In the present study, we have made three principal observations about the in vivo
function of Mint/X11 proteins in APP cleavage that may be important for our thinking of the biology of Mint/X11 proteins and about the pathogenesis of AD:
- Deletion of Mint proteins decreases plaque formation at 6-9 months of age with Mint2 deletion the most potent suppressor of Aβ production, most likely due to the relative expression levels of these proteins in the brain (Figs. -).
- Deletion of Mints decreases the extracellular cleavage of APP by β-secretase and possibly α-secretase in neurons, but does not appear to alter intramembranous cleavage of the APP-CTF by γ-secretase (Figs. -). The observed effects are primarily mediated by β-secretase because we measured in our experiments changes in Aβ peptides that are produced by β-secretase, and because C99 is affected more strongly than the combined C83 and C89 APP-CTFs which are produced by α- and β-cleavage, respectively.
- Deletion of Mints does not significantly alter γ-secretase activity since it does not dramatically increase APP-CTF levels in cultured neurons or adult Mint knockout mice ( and Suppl. Fig. 6-7) and it does not change the kinetics of APP-CTF cleavage ().
Our results differ from earlier conclusions that suggested that Mints regulate γ-secretase (see Suppl. Table 1
) and demonstrates a role for Mint proteins as intracellular adaptor proteins in regulating extracellular cleavage reaction. However, it is important to note that since β-cleavage of APP is predominantly intracellular in endosomes that perhaps Mint proteins may indeed affect intra-endosomal pathway in APP processing. The following conditions of our experiments ensure the specificity of our results. First, different from earlier studies, we employed both acute and constitutive deletions of Mints/X11s. Second, we used two different mouse models of AD to investigate the effects of Mint deletions. Third, we studied both intact brains and cultured neurons. Fourth, we measured a variety of parameters using a panoply of different antibodies. Lastly, we tested three different Mint isoforms in independent lines of mice, excluding genetic position effects.
Different from our results, previous studies suggested that Mints control γ-cleavage of APP-CTFs. Specifically, overexpression studies of Mints/X11suggested that overexpressed Mints inhibit γ-cleavage of APP-CTFs (Suppl. Table 1
), whereas RNAi knockdowns indicated that decreases in Mint1 or Mint2 levels also inhibit γ-cleavage of APP-CTFs and thereby decrease Aβ-production (Xie et al., 2005
). It is difficult to understand how overexpression and knockdowns of a protein has the same effects. Overexpressed proteins often assume functions incidental to those of the endogenous proteins, whereas RNAi occasionally exhibits off-target effects that need to be control for by rescue experiments. Furthermore, since γ-secretase has a major developmental role via
Notch cleavage, any change in γ-secretase by Mint/X11 deletions should have led to a developmental phenotype that was not observe in Mint-deficient mice (Ho et al., 2003
). Finally, a Mint1 and 2 knockout study (Sano et al., 2006
; Saito et al., 2008
) found an increase in endogenous mouse APP-CTFs and Aβ with a decrease in secreted APPs, changes that are difficult to reconcile but possibly due to strain differences and genetic background between our knockout mice. Our data currently points that Mint isoforms may indeed perform distinct functions in APP processing in brain due to their differential expression, but not due to intrinsic differences in their functions. Mint1 is expressed mostly in inhibitory neurons whereas Mint2 is expressed higher in excitatory neurons preferentially in the pyramidal neurons of the hippocampus and deletion of Mint1 and 2 have been shown to alter inhibitory and excitatory synaptic transmission, respectively (Ho et al., 2003
). It is possible that deletion of Mint proteins affects synaptic transmission thereby modulating the formation and secretion of amyloid peptides since neural activity has been shown to control APP processing (Kamenetz et al., 2003
What is the mechanism by which intracellular Mints modulate extracellular APP cleavage? The phenotype of the Mint knockouts described here is most consistent with an effect of Mints on APP trafficking, even though the steady-state distribution of APP and cell surface APP levels were not changed by deletion of Mints (Suppl. Fig. 8
). A role for Mints in protein trafficking is supported by two interactions that have been previously described. First, their binding to Munc18 proteins that are involved in plasma membrane fusion reactions, an interaction that is consistent with our previous Mint knockout analyses (Ho et al., 2003
), and second, their binding to arfs that are particularly important for Golgi membrane trafficking (Hill et al., 2003
). It also agrees with the co-localization of Mints with APP in the trans-Golgi network of neurons (Borg et al., 1996
; Sastre et al., 1998
; Biederer et al., 2002
). In neurons, the majority of APP is axonally transported to the synaptic terminal and upon endocytosis of cell surface APP directs it towards an amyloidogenic processing pathway (Nordstedt et al., 1993
; Ikin et al., 1996
; Marquez-Sterling et al., 1997
). However, the lack of APP enrichment in presynaptic terminals indicates that APP is not stably retained in the terminal. Therefore, it is conceivable that Mint proteins could alter APP trafficking either by altering APP secretory pathway or endocytotic trafficking of reinternalized APP thereby affecting APP processing, but further studies will be necessary to investigate this possibility.
The striking effects of Mint deletions on Aβ-production and Aβ-plaque formation suggest that even small changes in the expression of Mints, when present chronically, could have dramatic consequences for the production of Aβ, the deposition of Aβ-plaques, and the development of AD. Thus, increases in Mint expression, even if small, may be a risk factor for AD, and a possible therapeutic strategy to decrease Aβ-production could be to interfere with the Mint/APP interaction. If a pharmacological avenue to selectively block the Mint/APP interaction could be found, such a strategy would be attractive because of the relatively mild effects of deletions of either APP or individual Mints, and such a selective block would presumably not impair the other functions of Mints and APP.