain.">Prion-like spreading of pathological &#945;-synuclein in brain.</a> Brain. 136, 1128-1138 (2013).</li><li>Volpicelli-Daley, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exogenous+&#945;-Synuclein+Fibrils+Induce+Lewy+Body+Pathology+Leading+to+Synaptic+Dysfunction+and+Neuron+Death.">Exogenous &#945;-Synuclein Fibrils Induce Lewy Body Pathology Leading to Synaptic Dysfunction and Neuron Death.</a> Neuron. 72, 57-71 (2011).</li><li>Peelaerts, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#945;-Synuclein+strains+cause+distinct+synucleinopathies+after+local+and+systemic+administration.">&#945;-Synuclein strains cause distinct synucleinopathies after local and systemic administration.</a> Nature. 522, 340-344 (2015).</li><li>L&#225;zaro, D. F., Pavlou, M. A. S., Outeiro, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+models+as+tools+for+the+study+of+the+role+of+alpha-synuclein+in+Parkinson's+disease.">Cellular models as tools for the study of the role of alpha-synuclein in Parkinson's disease.</a> Exp. Neurol. 298, 162-171 (2017).</li><li>Lee, H. -. J., Patel, S., Lee, S. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravesicular+localization+and+exocytosis+of+alpha-synuclein+and+its+aggregates.">Intravesicular localization and exocytosis of alpha-synuclein and its aggregates.</a> J. Neurosci. Off. J. Soc. Neurosci. 25, 6016-6024 (2005).</li><li>Luk, K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebral+inoculation+of+pathological+&#945;-synuclein+initiates+a+rapidly+progressive+neurodegenerative+&#945;-synucleinopathy+in+mice.">Intracerebral inoculation of pathological &#945;-synuclein initiates a rapidly progressive neurodegenerative &#945;-synucleinopathy in mice.</a> J. Exp. Med. 209, 975-986 (2012).</li><li>Mougenot, A. -. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion-like+acceleration+of+a+synucleinopathy+in+a+transgenic+mouse+model.">Prion-like acceleration of a synucleinopathy in a transgenic mouse model.</a> Neurobiol. Aging. 33, 2225-2228 (2012).</li><li>Recasens, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lewy+body+extracts+from+Parkinson+disease+brains+trigger+&#945;-synuclein+pathology+and+neurodegeneration+in+mice+and+monkeys:+LB-Induced+Pathology.">Lewy body extracts from Parkinson disease brains trigger &#945;-synuclein pathology and neurodegeneration in mice and monkeys: LB-Induced Pathology.</a> Ann. Neurol. 75, 351-362 (2014).</li><li>Woerman, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagation+of+prions+causing+synucleinopathies+in+cultured+cells.">Propagation of prions causing synucleinopathies in cultured cells.</a> Proc. Natl. Acad. Sci. 112, 4949-4958 (2015).</li><li>Luk, K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exogenous+alpha-synuclein+fibrils+seed+the+formation+of+Lewy+body-like+intracellular+inclusions+in+cultured+cells.">Exogenous alpha-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells.</a> Proc. Natl. Acad. Sci. U. S. A. 106, 20051-20056 (2009).</li><li>Sacino, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intramuscular+injection+of+&#945;-synuclein+induces+CNS+&#945;-synuclein+pathology+and+a+rapid-onset+motor+phenotype+in+transgenic+mice.">Intramuscular injection of &#945;-synuclein induces CNS &#945;-synuclein pathology and a rapid-onset motor phenotype in transgenic mice.</a> Proc. Natl. Acad. Sci. U. S. A. 111, 10732-10737 (2014).</li><li>Lee, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+alpha-synuclein-harboring+familial+Parkinson's+disease-linked+Ala-53+-->+Thr+mutation+causes+neurodegenerative+disease+with+alpha-synuclein+aggregation+in+transgenic+mice.">Human alpha-synuclein-harboring familial Parkinson's disease-linked Ala-53 --> Thr mutation causes neurodegenerative disease with alpha-synuclein aggregation in transgenic mice.</a> Proc. Natl. Acad. Sci. U. S. A. 99, 8968-8973 (2002).</li><li>Colla, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+Reticulum+Stress+Is+Important+for+the+Manifestations+of+-Synucleinopathy+In+Vivo.">Endoplasmic Reticulum Stress Is Important for the Manifestations of -Synucleinopathy In Vivo.</a> J. Neurosci. 32, 3306-3320 (2012).</li><li>Fujiwara, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=alpha-Synuclein+is+phosphorylated+in+synucleinopathy+lesions.">alpha-Synuclein is phosphorylated in synucleinopathy lesions.</a> Nat. Cell Biol. 4, 160-164 (2002).</li><li>Colla, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxic+properties+of+microsome-associated+alpha-synuclein+species+in+mouse+primary+neurons.">Toxic properties of microsome-associated alpha-synuclein species in mouse primary neurons.</a> Neurobiol. Dis. 111, 36-47 (2018).</li><li>Colla, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accumulation+of+Toxic+-Synuclein+Oligomer+within+Endoplasmic+Reticulum+Occurs+in+-Synucleinopathy+In+Vivo.">Accumulation of Toxic -Synuclein Oligomer within Endoplasmic Reticulum Occurs in -Synucleinopathy In Vivo.</a> J. Neurosci. 32, 3301-3305 (2012).</li><li>Volpicelli-Daley, L. A., Luk, K. C., Lee, V. M. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Addition+of+exogenous+&#945;-synuclein+preformed+fibrils+to+primary+neuronal+cultures+to+seed+recruitment+of+endogenous+&#945;-synuclein+to+Lewy+body+and+Lewy+neurite-like+aggregates.">Addition of exogenous &#945;-synuclein preformed fibrils to primary neuronal cultures to seed recruitment of endogenous &#945;-synuclein to Lewy body and Lewy neurite-like aggregates.</a> Nat. Protoc. 9, 2135-2146 (2014).</li><li>Li, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggregation+promoting+C-terminal+truncation+of+alpha-synuclein+is+a+normal+cellular+process+and+is+enhanced+by+the+familial+Parkinson's+disease-linked+mutations.">Aggregation promoting C-terminal truncation of alpha-synuclein is a normal cellular process and is enhanced by the familial Parkinson's disease-linked mutations.</a> Proc. Natl. Acad. Sci. U. S. A. 102, 2162-2167 (2005).</li><li>Luk, K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+Biological+Compatibility+with+Host+Alpha-Synuclein+Influences+Fibril+Pathogenicity.">Molecular and Biological Compatibility with Host Alpha-Synuclein Influences Fibril Pathogenicity.</a> Cell Rep. 16, 3373-3387 (2016).</li><li>Volpicelli-Daley, L. A., Kirik, D., Stoyka, L. E., Standaert, D. G., Harms, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+can+rAAV-&#945;-synuclein+and+the+fibril+&#945;-synuclein+models+advance+our+understanding+of+Parkinson's+disease.">How can rAAV-&#945;-synuclein and the fibril &#945;-synuclein models advance our understanding of Parkinson's disease.</a> J. Neurochem. 139, 131-155 (2016).</li></ol>

