ABSTRACT
The ubiquitin/proteasome pathway (UPP) is the major proteolytic quality control system in cells and involves tightly regulated removal of unwanted proteins and retention of those that are essential. In addition to its function in normal protein degradation, the UPP plays a critical role in the quality control process by degrading mutated or abnormally folded proteins. The proteolytic component of the UPP is a multiprotein complex known as the proteasome. Many factors, including the aging process, can cause proteasome impairment leading to formation of abnormal ubiquitin-protein aggregates that are found in most progressive neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. In this chapter, we describe protocols to measure proteasome activity, evaluate its state of assembly, and assess the accumulation and aggregation of ubiquitinated proteins in two types of neuronal cultures: human neuroblastoma cells and rat primary cortical cultures. These protocols can be used with different types of neuronal cultures to estimate proteasome activity and the levels and aggregation of ubiquitinated proteins. In addition, they can be used to identify compounds potentially capable of preventing a decline in proteasome activity and formation of ubiquitin-protein aggregates associated with neurodegeneration.
Subject(s)
Neurons/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Multimerization , Ubiquitinated Proteins/chemistry , Ubiquitinated Proteins/metabolism , Animals , Blotting, Western , Cell Extracts , Cell Line, Tumor , Cell Separation , Centrifugation , Cerebral Cortex/cytology , Chymotrypsin/metabolism , Electrophoresis, Polyacrylamide Gel , Female , Fluorescent Antibody Technique , Glycerol/chemistry , Humans , Neurons/cytology , Pregnancy , Proteasome Endopeptidase Complex/isolation & purification , Protein Structure, Quaternary , Protein Structure, Secondary , Protein Subunits/isolation & purification , Protein Subunits/metabolism , RatsABSTRACT
Alzheimer's disease is a chronic, age-related neurodegenerative disorder. Neurofibrillary tangles are among the pathological hallmarks of Alzheimer's disease. Neurofibrillary tangles consist of abnormal protein fibers known as paired helical filaments. The accumulation of paired helical filaments is one of the most characteristic cellular changes in Alzheimer's disease. Tau protein, a microtubule-associated protein, is the major component of paired helical filaments. Tau in paired helical filaments is hyperphosphorylated, truncated, and aggregated. What triggers the formation of paired helical filaments is not known, but neuroinflammation could play a role. Neuroinflammation is an active process detectable in the earliest stages of Alzheimer's disease. The neuronal toxicity associated with inflammation makes it a potential risk factor in the pathogenesis of Alzheimer's disease. Determining the sequence of events that lead to this devastating disease has become one of the most important goals for the prevention and treatment of Alzheimer's disease. In this review, we focus on the pathological properties of tau thought to play a role in neurofibrillary tangle formation and summarize how central nervous system inflammation might be a critical contributor to the pathology of Alzheimer's disease. A better understanding of the mechanisms that cause neurofibrillary tangle formation is of clinical importance for developing therapeutic strategies to prevent and treat Alzheimer's disease. One of the major challenges facing us is singling out neuroinflammation as a therapeutic target for the prevention of Alzheimer's disease neurodegeneration. The challenge is developing therapeutic strategies that prevent neurotoxicity linked to inflammation without compromising its neuroprotective role.
