We have observed genetic association of ADAM10 with AD risk and identified two novel non-synonymous mutations in the ADAM10 prodomain in seven AD families; we also assessed their effects on ADAM10 function and APP processing. Using CHO-stable cell lines expressing ADAM10 and APP, we demonstrated that Q170H and R181G mutations significantly attenuate α-secretase activity of ADAM10, leading to decreased levels of sAPPα and C83, and increased levels of Aβ. Although the two novel mutations are located in the proximity of the conserved sequences for cysteine switch and proprotein convertase recognition, they did not affect either the maturation or biosynthesis of ADAM10.
The two novel ADAM10
prodomain mutations are rare, segregating in only seven AD families (three for one mutation and four for the other) out of 1004 AD families screened. For each mutation, there were two families in which one (or two) affected individuals were found to be non-carriers, suggesting that in these individuals disease onset was influenced by other genetic factors. Because multiple genetic and environmental factors likely contribute to risk for late-onset of the disorder, onset in one (or two) affected individuals in the absence of each ADAM10 mutation is not entirely unexpected. A similar situation has been observed for AD mutations observed in presenilin 2 (N141I (34
)) and presenilin 1 (A79V (35
)), most likely owing to high prevalence of AD in the elderly population. In addition, for each mutation, there was one family in which one unaffected subject carried each mutation, suggesting incomplete penetrance for late-onset AD.
Support for the potential pathogenicity of the ADAM10 Q170H and R181G mutations was derived from experiments assessing the functional impact of these missense mutations on ADAM10 activity. For this purpose, we tried to generate cell lines stably overexpressing both APP and ADAM10 using H4 neuroglioma cells, HEK293 cells and CHO cells. We were only able to select wild-type and mutant ADAM10-expressing clones in CHO cells, most likely due to cytotoxicity caused by overexpression of both ADAM10 (36
) and APP in the two human cell lines. In the CHO-APP-ADAM10 stable cells, the Q170H and R181G mutants caused a dramatic reduction in the generation of sAPPα and APP-C83 when compared with wild-type ADAM10 (Figs and ). However, western blot analysis did not reveal increases in sAPPβ and APP-CTFβ (C99 and C89) levels in the mutant cell lines (data not shown and Fig. ). This may be due to the relatively low sensitivity of our western blot analyses for sAPPβ and APP-CTFβ. Moreover, regulated α-secretase activity by TACE has previously been shown to directly compete with β-secretase activity for APP cleavage in TGN (38
). However, catalytically active ADAM10 is mainly localized in plasma membrane (20
); thus, concurrent changes in sAPPβ and APP-CTFβ levels may be difficult to detect upon ADAM10 overexpression using western blot analysis. Aβ levels, determined by a sensitive ELISA, were consistently increased in the presence of the two ADAM10 prodomain mutations. Collectively, these data indicate that both ADAM10 mutations lead to defective α-secretase activities and subsequently elevation in Aβ levels in vitro
, suggesting potentially pathogenic roles for these mutations, pending confirmation in vivo
The molecular mechanism underlying the effects of the two novel ADAM10 mutations likely involves the prodomain function. Both mutations are located near the proprotein convertase recognition sequence (RKKR), which is required for proteolytic activation of the zymogen (29
). However, since steady-state levels of mature and immature ADAM10s were not altered significantly (Fig. D), the mutations most likely do not affect ADAM10 maturation. The mutations may instead affect the intramolecular chaperone function of the ADAM10 prodomain. The role of prodomain in proper folding of ADAM proteins, particularly the metalloprotease domain, is also supported by studies of other members of ADAM protein family. For example, the secreted soluble form of ADAM 12 (ADAM12-S) lacking the prodomain remains in the early endomembrane system and is not secreted from cells, suggesting that the prodomain might be required in folding of the metalloprotease domain to a secretion-competent conformation (39
). Truncated forms of ADAM10 and TACE lacking the prodomain have been shown to be catalytically inactive (29
). Intracellular degradation of TACE lacking the prodomain and its rescue by prodomain expression in trans
strongly suggests a chaperone role for the prodomain. Moreover, the ability of the prodomain to hold the catalytic domain of TACE in a relatively open conformation that is inactive has also been shown (40
). In the case of ADAM10, the absence of the prodomain does not lead to defective biogenesis and secretion/trafficking of the protease. ADAM10 lacking the prodomain and expressed at high levels has been detected on the plasma membrane; however, it was proteolytically inactive (29
). Interestingly, as with TACE, the ADAM10 prodomain expressed in trans
was able to restore the catalytic activity of ADAM10 lacking the prodomain. Prodomain expression has also been shown to inhibit proteolytic activity of endogenous and overexpressed wild-type ADAM10, implying its direct interaction with mature ADAM10 (29
). Future studies will be necessary to test whether the Q170H and R181G mutations can affect the chaperone function of the ADAM 10 prodomain.
In summary, we have discovered two rare, partially penetrant, familial late-onset AD mutations in the ADAM10 gene that lead to defective α-secretase activity. The fact that these two mutations were both found in late- versus early-onset familial AD (average age of onset =69.5 years for both mutations) may reflect the relatively modest effects on Aβ accumulation relative to those of the early-onset familial mutations in APP, PSEN1 and PSEN2. Furthermore, since α-secretase activity is also exerted by TACE and ADAM9, one might expect a defect in ADAM10 activity to be compensated by molecular redundancy, possibly explaining the relatively late onset of AD in carriers of these two mutations and incomplete penetrance. The novel findings presented here provide the first genetic evidence in support of a possible role for the ADAM10 gene in the etiology and pathogenesis of late-onset AD. Moreover, given the location of these two mutations in ADAM 10, these data suggest that modulation of ADAM10 activity via the prodomain could represent a novel therapeutic target for the treatment and prevention of AD.