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 border="0" cellpadding="0" cellspacing="0" style="width:931px;" width="931">
	<colgroup>
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		<col />
		<col />
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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Sucrose&#160;</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td style="width:119px;">84097-1KG</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Hepes&#160;</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td>H0887-100ML</td>
			<td>1M pH=7-7.6</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:400px;">EDTA&#160;</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td style="width:119px;">0390-100ml</td>
			<td>pH=8 0.5M</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:400px;">MgCl2</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td>M8266-100G</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:400px;">NaCO3</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td>S7795-500G</td>
			<td></td>
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		<tr height="25">
			<td height="25" style="height:25px;width:400px;">NAHCO3</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td>S5761-500G</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Methanol</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td>322415-6X1L</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:400px;">KCl</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td>P9541-500G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:400px;">cOmplete Mini&#160;</td>
			<td style="width:245px;">Roche</td>
			<td style="width:119px;">11836170001</td>
			<td>protease inhibitor</td>
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			<td height="21" style="height:21px;width:400px;">PhosStop&#160;</td>
			<td style="width:245px;">Roche</td>
			<td style="width:119px;">4906837001</td>
			<td>phosphatase inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:400px;">BCA Protein Assay Kit</td>
			<td style="width:245px;">Euroclone</td>
			<td style="width:119px;">EMPO14500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Criterion TGX 4-20% Stain Free, 18 wells</td>
			<td style="width:245px;">Biorad</td>
			<td>5678094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Supported Nitrocellulose membrane&#160;</td>
			<td style="width:245px;">Biorad</td>
			<td>1620097</td>
			<td>0.2 &#956;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Blotting-Grade Blocker&#160;</td>
			<td style="width:245px;">Biorad</td>
			<td>1706404</td>
			<td>Non-fat dry milk</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:400px;">SuperSignal West Pico Chemiluminescent Substrate</td>
			<td style="width:245px;">Termo Fisher Scientific</td>
			<td style="width:119px;">34077</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Nitric acid&#160;</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td>1004411000</td>
			<td align="right">65%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Glass Coverslips&#160;</td>
			<td style="width:245px;">Termo Fisher Scientific</td>
			<td>1014355118NR1</td>
			<td>18 mm x&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Poly-D-Lysine</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td style="width:119px;">P7280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Hank&#39;s Balanced Salt Solution</td>
			<td style="width:245px;">Termo Fisher Scientific</td>
			<td style="width:119px;">14170-500 mL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Penicillin/Streptomycin&#160;</td>
			<td style="width:245px;">Termo Fisher Scientific</td>
			<td style="width:119px;">15140122</td>
			<td>10,000 U/mL, 100 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium&#160;</td>
			<td style="width:245px;">Termo Fisher Scientific</td>
			<td style="width:119px;">D5796-500 mL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Trypsin-EDTA&#160;</td>
			<td style="width:245px;">Termo Fisher Scientific</td>
			<td>15400054</td>
			<td align="right">0.50%</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:400px;">B27 Supplement&#160;</td>
			<td style="width:245px;">Termo Fisher Scientific</td>
			<td>17504044</td>
			<td>50X</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Glutamax&#160;</td>
			<td style="width:245px;">Termo Fisher Scientific</td>
			<td style="width:119px;">35050-038</td>
			<td>100x</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:400px;">DNAse</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td style="width:119px;">D5025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Fetal bovine serum</td>
			<td style="width:245px;">Euroclone</td>
			<td style="width:119px;">EC50182L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Glutamate</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td>1446600-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Gentamicin&#160;</td>
			<td style="width:245px;">Termo Fisher Scientific</td>
			<td style="width:119px;">15710</td>
			<td>10 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Neurobasal Medium&#160;</td>
			<td style="width:245px;">Termo Fisher Scientific</td>
			<td style="width:119px;">10888-022&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytosine arabinoside (AraC)</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td>C3350000&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:400px;">VECTASHIELD antifade mounting medium&#160;</td>
			<td style="width:245px;">Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:400px;">DAPI</td>
			<td style="width:245px;">Termo Fisher Scientific</td>
			<td>62247</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">90 Ti rotor</td>
			<td style="width:245px;">Beckman</td>
			<td style="width:119px;">N/A</td>
			<td>Ultracentrifuge rotor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optima L-90K Ultracentrifuge</td>
			<td style="width:245px;">Beckman</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Syn-1 antibody, clone 42&#160;</td>
			<td style="width:245px;">BD Biosciences</td>
			<td>610786</td>
			<td>anti-mouse WB: 1:5000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Syn303 antibody&#160;</td>
			<td style="width:245px;">BioLegend</td>
			<td style="width:119px;">824301</td>
			<td>anti-mouse IF: 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Tau antibody&#160;</td>
			<td style="width:245px;">Synaptic Systems</td>
			<td>314&#160;002</td>
			<td>anti-rabbit IF: 1:10,000</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:400px;">pser129-&#945;S antibody&#160;</td>
			<td style="width:245px;">A gift from Fujiwara et al, reference 19</td>
			<td style="width:119px;"></td>
			<td>anti-rabbit WB: 1:5000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:400px;">pser129-&#945;S antibody</td>
			<td style="width:245px;">Abcam</td>
			<td style="width:119px;">ab51253</td>
			<td>anti-rabbit&#160; IF: 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Mouse &#945;S (D37A6) XP&#160;</td>
			<td style="width:245px;">Cell Signaling</td>
			<td style="width:119px;">4179</td>
			<td>anti-rabbit IF 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Alexa fluor 555-conjugated anti-rabbit antibody&#160;</td>
			<td style="width:245px;">Termo Fisher Scientific</td>
			<td style="width:119px;">A27039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Alexa fluor 488-conjugated anti-mouse antibody&#160;</td>
			<td style="width:245px;">Termo Fisher Scientific</td>
			<td>A-11029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Microson XL-2000&#160;</td>
			<td style="width:245px;">Misonix</td>
			<td style="width:119px;"></td>
			<td>Sonicator&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra Bottles (Oakridge Bottles), PCB, 16x76mm, Assembly, Noryl Cap, Beckman-type</td>
			<td style="width:245px;">Science Service EU</td>
			<td style="width:119px;">S4484</td>
			<td>Ultracentrifuge tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AXIO Observer Inverted Light Microscope&#160;</td>
			<td style="width:245px;">Zeiss</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TCS SP2 laser scanning confocal microscope</td>
			<td style="width:245px;">Leica</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inverted epi-fluorescence microscope&#160;</td>
			<td style="width:245px;">Nikon</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Triton x-100</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td>X100-500ML</td>
			<td>Nonionic surfactant&#160;</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

