Both the amount and type of Aβ produced are important for AD pathogenesis. Thus, increased production of Aβ or increased production of Aβ1-42 relative to Aβ1-40 can accelerate the production of senile plaques and the development of AD. In addition, the subcellular sites where Aβ is produced may also play a role in AD pathogenesis. APP and the enzymes that cleave it colocalize throughout the secretory pathway, and Aβ can be produced at multiple intracellular sites. In neurons, APP undergoes fast anterograde transport to nerve terminals (Koo et al., 1990
; Ferreira et al., 1993
; Buxbaum et al., 1998
), and is metabolized into Aβ peptides that are released and deposited as amyloid plaques around nerve terminals (Lazarov et al., 2002
; Sheng et al., 2002
). Thus, the axonal/synaptic fractions of APP appear particularly important in the generation of Aβ species that are ultimately deposited in amyloid plaques.
To further explore the relationships between the quantitative, qualitative and spatial factors that influence Aβ production and deposition, we produced Tg mice that expressed increased levels of BACE. Surprisingly, an inverse relationship between BACE expression and Aβ production/deposition was found. Our efforts to understand this paradoxical result led us to discover that BACE overexpression shifted the sites of APP processing such that APP proteolysis occurred predominantly in neuronal perikarya rather than in axons and axon terminals (). This alteration of APP processing upon BACE overexpression, together with the reduction of Aβ accumulation, indicates that Aβ generated proximally in neuronal perikarya has a different fate than Aβ that is generated at or near the synapse.
Figure 9. Subcellular processing of APP and the development of Aβ amyloid pathology. In monogenic APP mice (red), APP is synthesized in the ER and trafficked to the Golgi apparatus acquiring N- and O-linked oligosaccharides within these organelles. APP (more ...)
Several lines of evidence were provided to demonstrate the depletion of APP before axonal transport. First, BACE overexpression reduced α-cleavage as seen by the reduction in C83. Since our previous studies have shown that α-secretase competes with BACE within the TGN (Skovronsky et al., 2000
), the reduction of C83 in APPxBACE mice suggests that β-cleavage of APP is enhanced within the TGN. Second, N
- and O
-glycosylated APP was reduced in BACE overexpressing mice indicating that APP is cleaved soon after carbohydrate processing in the Golgi apparatus. Third, although BACE overexpression increased C99 levels, the majority of C99 was not phosphorylated. In contrast, at least 50% of C99 was phosphorylated in monogenic APP mice. Because phosphorylation of threonine 668 of APP is a posttranslational modification found selectively within neuronal growth cones and neurites (Ando et al., 1999
; Iijima et al., 2000
), the lack of phospho-C99 indicates that APP cleavage in APPxBACE mice occurs in early compartments. Fourth, although mature full-length phospho-APP was readily detected from the corpus callosum, sciatic nerves and optic nerves of APP mice, almost no mature, phospho-APP was detected in APPxBACE bigenic mice demonstrating that APP was present only at very low levels in axons. Fifth, 32
P-labeled APP was dramatically reduced in optic nerves upon BACE overexpression, indicating that β-cleavage occurs before the phosphorylation and anterograde axonal transport of APP. Sixth, the reduction of APP accumulation upon sciatic nerve ligation supports the hypothesis that little intact APP is transported into axons in APPxBACE mice. Finally, pulse-labeling of spinal cords demonstrated that BACE overexpression increases APP turnover such that APP is not available for axonal transport. Notably, BACE overexpression did not alter APP synthesis, nor levels of immature ER-resident APP suggesting that APP cleavage is occurring within the Golgi after transit from the ER. Based on the evidence presented above, we concluded that BACE overexpression increases β-cleavage in proximal subcellular compartments, most likely within the Golgi apparatus, at the expense of axonal and synaptic APP ().
Despite increased β-cleavage, as evidenced by increased sAPPβ, sAPPβ′, sAPPβswe, and C99 levels in APPxBACE mice, Aβ levels were reduced by high BACE expression. This finding is contrary to cell culture models in which BACE overexpression leads to increased secretion of Aβ (Liu et al., 2002
; E.B. Lee et al., 2003
). One potential explanation of our results is that the fate of Aβ produced in neuronal perikarya and axonal terminals in the in vivo brain are different. For example, the microenvironment wherein Aβ is secreted may influence Aβ deposition. Synaptic zinc has been shown to play a role in Aβ aggregation and deposition, and depletion of synaptic zinc inhibits amyloid formation in vivo (Lee et al., 2002
) suggesting that the environment within synaptic vesicles or the microenvironment at synaptic terminals is crucial to Aβ amyloidogenesis.