作者没有什么可透露的。

通过添加αS Tg 动物模型分离纯化的微粒相关αS 骨料, 我们描述了一种从小鼠脑源性神经元培养中获得αS 包裹体的方法。
本协议的关键步骤如下: 微粒相关αS 集/神经元µg 的比值和αS 集料的来源。如结果会议所示, 最重要的是优化微粒相关αS 集/神经元数量的µg 比, 因为在次优条件下工作可能导致过早细胞死亡或太稀少的胞内聚集 (见代表性结果部分)。因此, 在开始治疗前, 评估 7 DIV (如图 1所示) 神经元培养的密度是非常重要的。此外, αS 骨料必须从患病的αS Tg 小鼠纯化, i. e。从以磷酸化和洗涤剂不溶αS 高分子量蛋白纤维为特征的动物模型中积累的 LB 状夹杂物。
对本议定书可能进行的修改涉及组织, 冷冻或新鲜, 微粒可以隔离和起始量。由于αS 不溶性骨料的含量高, 我们使用 Tg 小鼠的 SpC, 该协议适用于从任何组织中分离微粒相关的骨料, 前提是该地区的αS 骨料含量较高。冷冻样品也可以使用, 因为冷冻不影响微粒相关骨料的纯化步骤或骨料本身. 虽然推荐的组织起始重量约为 100-150 毫克, 但该协议适用于从低到50毫克的原料 (没有最大重量限制) 获得微粒。然而, 在低于100毫克的情况下, 适当的均匀化比将是 1:20 (瓦特/v), 以便有至少1毫升的上清 S10 装载在聚碳酸酯瓶为 ultracentrifuge 沉淀。事实上, 装入小于1毫升的体积可能导致管子的坍塌和样品损耗。增加均匀化体积将导致更稀释的上清液, 但微粒颗粒的浓度将保持不受影响。
本议定书的一项限制涉及在αS 包裹体的形成中缺乏效率的交叉播种, 最近据报在αS PFFs 的病例管理中对小鼠的神经元培养, 而不是老鼠αS PFFs24。由于增加外源αS 纤维的数量给小鼠的文化可以绕过这个问题, 我们建议微调的数量, 微粒相关的骨料的情况下, 从其他αS 变种或从不同的纤维管理物种比我们所描述的小鼠神经细胞培养。
添加到区域性介质中的外源αS 聚合可以来自不同的源。体外αS PFFs 以前被用作细胞培养、原代神经元和动物模型3、7、8、15中胞内αS 聚集的种子模板。与我们的方法相比, 微粒相关的αS 聚集体可以在几个小时内分离, PFFs 的形成是漫长而费力的, 需要多步骤的纯化, 其次是额外的化验, 以检查αS 骨料 confomations25.此外, PFFs 是从细菌表达的人或小鼠αS,即缺乏翻译后修改典型的真核生物, 可以呈现不同的构象, 具有选择性播种和致病性质, 根据所遵循的成核协议 (即丝带 vs 纤维)5,8导致不同的结果和结论。相反,在体内纯化的αS 聚集物的单一管理保证了更真实的致病模板的传播, 密切模仿动物模型和 PD 患者的αS 包裹体的形成过程。
作为这项技术的未来应用, 我们认为该协议可以成功地用于隔离 PD 患者或其他αS Tg 动物模型的大脑中的αS 致病种子, 前提是微粒被隔离的病区丰富αS包裹 体。
根据我们的知识, 这是第一种方法, 允许纯化的本地有毒物种的αS 从体内PD 模型被用作种子模板, 以获得形成的αS 包裹体在原代神经元。
我们认为这种方法是非常多才多艺的, 可以提供一个特殊的细胞模型, 以研究αS 聚集的不同方面及其对细胞病理生理学的影响。由于αS 包裹体的形成是一个复杂的过程, 在培养细胞中很难复制, 我们希望这个模型能为急性致病机制提供深刻的洞察力, 难以在慢性和更精细的鉴别诸如动物模型之类的系统。

