Alzheimer's Research Unit, MassGeneral Institute for Neurodegenerative Disease, MGH, Harvard Medical School, CNY114, 16th St., Charlestown, MA 02129, USA.
Methods. 2011 Mar;53(3):194-200. doi: 10.1016/j.ymeth.2010.12.032. Epub 2010 Dec 25.
Understanding how specific proteins are degraded by neurons in living animals is a fundamental question with relevance to many neurodegenerative diseases. Dysfunction in the ubiquitin-proteasome system (UPS) specifically has been implicated in several important neurodegenerative diseases including Alzheimer's Disease, Parkinson's Disease, and amyotrophic lateral sclerosis. Research in this area has been limited by the fact that many inhibitors of the UPS given systemically do not cross the blood-brain barrier (BBB) in appreciable levels. This limits the ability to easily test in vivo specific hypotheses generated in reduced systems, like brain slice or dissociated cell culture, about whether the UPS may degrade a particular protein of interest. Although several techniques including intracerebral application via direct syringe injection, catheter-pump systems and drug-eluting beads are available to introduce BBB-impermeant drugs into brain they each have certain limitations and new approaches could provide further insights into this problem. In order to test the role of the UPS in protein degradation in vivo we have developed a strategy to treat mouse cortex with the UPS inhibitor clasto-lactacystin beta-lactone (CLBL) via a "cranial window" and recover the treated tissue for immunoblot analysis. This approach can be used in several different cranial window configurations including single window and double hemi-window arrangements that are tailored for different applications. We have also developed two different strategies for recovering treated cortical tissue including a vibratome/laser capture microscopy (LCM)-based and a vibratome only-based approach, each with its own specific advantages. We have documented UPS inhibition >600μm deep into the cortex with this strategy. This set of techniques in the living mammalian brain is complementary to previously developed approaches and extends the repertoire of tools that can be used to the study protein degradation pathways relevant to neurodegenerative disease.
了解特定蛋白质在活体动物神经元中降解的机制是一个具有重要意义的基础问题，与许多神经退行性疾病都相关。特别是泛素-蛋白酶体系统（UPS）的功能障碍与几种重要的神经退行性疾病有关，包括阿尔茨海默病、帕金森病和肌萎缩侧索硬化症。该领域的研究受到限制，因为许多全身性给予的 UPS 抑制剂并不能以可观的水平穿过血脑屏障（BBB）。这限制了在体内测试特定假说的能力，这些假说在减少系统中产生，如脑片或分离细胞培养，关于 UPS 是否可以降解特定的感兴趣的蛋白质。尽管有几种技术，包括通过直接注射器注射、导管-泵系统和载药珠，可将不透血脑屏障的药物引入大脑，但每种技术都有一定的局限性，新的方法可以为这个问题提供进一步的见解。为了测试 UPS 在体内蛋白质降解中的作用，我们开发了一种通过“颅窗”用 UPS 抑制剂 clasto-lactacystin beta-lactone（CLBL）处理小鼠皮层的策略，并回收处理过的组织进行免疫印迹分析。这种方法可以在几种不同的颅窗配置中使用，包括用于不同应用的单窗和双半窗排列。我们还开发了两种回收处理过的皮层组织的策略，包括基于振动切片/激光捕获显微镜（LCM）和仅基于振动切片的方法，每种方法都有其自身的特定优势。我们已经记录了这种策略下 UPS 抑制作用可深入皮层 600μm 以上。这组技术在活体哺乳动物大脑中与以前开发的方法互补，并扩展了可用于研究与神经退行性疾病相关的蛋白质降解途径的工具库。
