Assessing the degradation of tau in primary neurons: The role of autophagy.

ABSTRACT

Tau is a neuronal cytosolic, highly regulated protein. Although first identified as a protein that binds and stabilizes microtubules, it is now clear that tau plays numerous other roles in neurons. In addition to its key physiological roles in neuronal structure and function, tau is also involved in the pathogenesis of Alzheimer's disease and numerous other neurodegenerative disorders. In all tauopathies, there are pathogenic accumulations of tau. Given that tau homeostasis requires a balance of synthesis and degradation, understanding the pathways that mediate tau clearance and regulate this process in the disease state is of fundamental importance. In neurons, macroautophagy (referred to as autophagy in this chapter) plays a pivotal role in clearing damaged or misfolded proteins under normal conditions. However, in the disease state autophagy is impaired and tau may not be efficiently targeted for degradation which contributes to the increases in pathological tau species. Therefore, establishing model systems that allow for the analysis of tau clearance by autophagy and quantitative assessment of interventions that increase autophagy and tau clearance are needed. Of particular importance is the use of primary neurons as a model system, as they are more reflective of the relevant in vivo autophagy pathway than clonal or immortalized cell models. In this chapter we present detailed methods for the preparation of neurons, immunoblotting and imaging analyses, genetic and pharmacological manipulation of autophagy with analyses, and methods to quantitatively measure changes in tau and phospho-tau levels.