synuclein (αS) 蛋白质包裹体的积累是帕金森病 (PD) 和阿尔法-synulceinopathies1的突出和重要特征。不幸的是, 虽然动物模型能够提供足够的细胞和生物化学环境, 以诱导形成蛋白质纤维2所必需的聚集步骤, 复制形成复杂的 Lewy 体 (LB) 样骨料在细胞培养是困难和挑战性的。
在这里我们描述了一种诱导αS 包裹体形成的方法, 类似于动物模型和 PD 患者在培养细胞中获得的蛋白质聚集物, 利用脑孤立的小鼠原代神经元。我们的协议是基于外源管理的微粒相关的αS 聚集从αS 症状的转基因 (Tg) 小鼠, 以小鼠海马或皮质原代神经元。该方法利用αS 有毒物种的传播和传播能力, 一旦添加到培养基中, 就能形成内化, 并诱导成熟的αS-阳性骨料3,4, 5,6,7,8。
最初, 在细胞培养中获得αS 纤维形成的标准方法是基于相应的αS cDNA 通过常规转染协议或病毒介导的感染9的过度表达。在第一种情况下, 获得 LB 样的αS 集是偶然的, 显示效率低下, 并依赖于细胞类型, 第二个协议导致不溶性纤维的形成, 包括高分子量 (蛋白) 的物种在 24-48 小时的感染10. 在这些方法中, 聚集物的形成可能是由于αS 蛋白量的过度和不平衡而变得不溶, 而不是αS 构象的病态转换, 它决定了聚合。相反, 我们在这里展示的技术并没有改变αS 的表达水平, 而是由于外源纤维的内化导致了广泛的蛋白质聚集。此外, 通过对外源纤维的管理, αS 骨料的形成是一个漫长的过程, 需要数天或数周的时间, 使我们能够以过时的方式研究αS 包裹体形成的早期和中期阶段, 并把它与细胞生物化学的变化联系起来。因此, 我们的方法是一个有价值的应用, 创建αS 聚集的细胞模型, 有助于研究αS 纤维形成微观上与细胞病理生理学有关。
此外, 虽然从患病的αS 转基因 (Tg) 小鼠11,12或人 PD 大脑6,13可以诱发αS 沉积的 Tg 或野生型 (动物) 的原始脑提取物, 应用同样的程序, 细胞培养还没有被证明是成功的, 可能是因为在所用样品中的聚合量很低, 并且缺乏一个标准程序来隔离本αS 有毒物种14。正因为如此,体外预制纤维 (PFFs) 的αS 一直是聚合源的选择, 直到现在的αS 包裹体诱导细胞和动物模型3,4,6,7 ,15,16。然而, 通过我们的协议, 我们表明, 从αS Tg 小鼠分离的微粒相关的αS 聚集物种可以有效地诱导细胞内的 LB 样αS 包裹体在原代神经元中的积累。
在我们的实验室中, 微粒相关的αS 聚集物种是从患病的 Tg 小鼠的脊髓 (SpC) 组织中分离出来的, 表达人类 A53T αS 基因控制下的鼠朊蛋白 (prp) 启动子 [prp 人类 A53T αS Tg 小鼠, 行 G2-317]。这些小鼠显示了年龄依赖性神经退行性表型, 包括强健的运动功能障碍和形成的包裹体在中枢神经系统由磷酸化, 泛素化, 和不溶αS, 开始后9月的年龄。一旦出现运动神经功能障碍, 表型迅速演变为瘫痪, 从后肢开始, 导致 2-3 周死亡。αS 聚集与疾病表现相似。在运动功能障碍发生时牺牲的小鼠表现出在 SpC、脑干和小脑中αS 聚集的强健程度。没有必要等到瘫痪后才会牺牲老鼠。Presymptomatic 老鼠是在9月的动物, 不显示运动障碍。

