Amyloid beta (Abeta), a key pathogenic factor in Alzheimer's disease (AD), accumulates and forms toxic oligomers in the brain as a result of overproduction or inefficient clearance. Thus, pathways regulating Abeta degradation and clearance are prime candidates for therapeutic intervention. We discovered that cathepsin B (CatB), a cysteine protease, degrades Abeta in vitro and in vivo. In more recent studies, we showed that the Abeta -degrading activity of CatB is inhibited by its endogenous inhibitor, cystatin C (CysC), and that reducing CysC enhances CatB- induced Abeta degradation and protects against Abeta -associated synaptic and behavioral deficits. However, unlike some other Abeta degradation enzymes, such as neprilysin (NEP, an endopeptidase with optimal activity at a neutral pH), CatB truncates Abeta at the C-terminus with optimal activity at acidic pHs. CatB also appears to be more effective than NEP in reducing higher orders of Abeta assemblies in vivo. These observations suggest that CatB and NEP may play complementary roles in Abeta degradation. The objectives of this proposal are to determine the cellular mechanisms of the CatB-CysC axis and how it and NEP might work together to regulate Abeta degradation. In Specific Aim 1, we will assess the effects of the neuronal CatB-CysC axis on Abeta degradation and neuronal synaptic function in vivo. The effects of microglia- and neuron-derived CatB will be compared. In Specific Aim 2, we will determine if neuronal CatB-CysC axis acts in the endosomal-lysosomal pathway to regulate Abeta degradation. Understanding the subcellular mechanisms of CatB-CysC axis is a prerequisite for the development of therapeutic strategies that target this newly identified pathway. In Specific Aim 3, we will examine the role of NEP in CatB-induced Abeta degradation and the role of CatB in NEP-induced Abeta degradation. By defining the interplay between the CatB-CysC axis and NEP, new insights into molecular mechanisms regulating Abeta -degradation will likely emerge. Confirmation that the CatB-CysC axis action acts in a complementary manner with NEP would lay the foundation for designing effective Abeta clearance strategies.