Analysis of the Concerted Functions of Major Cathepsin Proteases and their Role in Neurodegenerative Diseases

Cathepsins (CTS) are acid lysosomal proteases classified by their structure and catalytic mechanisms. Impairment of CTSs is associated with lysosomal storage diseases, neurodegeneration and cancer. Despite their role for lysosomal proteolysis, there is a need to understand how CTS are functionally embedded in the network of lysosomal proteases and protein degradation pathways.

This study focused on major CTSs expressed in human neuroblastoma-derived SH-SY5Y cells. Using CRISPR/Cas9 editing, CTSB-, CTSD-, CTSL and CTSBDL-triple deficient (KO) SH-SY5Y cells were generated. The investigations of these cell lines revealed significantly enlarged lysosomes filled with storage material in multiple CTS-KO cell lines. Despite the lack of the three major CTs autophagic flux assays pointed towards an only mild impairment. To obtain insight into the substrate usage of the different CTSs, proteome analysis of whole-cell lysates from wildtype and CTS-depleted cells was performed. The proteome analysis revealed overexpressed proteins connected to the autophagy system. In addition, amyloid precursor protein (APP) and APP fragments, which are linked to amyloid-ß (Aß) peptide production later forming plaques in Alzheimer's Disease (AD), are processed through CTS-mediated cleavages.

Hence, another focus of this work was the analysis of the possible beneficial function of CTSD in AD. Here, it was aimed to delay the Aß plaque burden, neuroinflammation and microgliosis by boosting the lysosomal protein turnover with recombinant human pro-CTSD (rhpro-CTSD). To deliver high concentrations of CTSD to lysosomes of cells within the mouse brain, rhpro-CTSD was intracranially injected. Careful monitoring revealed uptake and maturation of the pro-CTSD enzyme. However, no evidence of CTSD having therapeutic effects on APP cleavage, Aß burden, neuroinflammation and microgliosis was found, ruling CTSD out as suitable drug target for AD.


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