<table><tbody><tr><td>Sucrose&amp;nbsp;</td><td>Sigma-Aldrich</td><td>84097-1KG</td><td></td></tr><tr><td>Hepes&amp;nbsp;</td><td>Sigma-Aldrich</td><td>H0887-100ML</td><td>1M pH=7-7.6</td></tr><tr><td>EDTA&amp;nbsp;</td><td>Sigma-Aldrich</td><td>0390-100ml</td><td>pH=8 0.5M</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M8266-100G</td><td></td></tr><tr><td>NaCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S7795-500G</td><td></td></tr><tr><td>NAHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S5761-500G</td><td></td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>322415-6X1L</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9541-500G</td><td></td></tr><tr><td>cOmplete Mini&amp;nbsp;</td><td>Roche</td><td>11836170001</td><td>protease inhibitor</td></tr><tr><td>PhosStop&amp;nbsp;</td><td>Roche</td><td>4906837001</td><td>phosphatase inhibitor</td></tr><tr><td>BCA Protein Assay Kit</td><td>Euroclone</td><td>EMPO14500</td><td></td></tr><tr><td>Criterion TGX 4-20% Stain Free, 18 wells</td><td>Biorad</td><td>5678094</td><td></td></tr><tr><td>Supported Nitrocellulose membrane&amp;nbsp;</td><td>Biorad</td><td>1620097</td><td>0.2 &amp;mu;m</td></tr><tr><td>Blotting-Grade Blocker&amp;nbsp;</td><td>Biorad</td><td>1706404</td><td>Non-fat dry milk</td></tr><tr><td>SuperSignal West Pico Chemiluminescent Substrate</td><td>Termo Fisher Scientific</td><td>34077</td><td></td></tr><tr><td>Nitric acid&amp;nbsp;</td><td>Sigma-Aldrich</td><td>1004411000</td><td>65%</td></tr><tr><td>Glass Coverslips&amp;nbsp;</td><td>Termo Fisher Scientific</td><td>1014355118NR1</td><td>18 mm x&amp;nbsp;</td></tr><tr><td>Poly-D-Lysine</td><td>Sigma-Aldrich</td><td>P7280</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution</td><td>Termo Fisher Scientific</td><td>14170-500 mL</td><td></td></tr><tr><td>Penicillin/Streptomycin&amp;nbsp;</td><td>Termo Fisher Scientific</td><td>15140122</td><td>10,000 U/mL, 100 mL</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium&amp;nbsp;</td><td>Termo Fisher Scientific</td><td>D5796-500 mL</td><td></td></tr><tr><td>Trypsin-EDTA&amp;nbsp;</td><td>Termo Fisher Scientific</td><td>15400054</td><td>0.50%</td></tr><tr><td>B27 Supplement&amp;nbsp;</td><td>Termo Fisher Scientific</td><td>17504044</td><td>50X</td></tr><tr><td>Glutamax&amp;nbsp;</td><td>Termo Fisher Scientific</td><td>35050-038</td><td>100x</td></tr><tr><td>DNAse</td><td>Sigma-Aldrich</td><td>D5025</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Euroclone</td><td>EC50182L</td><td></td></tr><tr><td>Glutamate</td><td>Sigma-Aldrich</td><td>1446600-1G</td><td></td></tr><tr><td>Gentamicin&amp;nbsp;</td><td>Termo Fisher Scientific</td><td>15710</td><td>10 mg/ml</td></tr><tr><td>Neurobasal Medium&amp;nbsp;</td><td>Termo Fisher Scientific</td><td>10888-022&amp;nbsp;</td><td></td></tr><tr><td>Cytosine arabinoside (AraC)</td><td>Sigma-Aldrich</td><td>C3350000&amp;nbsp;</td><td></td></tr><tr><td>VECTASHIELD antifade mounting medium&amp;nbsp;</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr><tr><td>DAPI</td><td>Termo Fisher Scientific</td><td>62247</td><td></td></tr><tr><td>90 Ti rotor</td><td>Beckman</td><td>N/A</td><td>Ultracentrifuge rotor</td></tr><tr><td>Optima L-90K Ultracentrifuge</td><td>Beckman</td><td>N/A</td><td></td></tr><tr><td>Syn-1 antibody, clone 42&amp;nbsp;</td><td>BD Biosciences</td><td>610786</td><td>anti-mouse WB: 1:5000</td></tr><tr><td>Syn303 antibody&amp;nbsp;</td><td>BioLegend</td><td>824301</td><td>anti-mouse IF: 1:1000</td></tr><tr><td>Tau antibody&amp;nbsp;</td><td>Synaptic Systems</td><td>314&amp;nbsp;002</td><td>anti-rabbit IF: 1:10,000</td></tr><tr><td>pser129-&amp;alpha;S antibody&amp;nbsp;</td><td>A gift from Fujiwara et al, reference 19</td><td></td><td>anti-rabbit WB: 1:5000</td></tr><tr><td>pser129-&amp;alpha;S antibody</td><td>Abcam</td><td>ab51253</td><td>anti-rabbit&amp;nbsp; IF: 1:1000</td></tr><tr><td>Mouse &amp;alpha;S (D37A6) XP&amp;nbsp;</td><td>Cell Signaling</td><td>4179</td><td>anti-rabbit IF 1:200</td></tr><tr><td>Alexa fluor 555-conjugated anti-rabbit antibody&amp;nbsp;</td><td>Termo Fisher Scientific</td><td>A27039</td><td></td></tr><tr><td>Alexa fluor 488-conjugated anti-mouse antibody&amp;nbsp;</td><td>Termo Fisher Scientific</td><td>A-11029</td><td></td></tr><tr><td>Microson XL-2000&amp;nbsp;</td><td>Misonix</td><td></td><td>Sonicator&amp;nbsp;</td></tr><tr><td>Ultra Bottles (Oakridge Bottles), PCB, 16x76mm, Assembly, Noryl Cap, Beckman-type</td><td>Science Service EU</td><td>S4484</td><td>Ultracentrifuge tubes</td></tr><tr><td>AXIO Observer Inverted Light Microscope&amp;nbsp;</td><td>Zeiss</td><td>N/A</td><td></td></tr><tr><td>TCS SP2 laser scanning confocal microscope</td><td>Leica</td><td>N/A</td><td></td></tr><tr><td>Inverted epi-fluorescence microscope&amp;nbsp;</td><td>Nikon</td><td>N/A</td><td></td></tr><tr><td>Triton x-100</td><td>Sigma-Aldrich</td><td>X100-500ML</td><td>Nonionic surfactant&amp;nbsp;</td></tr></tbody></table>

exogenous administration of microsomes-associated alpha-synuclein aggregates to primary neurons as a powerful cell model of fibrils formation

