治疗SCA1小鼠水溶性化合物，以非特异性升压线粒体功能
Summary
We present a biochemical and behavioral protocol to evaluate the efficacy of mitochondria-targeted water-soluble compounds for the treatment of Spinocerebellar ataxia type 1 (SCA1) and other cerebellar neurodegenerative diseases.
Abstract
线粒体功能障碍起着老化过程，并在神经退行性疾病，包括遗传性的几个小脑萎缩症和小脑进行性变性标记等运动障碍一个显著的作用。该协议的目的是评估在脊髓小脑性共济失调1型（SCA1）线粒体功能障碍，并通过该水溶性化合物琥珀酸评估代谢呼吸药理定位的功效减缓疾病进展。这种方法也适用于其他小脑疾病和可适应的水溶性治疗的宿主。
线粒体呼吸的离体分析被用于检测和量化在线粒体功能疾病相关的变化。遗传证据（未发表的数据），并在SCA1小鼠模型线粒体功能障碍蛋白质组学的证据，我们评价与水溶性代谢增压待遇的功效uccinic酸由该化合物直接溶解到笼子的饮用水。药物的通过血脑屏障的能力可使用高效液相色谱（HPLC）来推导。这些化合物的疗效然后可以使用多种行为范式，包括加速转棒，平衡木试验和足迹分析测试。小脑Cytoarchitectural完整性可以使用检测浦肯野细胞的细胞核和浦肯野细胞树突和胞体免疫荧光法进行评估。这些方法是用于确定线粒体功能障碍和治疗与小脑的神经变性疾病的水溶性化合物的功效健壮的技术。
Introduction
线粒体是三磷酸腺苷（ATP），为细胞能量一种必需的辅酶的关键生产，大多数使用电子传递链通过氧化磷酸化（OXPHOS）产生的线粒体ATP。脑，鉴于其高代谢需求及对氧化磷酸化进行供电的神经活动的依赖，是高度敏感的线粒体功能障碍。其结果是，线粒体功能障碍是在老化过程中触发 1，并在多个神经变性疾病2，3，4的发病机制有关。因此，它遵循线粒体是神经变性吸引力的治疗目标。
在这个协议中，我们已经采用脊髓小脑性共济失调1型（SCA1）作为模型神经退行性疾病线粒体的研究升功能障碍和线粒体靶向疗法的开发。 SCA1是通过在共济失调蛋白-1基因产物触发小脑浦肯野神经元和神经元其他脑区域中的进行性变性一个多聚谷氨酰胺（的polyQ）重复扩张突变引起的。此处所用的转基因小鼠系（指定为SCA1小鼠），它表达了浦肯野细胞特异性启动子的控制下的polyQ突变共济失调蛋白-1基因，允许SCA1 5的浦肯野细胞成分的针对性的分析。 SCA1小鼠进行逐步浦肯野细胞变性和发展共济失调步态6。
线粒体复合物功能障碍和线粒体靶向治疗功效可与分子和行为分析的电池进行评价。线粒体复合物功能障碍是测定体外通过检测在小脑组织内改变氧消耗呼吸测定存在电子传递链底物和抑制剂7。呼吸检测先前已经用于透化组织，线粒体分离物，和全组织7，8，9。它们允许不同形态的数据收集方法，如透射电子显微镜或免疫荧光染色线粒体功能的直接评估。利用整个组织，而不是孤立的线粒体防止在分离过程7可能出现的健康线粒体的偏倚选择。当如图适于协议，所述呼吸检测是在小脑的神经变性疾病状态检测线粒体功能障碍的有价值的方法。
代谢的非特异性活化剂可以用来推断线粒体功能障碍的神经变性diseas的转基因小鼠模型e和在新疗法的发展援助。槲皮素，辅酶Q10和肌酸都被证明改善神经变性疾病的病理的患者，并在神经变性疾病10，11，12，13，14，15，16的动物模型。这里，我们提出了一种新的代谢活化剂，琥珀酸，刺激代谢，促进线粒体功能中的神经变性疾病。以确保该活化剂穿越血脑屏障，高效液相色谱法检测在处理的小鼠17递送至神经组织。
为了评价如琥珀酸代谢靶向水溶性化合物的治疗效果，可以使用行为范例和免疫病理研究的电池。杜E要在小脑的神经变性疾病，足迹跑道试验，梁法和加速旋转杆实验发现，运动协调障碍是用来检测行为病理6，18，19日抢救。这些措施是由一个定义的小脑组织6，20，21的小叶内评估分子层的厚度（定义为浦肯野细胞树突长度）和浦肯野细胞胞体计数补充有小脑细胞构筑的免疫病理评估。在这里，我们提出了诊断和治疗的线粒体功能障碍与代谢有针对性的水溶性化合物多的神经病理学和行为的方法。
我们使用线粒体呼吸体外分析来分析线粒体功能障碍在SCA1 TRANsgenic鼠标。此外，我们表明，疾病的症状和病理学由水溶性线粒体增压琥珀酸改进，还牵连于SCA1疾病进展的线粒体功能障碍。
Protocol
Representative Results
Discussion
如果如所述使用这些方法，它们是能够检测和小脑的神经变性疾病的小鼠模型减轻氧化磷酸化介导的线粒体功能障碍。将合并的生物化学和行为测定是用于确定对小脑的神经变性疾病的病理线粒体贡献程度多方面的方法。通过与琥珀酸治疗小鼠，刺激新陈代谢，促进线粒体功能，我们能够显示小脑行为缺陷和小脑变性的救援。
这里提出的方法可以用于测试多种水溶性代谢靶?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
We would like to thank Dr. Harry Orr at the University of Minnesota for his generous gift of transgenic mice. We would also like to thank the following Skidmore College alum for their work performing the preceding experiments: Monica Villegas, Porter Hall, Mitchell Spring, Nicholas Toker, Jenny Zhang, Chloe Larson and Cheyanne Slocum. Furthermore, we would like to thank Skidmore College for funding the development of these methods.
