Studying protein degradation pathways in vivo using a cranial window-based approach

摘要: Understanding how specific proteins are degraded by neurons in living animals is a fundamental question with relevance to many neurodegenerative diseases. Dysfunction the ubiquitin-proteasome system (UPS) specifically has been implicated several important diseases including Alzheimer's Disease, Parkinson's and amyotrophic lateral sclerosis. Research this area limited fact that inhibitors of UPS given systemically do not cross blood-brain barrier (BBB) appreciable levels. This limits ability easily test vivo hypotheses generated reduced systems, like brain slice or dissociated cell culture, about whether may degrade particular protein interest. Although techniques intracerebral application via direct syringe injection, catheter-pump systems drug-eluting beads available introduce BBB-impermeant drugs into they each have certain limitations new approaches could provide further insights problem. In order role degradation we developed strategy treat mouse cortex inhibitor clasto-lactacystin beta-lactone (CLBL) "cranial window" recover treated tissue for immunoblot analysis. approach can be used different cranial window configurations single double hemi-window arrangements tailored applications. We also two strategies recovering cortical vibratome/laser capture microscopy (LCM)-based vibratome only-based approach, its own advantages. documented inhibition >600μm deep strategy. set mammalian complementary previously extends repertoire tools study pathways relevant disease.