多年来, 细胞培养中不溶性α-synuclein (αS) 包裹体的复制能力一直是帕金森病 (PD) αS 聚集研究的一大局限性。最近, 通过对αS 转基因小鼠或 PD 患者的脑提取物进行外源接种, 开发出新的动物模型, 为建立更充分的αS 聚集细胞模型提供了新的希望。不幸的是, 当涉及到细胞在文化中, 原始的大脑提取物的管理并没有被证明是成功的, 如在小鼠和外源团聚体的选择来源仍然是预制αS 纤维。
我们已经开发了一种方法, 以诱导形成细胞内αS 包裹体的原发神经元通过外源管理的本地微粒相关的αS 集, 一个高度毒性αS 物种从疾病地区的转基因小鼠。这一小部分的αS 聚集, 是与微粒囊, 有效内化, 并诱导形成细胞内夹杂阳性的聚合和磷酸化αS。与重组αS 制成的体外预制纤维相比, 我们的方法较快, 保证了致病性播种是由帕金森病动物模型提取的正宗αS 骨料, 模仿更紧密的类型在体内获得的包裹体。因此, 富含αS 包裹体的组织的可用性是强制性的。
我们相信, 这种方法将提供一个多才多艺的细胞模型来研究αS 聚集的微观方面和相关的细胞病理生理学在体内, 将是创建更准确和复杂的细胞的起点PD 的范例。

按照上面描述的协议并在图 2中总结, 我们从三只患病的 A53T αS Tg 小鼠中纯化微粒相关的αS 聚合体 (图 3)。微粒是含有内质网、高尔基和小突触泡的粗膜颗粒。与其他分数相比, 微粒颗粒的纯度比以前用特定的细胞器标记18进行了评估。
一旦分离, αS 集料的生化特征通过变性 SDS 页评估, 其次是 Syn-1 或磷酸化αS 在丝氨酸 129 (pSer129-αS) 抗体的孵化。与 presymptomatic (压力) 和年龄匹配的非 Tg (nTg) 小鼠相比, 微粒从病鼠与αS 团聚体共沉淀。微粒相关的αS 物种显示αS 聚合体的典型特征, 如积累高分子量抗洗涤剂的种类, 磷酸化在丝氨酸 12919和 C 和 N 末端截断片段 (为充分的描述微粒伴生的αS 合计参见参考18,20,21)。这些都是基本要求, 因为单体αS 没有得到有效内化, 不诱导αS 沉积7,15,22。重要的是不要使用任何洗涤剂 (离子或非离子) 并用重悬 P100 丸, 因为它可能是有害的细胞。此外, 为了避免样本变异性, 微粒相关的αS 聚集从三个不同的患病小鼠将汇集为神经元治疗。
从患病 A53T 小鼠到皮质或海马神经元培养基的1µg 共微粒相关αS 聚集, 诱导了αS 包裹体的时间依赖性形成, 阳性为集料特定的αS 抗体, 如Syn303 (图 4、 5) 或 pser129-αS (图 5)。经过两天 (2d) 的治疗后, 这些骨料出现的小, 分散的 puncta, 将变得更加丰富, 在以后的时间点。两周后, αS 夹杂物类似于长而成熟的珠子状结构, 在整个神经元培养过程中大量传播, 遵循突起模式, 并与突触前和突起标记部分协同本地化 (图 5)。偶尔, 新形成的αS 包裹体可以被看到, 以共同本地化和染色细胞体细胞或覆盖整个过程, 类似坏死突起。
尽管微粒相关的αS 聚合分数可以有效地在神经元培养中传播, 但它们的数量必须精确地调整到被镀神经元的数量 (图 1)。事实上, 超过建议的比例, µg 微粒相关的聚合体: 神经元的数量, 将导致过早细胞死亡在几天内 (图 6), 而微粒相关αS 总量不足将导致在两周的治疗后, 夹杂物的稀少和减少数量, 类似于在较早的时间点获得的 (图 4A)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57884/57884fig1.jpg"> 图 1.皮质神经元培养。代表的图像显示密度在7的皮层神经元培养。图像采用倒置光显微镜, 10X 目标。刻度条 = 100 µm.<a href="/files/ftp_upload/57884/57884fig1large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57884/57884fig2.jpg"> 图 2.从小鼠 SpC 中分离微粒相关的αS 聚合体.微粒相关αS 集料从病鼠 SpC 中纯化的流程图。<a href="/files/ftp_upload/57884/57884fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57884/57884fig3.jpg"> 图 3.从病、presymptomatic A53T αS Tg 和 nTg 小鼠中分离微粒组分.西方印迹分析显示纯化的微粒相关αS 聚集从三病 (病), presymptomatic (压力) 和老年匹配 nTg 小鼠。患病小鼠是 A53T αS Tg 小鼠, 显示电机和神经功能障碍, 包括αS 包裹体的积累, 而 presymptomatic 动物是健康 A53T αS 9 月的年龄, 并没有显示任何 Tgs 病理相关表型。nTg 小鼠窝不携带αS 转基因的病鼠, 因此不发育αS 诱发病理。1µg 的每一个纯化的分数是运行在变性 SDS 页上, 转移到硝化膜和涂抹 Syn-1 或 pSer129-αS 抗体。只有从患病小鼠中分离出的微粒分数含有高分子量的抗αS 聚合蛋白, 在丝氨酸129中磷酸化, 显示 C 和 N 末端截断。这个数字已经从科亚等。18.<a href="/files/ftp_upload/57884/57884fig3large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57884/57884fig4.jpg"> 图 4.微粒相关αS 集料后αS 沉积的时间依赖性诱导。(A)用1µg 微粒相关的αS 集料从三种不同的患病小鼠中分离出的原发性海马神经元免疫荧光。神经元被固定在2天 (2d), 1 周 (1w) 或2周 (2w) 的治疗和 immunostained 与 syn303 (S303, 1:1000), 抗体特异的氧化和聚集αS。细胞与 DAPI 复染。共焦图像采用激光扫描共聚焦显微镜, 63X 目标。刻度条 = 50 µm (B)利用图像 J 软件的粒子计数插件, 对背景减法后的总ﬂuorescence 进行定量分析。值被规范化的每个领域的核数 (DAPI 计数), 并表示为 S303 荧光信号的百分比在2D。值作为平均值给出 (n = 5)。** p < 0.001, *** p < 0.00001, 单向方差分析, 其次是费舍尔的迷幻药后测试。<a href="/files/ftp_upload/57884/57884fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57884/57884fig5.jpg"> 图 5.细胞内αS 包裹体 colocalize 皮质突起网络。用微粒相关的αS 集料处理的脑皮质神经元的代表性共焦图像。经过2周的治疗神经元固定和双重染色的聚合特异抗体 [S303 (A, B)或 pser129-αS, 1:1, 000 (C)] 和突起标记 [鼠标αS, 1:200 (A, B) 或头, 1:10,000 (C)]。荧光信号的联合标记显示了新形成的αS 珠状结构与突起网络的局部协同定位。偶尔αS 包裹体积累在神经元躯体 (A, B, 箭头)。用激光扫描共聚焦显微镜, 63X 目标采集堆积图像。刻度条 = 50 µm。这个数字已经从科亚等。20.<a href="/files/ftp_upload/57884/57884fig5large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57884/57884fig6.jpg"> 图 6.增加微粒相关αS 集料的次优量对神经元是有毒的。随着微粒相关αS 聚集物浓度的增加, 神经元的 DAPI 染色导致细胞死亡。(A)皮质神经元用 1, 2 µg 微粒相关的αS 聚集物从患病小鼠身上提取, 或仅有不含聚集物(B)并沾有 DAPI 的缓冲液。利用20X 目标, 用荧光显微镜获得荧光图像。刻度条 = 100 µm(B)使用图像 J 软件计算 DAPI 阳性细胞。该图显示了在神经元介质中添加的微粒相关αS 骨料浓度增加的细胞核数目减少。值表示为% 的 b, 并给出的平均值 (n = 5), ** p < 0.0001, 单向方差分析, 其次是费舍尔的迷幻后的测试。<a href="/files/ftp_upload/57884/57884fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation at JoVE.com