Universidad de Cartagena UDEC

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

Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, the olfactory bulb (OB), the dorsal motor nucleus of the vagus (DMV)3, the intermediolateral nucleus of the spinal cord 4 and the substantia nigra, providing the basis for the neuropathological staging of the disease4,5. The ENS and the OB are the most exposed nervous structures and the first ones to be affected. Interestingly, PD has been related to pesticide exposure6-8. Here we show in detail two methods used in our previous study 9. In order to analyze the effects of rotenone acting locally on the ENS, we administered rotenone using a gavage to one-year old C57/BL6 mice. Rotenone is a widely used pesticide that strongly inhibits mitochondrial Complex I 10. It is highly lipophylic and poorly absorbed in the gastrointestinal tract 11. Our results showed that the administration of 5 mg/kg of rotenone did not inhibit mitochondrial Complex I activity in the muscle or the brain. Thus, suggesting that using our administration method rotenone did not cross the hepatoportal system and was acting solely on the ENS. Here we show a method to administer pesticides using a gavage and the image analysis protocol used to analyze the effects of the pesticide in alpha-synuclein accumulation in the ENS. The first part shows a method that allows intragastric administration of pesticides (rotenone) at a desired precise concentration. The second method shows a semi-automatic image analysis protocol to analyze alpha-synuclein accumulation in the ENS using an image analysis software.

Parkinson's disease has been related to the exposure to pesticides. Here we show a method to deliver pesticides using a gastric tube at the desired concentration and a method to analyze their effect in alpha-synuclein accumulation in the enteric nervous system.

鱼藤酮灌胃和图像分析α- synuclein的夹杂物在肠道神经系统的一个使用口服

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, ...

科研

神经科学

Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis

Antibodies provide the ability to gain novel insight into various events taking place in living systems. The ability to produce highly specific antibodies to target proteins has allowed for very precise biological questions to be addressed. Importantly, antibodies have been implicated in the pathogenesis of a number of human diseases including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), paraneoplastic syndromes, multiple sclerosis (MS) and human T-lymphotropic virus type 1 (HTLV-1) associated myelopathy/tropical spastic paraparesis (HAM/TSP) 1-9. How antibodies cause disease is an area of ongoing investigation, and data suggests that interactions between antibodies and various intracellular molecules results in inflammation, altered cellular messaging, and apoptosis 10. It has been shown that patients with MS and HAM/TSP produce autoantibodies to the intracellular RNA binding protein heterogeneous ribonuclear protein A1 (hnRNP A1) 3, 5-7, 9, 11. Recent data indicate that antibodies to both intra-neuronal and surface antigens are pathogenic 3, 5-9, 11. Thus, a procedure that allows for the study of intracellular antibody:protein interactions would lend great insight into disease pathogenesis.
Genes are commonly transfected into primary cells and cell lines in culture, however transfection of antibodies into cells has been hindered by alteration of antibody structure or poor transfection efficiency 12. Other methods of transfection include antibody transfection based on cationic liposomes (consisting of DOTAP/DOPE) and polyethylenimines (PEI); both of which resulted in a ten-fold decrease in antibody transfection compared to controls 12. The method performed in our study is similar to cationic lipid-mediated methods and uses a lipid-based mechanism to form non-covalent complexes with the antibodies through electrostatic and hydrophobic interactions 13. We utilized Ab-DeliverIN reagent, which is a lipid formulation capable of capturing antibodies through non-covalent electrostatic and hydrophobic interactions and delivering them inside cells. Thus chemical and genetic couplings are not necessary for delivery of functional antibodies into living cells. This method has enabled us to perform various antibody tracing and protein localization experiments, as well as the analyses of the molecular consequences of intracellular antibody:protein interactions 9.
In this protocol, we will show how to transfect antibodies into neurons rapidly, reproducibly and with a high degree of transfection efficiency. As an example, we will use anti-hnRNP A1 and anti-IgG antibodies. For easy quantification of transfection efficiency we used anti-hnRNP A1 antibodies labelled with Atto-550-NHS and FITC-labeled IgG. Atto550 NHS is a new label with high molecular absorbtion and quantum yield. Excitation source and fluorescent filters for Atto550 are similar to Cy3 (Ex. 556 Em. 578). In addition, Atto550 has high photostability. FITC-labeled IgG were used as a control to show that this method is versatile and not dye dependent. This approach and the data that is generated will assist in understanding of the role that antibodies to intracellular target antigens might play in the pathogenesis of human diseases.

