Person:

Hemming, Matthew

The presenilin/γ-secretase complex, an unusual intramembrane aspartyl protease, plays an essential role in cellular signaling and membrane protein turnover. Its ability to liberate numerous intracellular signaling proteins from the membrane and also mediate the secretion of amyloid-β protein (Aβ) has made modulation of γ-secretase activity a therapeutic goal for cancer and Alzheimer disease. Although the proteolysis of the prototypical substrates Notch and β-amyloid precursor protein (APP) has been intensely studied, the full spectrum of substrates and the determinants that make a transmembrane protein a substrate remain unclear. Using an unbiased approach to substrate identification, we surveyed the proteome of a human cell line for targets of γ-secretase and found a relatively small population of new substrates, all of which are type I transmembrane proteins but have diverse biological roles. By comparing these substrates to type I proteins not regulated by γ-secretase, we determined that besides a short ectodomain, γ-secretase requires permissive transmembrane and cytoplasmic domains to bind and cleave its substrates. In addition, we provide evidence for at least two mechanisms that can target a substrate for γ cleavage: one in which a substrate with a short ectodomain is directly cleaved independent of sheddase association, and a second where a substrate requires ectodomain shedding to instruct subsequent γ-secretase processing. These findings expand our understanding of the mechanisms of substrate selection as well as the diverse cellular processes to which γ-secretase contributes.

Background: Understanding the mechanisms of amyloid-β protein (Aβ) production and clearance in the brain has been essential to elucidating the etiology of Alzheimer disease (AD). Chronically decreasing brain Aβ levels is an emerging therapeutic approach for AD, but no such disease-modifying agents have achieved clinical validation. Certain proteases are responsible for the catabolism of brain Aβ in vivo, and some experimental evidence suggests they could be used as therapeutic tools to reduce Aβ levels in AD. The objective of this study was to determine if enhancing the clearance of Aβ in the brain by ex vivo gene delivery of an Aβ-degrading protease can reduce amyloid plaque burden. Methods and Findings: We generated a secreted form of the Aβ-degrading protease neprilysin, which significantly lowers the levels of naturally secreted Aβ in cell culture. We then used an ex vivo gene delivery approach utilizing primary fibroblasts to introduce this soluble protease into the brains of β-amyloid precursor protein (APP) transgenic mice with advanced plaque deposition. Brain examination after cell implantation revealed robust clearance of plaques at the site of engraftment (72% reduction, p = 0.0269), as well as significant reductions in plaque burden in both the medial and lateral hippocampus distal to the implantation site (34% reduction, p = 0.0020; and 55% reduction, p = 0.0081, respectively). Conclusions: Ex vivo gene delivery of an Aβ-degrading protease reduces amyloid plaque burden in transgenic mice expressing human APP. These results support the use of Aβ-degrading proteases as a means to therapeutically lower Aβ levels and encourage further exploration of ex vivo gene delivery for the treatment of Alzheimer disease.