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

本协议的目标是提供一个基于细胞的系统,在体内复制α synuclein 聚合体的形成。细胞内阿尔法-synuclein 包裹体的种子在原代神经元的内化和传播的外源管理的原生微粒相关的阿尔法-synuclein 集分离的病阿尔法 synuclein 转基因小鼠。

微粒相关α synuclein 聚合体对原代神经元的外源性管理--纤维形成的强大细胞模型

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

exogenous-administration-microsomes-associated-alpha-synuclein

Research

JoVE Journal

Neuroscience

7.5K 次观看.  Scuola Normale Superiore. 本协议的目标是提供一个基于细胞的系统,在体内复制α synuclein 聚合体的形成。细胞内阿尔法-synuclein 包裹体的种子在原代神经元的内化和传播的外源管理的原生微粒相关的阿尔法-synuclein 集分离的病阿尔法 synuclein 转基因小鼠。

视频: Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, &#945;-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated &#945;-syn is a neuropathological feature of Parkinson&#39;s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble &#945;-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of &#945;-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated &#945;-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal &#945;-syn biology in transgenic animal models harbouring different &#945;-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.

Here, we present a protocol for the isolation of increasingly insoluble/aggregated alpha-synuclein (&#945;-syn) from post-mortem human brain tissue. Through the utilization of buffers with increasing detergent strength and high-speed ultracentrifugation techniques, the variable properties of &#945;-syn aggregation in diseased and non-diseased tissue can be examined.

可溶和不可溶α-突触核蛋白从帕金森脑连续提取

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, ...

科研

神经科学

Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein prionoids, which cause neuropathology within the central nervous system (CNS). Here we describe that intraglossal or intraperitoneal injection of alpha-synuclein fibrils into bigenic Tg(M83+/-:Gfap-luc+/-) mice, which overexpress human alpha-synuclein with the A53T mutation from the prion protein promoter and firefly luciferase from the promoter for glial fibrillary acidic protein (Gfap), is sufficient to induce neuropathologic disease. In comparison to homozygous Tg(M83+/+) mice that develop severe neurologic symptoms beginning at an age of 8 months, heterozygous Tg(M83+/-:Gfap-luc+/-) animals remain free of spontaneous disease until they reach an age of 22 months. Interestingly, injection of alpha-synuclein fibrils via the intraperitoneal route induced neurologic disease with paralysis in four of five Tg(M83+/-:Gfap-luc+/-) mice with a median incubation time of 229 &#177;17 days. Diseased animals showed severe deposits of phosphorylated alpha-synuclein in their brains and spinal cords. Accumulations of alpha-synuclein were sarkosyl-insoluble and colocalized with ubiquitin and p62, and were accompanied by an inflammatory response resulting in astrocytic gliosis and microgliosis. Surprisingly, inoculation of alpha-synuclein fibrils into the tongue was less effective in causing disease with only one of five injected animals showing alpha-synuclein pathology after 285 days. Our findings show that inoculation via the intraglossal route and more so via the intraperitoneal route is suitable to induce neurologic illness with relevant hallmarks of synucleinopathies in Tg(M83+/-:Gfap-luc+/-) mice. This provides a new model for studying prion-like pathogenesis induced by alpha-synuclein prionoids in greater detail.