A rapid approach to investigate interactions and effects on molecular mechanisms related to the presence of antibodies in an intracellular environment is described. The method involves transfection of antibodies into live cells using a non-covalent complex formation based on a lipid formulation. The technique is adaptable to immortalized cell lines and primary cells.

抗体转染神经元作为一种工具来研究疾病的发病机制

Antibodies provide the ability to gain novel insight into various events taking place in living systems.  The ability to produce highly specific ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

原代海马神经元的磷酸钙转染

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

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, ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

器官海马切片培养模型研究肿瘤细胞的神经保护和侵袭性

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

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 ...

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), Huntington&#39;s disease (HD), and amyotrophic lateral sclerosis (ALS). Protein aggregates are closely associated with neuropathology in these diseases, although the exact mechanism by which aberrant protein aggregation disrupts normal cellular homeostasis is not known. Emerging data provide strong support for the hypothesis that pathogenic aggregates in AD, PD, HD, and ALS have many similarities to prions, which are protein-only infectious agents responsible for the transmissible spongiform encephalopathies. Prions self-replicate by templating the conversion of natively-folded versions of the same protein, causing spread of the aggregation phenotype. How prions and prion-like proteins in AD, PD, HD, and ALS move from one cell to another is currently an area of intense investigation. Here, a Drosophila melanogaster model that permits monitoring of prion-like, cell-to-cell transmission of mutant huntingtin (Htt) aggregates associated with HD is described. This model takes advantage of powerful tools for manipulating transgene expression in many different Drosophila tissues and utilizes a fluorescently-tagged cytoplasmic protein to directly report prion-like transfer of mutant Htt aggregates. Importantly, the approach we describe here can be used to identify novel genes and pathways that mediate spreading of protein aggregates between diverse cell types in vivo. Information gained from these studies will expand the limited understanding of the pathogenic mechanisms that underlie neurodegenerative diseases and reveal new opportunities for therapeutic intervention.

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Accumulating evidence supports the idea that pathogenic protein aggregates associated with neurodegenerative diseases spread between cells with prion-like properties. Here, we describe a method that enables visualization of cell-to-cell spreading of prion-like aggregates in the model organism, Drosophila melanogaster.

监测果蝇中朊病毒样蛋白聚集物的细胞间传输

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), ...

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity &#947;-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

自体影像学作为一种简单而有力的药理靶标可视化和表征方法

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...

Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the diagnosis still relies only on clinical signs of motor involvement that appear later on in the disease course, when most of the dopaminergic neurons are already lost. Hence, there is a strong need for a biomarker that can identify patients at the beginning of disease or at the risk of developing it. Over the last few years, skin biopsy has proved to be an excellent research and diagnostic tool for peripheral nerve diseases such as small fiber neuropathy. Interestingly, a small fiber neuropathy and alpha synuclein (&#945;Syn) neural deposits have been shown by skin biopsy in PD patients. Indeed, skin biopsy has the great advantage of being an easily accessible, minimally invasive and painless procedure that allows the analysis of peripheral nervous tissue prone to the pathology. Moreover, the possibility of repeating the skin biopsy in the course of the follow-up of the same patient allows studying the longitudinal correlation with the disease progression. We set up a standardized reliable protocol to investigate the presence of &#945;Syn aggregates in skin nerve fibers of the PD patient. This protocol involves few short fixation steps, a cryotome sectioning and then a free-floating immunofluorescence double-staining with two specific antibodies: anti Protein Gene Product 9.5 (PGP9.5) to mark the cutaneous nerve fibers and anti 5G4 for detecting &#945;Syn aggregates. It is a versatile, sensitive and easy to perform protocol that can also be applied for targeting other proteins of interest in skin nerves. The ability to mark &#945;Syn aggregates is another step forward to the use of skin biopsy as a tool for establishing a pre-mortem histopathological diagnosis of PD.

Here, we present a protocol for a free-floating indirect immunofluorescence assay on skin biopsy sections that allows for the identification of disease specific conformation variants of alpha synuclein involved in Parkinson disease and multiple proteins of the peripheral nervous system.

通过自由浮动免疫荧光测定,在皮外神经纤维中定位阿尔法核糖核酸聚合体

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the ...

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 ...