Materials
| Adenosine diphosphate | Sigma Aldrich | A2754 | ADP |
| Ascorbate | Sigma Aldrich | A7631 | |
| Bovine serum albumin | Sigma Aldrich | A2153 | BSA |
| 4',6-Diamidino-2-phenylindole | Sigma Aldrich | D9542 | DAPI |
| Digitonin | Sigma Aldrich | D141 | |
| Dithiothreitol | Sigma Aldrich | D0632 | DTT |
| Donkey serum | Sigma Aldrich | D9663 | |
| Glutamate | Sigma Aldrich | 1446600 | |
| Malate | Sigma Aldrich | 6994 | |
| Mannitol | Sigma Aldrich | M4125 | |
| Paraformaldehyde | Sigma Aldrich | P6148 | |
| Potassium-lactobionate | Bio-Sugars | 69313-67-3 | |
| Rotenone | Sigma Aldrich | R8875 | |
| Saponin | Sigma Aldrich | 47036 | |
| Succinic Acid | Sigma Aldrich | S3674 | |
| N,N,N′,N′-Tetramethyl-p-phenylenediamine | Sigma Aldrich | T7394 | TMPD |
| Triton X-100 | Sigma Aldrich | T9284 | |
| Urea | Sigma Aldrich | U0631 | |
| Vectashield mounting medium | Vector Labs | H-1000 | |
| Antibodies | |||
| 11NQ antibody (anti-ataxin-1 ) | Servadio, et al. 1995, PMID: 7647801 | ||
| Alexa Fluor 488 anti-mouse secondary antibody | Life Technologies | A-11015 | |
| Alexa Fluor 594 anti-rabbit secondary antibody | Life Technologies | A-11012 | |
| Calbindin antibody (goat) | Santa Cruz | C-20 | |
| Animals | |||
| Control transgenic mice | Harry Orr, Ph.D. | A02 | Burright, et al. 1997, PMID: 9217978 |
| SCA1 mice | Harry Orr, Ph.D. | B05 | Burright, et al. 1997, PMID: 9217978 |
| Wildtype mice | The Jackson Laboratory | 001800 | |
| Equipment | |||
| ESM-100L microtome | ERMA | Sledge microtome | |
| Fluoview FV1200 Confocal Microscope | Olympus | ||
| Glycerol-gelatin slides | FD Neuro Technologies | PO101 | |
| Hamilton syringe | Sigma Aldrich | VCAT 80465 | |
| OXYT1 Oxytherm Electrode Control Unit | Hansatech Instruments | ||
| P.T.F.E. paper | Cole-Parmer | UX-08277-15 | |
| Rotallion Rotarod | PPP&G | contact corresponding author for information | |
| Ultimate 3000 HPLC | Dionex | ||
| Software | |||
| ImageJ | National Institute of Health | http://imagej.nih.gov/ij/ | |
| Cell counter plugin (for ImageJ) | National Institute of Health | http://rsb.info.nih.gov/ij/plugins/cell-counter.html | |
| 3P&G Rota-Rod v3.3.3 (rotarod software) | PPP&G | contact corresponding author for information | |
| Phidget21.dll (required for rotarod software) | DLL-Files.com | https://www.dll-files.com/phidget21.dll.html |
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