Alternatively, the differential fates of Aβ may be related to the localization of Aβ degrading activities in brain. Neprilysin is found predominantly in synapses and axons of smaller interneurons (Fukami et al., 2002
). The localization and enzymatic properties of neprilysin are consistent with Aβ degrading activity within secretory vesicles and the plasma membrane (Iwata et al., 2000
). In contrast, endothelin-converting enzyme is likely to degrade intracellular Aβ within acidic organelles such as the TGN (Schweizer et al., 1997
; Eckman et al., 2001
). Finally, cell surface and secreted forms of insulin-degrading enzyme (IDE) have been implicated in Aβ catabolism (Qiu et al., 1998
; Vekrellis et al., 2000
). Genetic ablation of IDE results in accumulation of unphosphorylated APP fragments without altering phosphorylated fragments, suggesting that IDE activity is localized predominantly near the cell soma (Farris et al., 2003
). All three of these enzymes have been shown to influence steady-state levels of Aβ in vivo, and may serve complimentary roles in Aβ catabolism (Iwata et al., 2001
; Eckman et al., 2003
; Farris et al., 2003
; Miller et al., 2003
). Thus, altering the subcellular localization of β-cleavage may disrupt the normal catabolic pathways of Aβ, thereby accounting for the different fates of somatic and synaptic Aβ.
In support of the view that the ability of certain Aβ peptides to deposit into amyloid plaques is related to their susceptibility to degradation, Aβ peptides of different lengths were differentially affected by BACE overexpression. The rate-limiting step of Aβ degradation in vivo is the production of Aβ10-37 (Iwata et al., 2000
), suggesting that NH2
-terminal truncations may render Aβ peptides more prone to degradation. We found that minimal amounts of the NH2
-terminally truncated Aβ11-40/42 peptide could be detected upon BACE overexpression despite the increase in C89 levels, supporting the hypothesis that this NH2
-terminal truncated variant is easily degraded in vivo.
In APPxBACE bigenic mice overexpressing the lowest levels of BACE, we observed an increase in Aβ deposition in neocortex, supporting the idea that slight elevations in BACE expression and activity may facilitate the development of AD (Fukumoto et al., 2002
; Holsinger et al., 2002
). Furthermore, lower levels of BACE overexpression than those reported here increase steady-state Aβ levels in Tg mice (Bodendorf et al., 2002
). Nonetheless, the effects of BACE overexpression were not specific to APP harboring the Swedish mutation as BACE overexpression resulted in similar reductions of endogenous and exogenous mature APP in non-Tg mice, APPΔI Tg mice, and human NTera2 cells (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200407070/DC1
Regardless of the expression level, Aβ deposition was inhibited in the hippocampus. Even at 20 mo old when APPxBACE-M mice accumulate a large amount of brain Aβ deposits, hippocampal Aβ deposits are markedly reduced (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200407070/DC1
). We postulate that the region-specific effects of BACE overexpression on Aβ pathology are related to qualitative or quantitative differences in metabolic pathways for APP intrinsic to specific subsets of neurons. For example, mild increases in BACE activity may increase synaptic Aβ in smaller cortical interneurons with relatively short axonal processes. However, due to the length of both the perforant pathway and the mossy fiber pathway, slight elevations in perikaryal BACE activity may preclude synaptic processing of APP in the hippocampus.
Similar region-specific amyloid plaque formation has been observed in the brain of AD patients. For example, whereas association cortices and the limbic system are prone to Aβ amyloid, other regions such as primary sensory/motor neocortices, striatum, brainstem, and spinal cord are relatively unaffected (Braak and Braak, 1991
) despite the widespread expression of APP (Tanzi et al., 1987
). Interestingly, we found that layer IV neurons within regions of the mouse somatosensory cortex were spared from Aβ pathology in both APP and APPxBACE-M mice (Fig. S3). These layer IV neurons receive a large proportion of their synaptic input from spatially distant thalamic neurons. Although speculative at this point, our results suggest that distinct subsets of neurons and/or the length and number of their efferent inputs may be significant factors that in part determine the regional differences in amyloid pathology found in AD.
In conclusion, although the reduction of Aβ deposition upon BACE overexpression was unexpected, our finding that synaptic Aβ is crucial to the development of amyloid plaques offers several new avenues of research that may improve our understanding of the pathogenesis of amyloid plaques. Thus, further progress toward understanding APP transport, Aβ aggregation within axonal or synaptic vesicles, and the distribution of Aβ degrading enzymes may yield insights which may prove to be clinically relevant.