Peripheral injection of alpha-synuclein fibrils into the peritoneum or tongue of Tg(M83+/-:Gfap-luc+/-) mice, which express human alpha-synuclein with the familial A53T mutation and firefly luciferase, can induce neuropathology, including neuroinflammation, in their central nervous system.

在转基因小鼠神经炎症的生物发光成像的α-共核蛋白纤丝的外围接种后

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein ...

Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model Parkinson&#39;s disease synucleinopathy and nigrostriatal degeneration. The standardization of &#945;-syn PFF generation and in vivo application is critical in order to ensure consistent, robust &#945;-syn pathology. Here, we present a detailed protocol for the generation of fibrils from monomeric &#945;-syn, post-fibrilization quality control steps, and suggested parameters for successful neurosurgical injection of &#945;-syn PFFs into rats or mice. Starting with monomeric &#945;-syn, fibrilization occurs over a 7-day incubation period while shaking at optimal buffer conditions, concentration, and temperature. Post-fibrilization quality control is assessed by the presence of pelletable fibrils via sedimentation assay, the formation of amyloid conformation in the fibrils with a thioflavin T assay, and electron microscopic visualization of the fibrils. Whereas successful validation using these assays is necessary for success, they are not sufficient to guarantee PFFs will seed &#945;-syn inclusions in neurons, as such aggregation activity of each PFF batch should be tested in cell culture or in pilot animal cohorts. Prior to use, PFFs must be sonicated under precisely standardized conditions, followed by examination using electron microscopy or dynamic light scattering to confirm fibril lengths are within optimal size range, with an average length of 50 nm. PFFs can then be added to cell culture media or used in animals. Pathology detectable by immunostaining for phosphorylated &#945;-syn (psyn; serine 129) is apparent days or weeks later in cell culture and rodent models, respectively.&#160;

The goal of this article is to outline the steps required for the generation of fibrils from monomeric alpha-synuclein, subsequent quality control, and use of the preformed fibrils in vivo.

从单体和体内使用的Α-syr核 in 预成型纤维的产生

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model ...

Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on &#945;-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson&#8217;s disease, Lewy bodies (LB), is mimicked by the addition of &#945;-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated &#945;-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing &#945;-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-&#945;-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated &#945;-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of &#945;-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.

Here, we present a detailed protocol to study neuronal &#945;-synuclein accumulation in primary mouse dopamine neurons. Phosphorylated &#945;-synuclein aggregates in neurons are induced with pre-formed &#945;-synuclein fibrils. Automated imaging of immunofluorescently labeled cells and unbiased image analysis make this robust protocol suitable for medium-to-high throughput screening of drugs that inhibit &#945;-synuclein accumulation.

研究原发性胚胎小鼠中脑多巴胺神经元中预制纤维诱导β-合成素积累

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with ...

Stereotaxic Intracranial Delivery of Chemicals, Proteins or Viral Vectors to Study Parkinson's Disease

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in the substantia nigra. Affected brain areas display intraneuronal fibrillar inclusions consisting mainly of alpha-synuclein (asyn) proteins. No animal model thus far has recapitulated all characteristics of this disease. Here, we describe the use of stereotaxic injection to deliver chemicals, proteins, or viral vectors intracranially in order to mimic various aspects of PD. These methods are well-established and widely used throughout the PD field. Stereotaxic injections are incredibly flexible; they can be adjusted in concentration, age of animal used for injection, brain area targeted and in animal species used. Combinations of substances allow for rapid variations to assess treatments or alter severity of the pathology or behavioral deficits. By injecting toxins into the brain, we can mimic inflammation and/or a severe loss of dopaminergic neurons resulting in substantial motor phenotypes. Viral vectors can be used to transduce cells to mimic genetic or mechanistic aspects. Preformed fibrillar asyn injections best recapitulate the progressive phenotype over an extended period of time. Once these methods are established, it can be economical to generate a new model compared to creating a new transgenic line. However, this method is labor intensive as it requires 30 minutes to four hours per animal depending on the model used. Each animal will have a slightly different targeting and therefore create a diverse cohort which on one hand can be challenging to interpret results from; on the other hand, help mimic a more realistic diversity found in patients. Mistargeted animals can be identified using behavioral or imaging readouts, or only after being sacrificed leading to smallercohort size after the study has already been concluded. Overall, this method is a rudimentary but effective way to assess a diverse set of PD aspects.

We describe how to successfully inject solutions into specific brain areas of rodents using a stereotaxic frame. This survival surgery is a well-established method used to mimic various aspects of Parkinson's disease.

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in ...

