Neuroprotective functions of the APPI domain of amyloid precursor protein (APP): Reducing KLK6-mediated APP cleavage...

Amyloid beta 42 (Aβ42) and amyloid precursor protein inhibitor (APPI) are amyloid precursor protein (APP) domains. As fragments, Aβ42 is neurotoxic and APPI inhibits kallikrein-related peptidase 6 (KLK6). While KLK6-dependent cleavage of APP695 (an APP isoform that lacks APPI) has been shown to result in Aβ production, its proteolytic activity on APPI-containing isoforms (APP751 and APP770) and the molecular function of APPI in APP proteolysis have not been explored. In addressing this knowledge gap, we show that APP695 serves as a KLK6 substrate in SH-SY5Y neuroblastoma cells. We also show that APPI, as either a soluble fragment or an APP751 domain, strongly inhibits KLK6 catalytic activity and reduces KLK6-mediated APP695 cleavage in a dose-dependent manner. Moreover, the soluble extracellular APPI fragment reduces Aβ42 aggregation and fibril length, thereby favoring the formation of Aβ42 oligomers that cannot permeate into cells and thus significantly reducing apoptosis in SH-SY5Y neuroblastoma cells. In a similar way, the expression of both intracellular APPI and Aβ42 (vs. Aβ42 alone) reduces Aβ42 aggregate formation and increases neuronal cell viability. Our results provide an explanation for the presence of alternatively spliced APP isoforms in the brains of patients with Alzheimer's disease (AD) and reveal the roles of APPI in APP cleavage and in the formation, aggregation and toxicity of both extra- and intracellular Aβ peptide fragments. Our results suggest that APPI may serve as a dual inhibitor, disrupting both KLK6 proteolytic activity and Aβ42 aggregation, thereby offering a potential therapeutic agent for reducing amyloid beta toxicity in AD.