Analyzing the Parkinson's Disease Mouse Model Induced by Adeno-associated Viral Vectors Encoding Human &#945;-Synuclein

Parkinson&#39;s disease is a neurodegenerative disorder that involves the death of the dopaminergic neurons of the nigrostriatal pathway and, consequently, the progressive loss of control of voluntary movements. This neurodegenerative process is triggered by the deposition of protein aggregates in the brain, which are mainly constituted of &#945;-synuclein. Several studies have indicated that neuroinflammation is required to develop the neurodegeneration associated with Parkinson&#39;s disease. Notably, the neuroinflammatory process involves microglial activation as well as the infiltration of peripheral T cells into the substantia nigra (SN). This work analyzes a mouse model of Parkinson&#39;s disease that recapitulates microglial activation, T-cell infiltration into the SN, the neurodegeneration of nigral dopaminergic neurons, and motor impairment. This mouse model of Parkinson&#39;s disease is induced by the stereotaxic delivery of adeno-associated viral vectors encoding the human wild-type &#945;-synuclein (AAV-h&#945;Syn) into the SN. The correct delivery of viral vectors into the SN was confirmed using control vectors encoding green fluorescent protein (GFP). Afterward, how the dose of AAV-h&#945;Syn administered in the SN affected the extent of h&#945;Syn expression, the loss of nigral dopaminergic neurons, and motor impairment were evaluated. Moreover, the dynamics of h&#945;Syn expression, microglial activation, and T-cell infiltration were determined throughout the time course of disease development. Thus, this study provides critical time points that may be useful for targeting synuclein pathology and neuroinflammation in this preclinical model of Parkinson&#39;s disease.

This work analyzes the vector dose and exposure time required to induce neuroinflammation, neurodegeneration, and motor impairment in this preclinical model of Parkinson&#39;s disease. These vectors encoding the human &#945;-synuclein are delivered into the substantia nigra&#160;to recapitulate the synuclein pathology associated with Parkinson&#39;s disease.

分析编码人类α突触核蛋白的腺相关病毒载体诱导的帕金森病小鼠模型

Parkinson&#39;s disease is a neurodegenerative disorder that involves the death of the dopaminergic neurons of the nigrostriatal pathway and, ...

<meta charset="utf8"></head><body>α-突触核蛋白繁殖:帕金森病的鼻内给药

Technique for Intranasal Administration of &#945;-Synuclein Aggregates

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the presence of Lewy bodies, which are aggregates of &#945;-synuclein (&#945;-Syn). Recently, the disease was proposed to develop and progress through the prion-like propagation of &#945;-Syn aggregates from the olfactory bulb (OB) or dorsal nucleus of the vagus nerve. Although the origin of &#945;-Syn aggregates in the OB remains unclear, their propagation from the olfactory mucosa has been recently suggested. We previously showed that intranasal administration of &#945;-Syn aggregates in a mouse model induced &#945;-Syn pathology in the OB of mice. In this study, we present a method of intranasal administration of &#945;-Syn aggregates that induced &#945;-Syn pathology in the OB of mice. Intranasal administration of &#945;-Syn aggregates is a very simple and straightforward method, and we believe it will be a useful tool in the research for elucidating the origin of &#945;-Syn pathology in the OB and the pathway of &#945;-Syn propagation through the olfactory system.

<meta charset="utf8"></head><body>α-突触核蛋白聚集体的鼻内给药技术

The present protocol describes a method for intranasal administration of &#945;-synuclein aggregates. This method provides insights into &#945;-synuclein propagation from the olfactory mucosa to the olfactory bulb in Parkinson's disease.

α-突触核蛋白聚集体的鼻内给药技术

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the presence of Lewy bodies, which are aggregates of &#945;-synuclein ...

A Method to Study &#945;-Synuclein Toxicity and Aggregation Using a Humanized Yeast Model

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by progressive cell death caused by the formation of Lewy bodies containing misfolded and aggregated &#945;-synuclein. &#945;-synuclein is an abundant presynaptic protein that regulates synaptic vesicle trafficking, but the accumulation of its proteinaceous inclusions results in neurotoxicity. Recent studies have revealed that various genetic factors, including bacterial chaperones, could reduce the formation of &#945;-synuclein aggregates in vitro. However, it is also important to monitor the anti-aggregation effect in the cell to apply this as a potential treatment for the patients. It would be ideal to use neuronal cells, but these cells are difficult to handle and take a long time to exhibit the anti-aggregation phenotype. Therefore, a quick and effective in vivo tool is required for the further evaluation of in vivo anti-aggregation activity. The method described here was used to monitor and analyze the anti-aggregation phenotype in the humanized yeast Saccharomyces cerevisiae, which expressed human &#945;-synuclein. This protocol demonstrates in vivo tools that could be used for monitoring &#945;-synuclein-induced cellular toxicity, as well as the formation of &#945;-synuclein aggregates in cells.

An in vivo physiological model of &#945;-synuclein is required to study and understand the pathogenesis of Parkinson's disease. We describe a method to monitor the cytotoxicity and aggregate formation of &#945;-synuclein using a humanized yeast model.

一种基于人源酵母模型研究α-突触核蛋白毒性和聚集的方法

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by progressive cell death caused by the formation of Lewy ...

微粒相关α synuclein 聚合体对原代神经元的外源性管理--纤维形成的强大细胞模型

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在转基因小鼠神经炎症的生物发光成像的α-共核蛋白纤丝的外围接种后

从单体和体内使用的Α-syr核 in 预成型纤维的产生

研究原发性胚胎小鼠中脑多巴胺神经元中预制纤维诱导β-合成素积累

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α-突触核蛋白聚集体的鼻内给药技术

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