Recent studies have revealed BACE as a stress-related protease that is upregulated in the brains of AD patients (Fukumoto et al., 2002
; Holsinger et al., 2002
; Li et al., 2004a
; Tyler et al., 2002; Yang et al., 2003
). BACE levels have also been shown to increase following ischemia (Wen et al., 2004
); however, the molecular mechanism responsible has remained elusive. Here, we show that caspase activation elevates BACE levels and β-secretase activity owing to post-translational stabilization of BACE. Using a rat ischemia model, we found that levels of GGA3, an adaptor molecule implicated in BACE trafficking, are reduced in a temporally coordinated manner with caspase activation and increases in BACE proteins levels. In cell-based studies, RNAi silencing of GGA3 directly led to increased BACE protein levels and β-secretase activity as evidenced by enhanced APP-C99 and Aβ levels. Together, these data suggest a model in which apoptosis, e.g. induced by ischemia, drives the depletion of GGA3, which, in turn, leads to the stabilization of BACE and increased β-secretase activity. We also analyzed brain samples of AD patients in which BACE levels and β-secretase activity have previously been shown to be elevated in the temporal cortx. In further support of our model of GGA3-dependent degradation/stabilization of BACE, in the brains of AD patients, GGA3 protein levels were significantly decreased. Moreover, this decrease was inversely correlated with increased BACE levels in the temporal cortex.
Decreased levels of GGA3 most likely engender an increase in BACE protein levels by interfering with the sorting of BACE to lysosomes where it is degraded (Koh et al., 2005
). GGA3 has previously been demonstrated to target cargo (e.g. EGFR) to lysosomes (Puertollano and Bonifacino, 2004
). RNAi silencing of GGA3 resulted in the accumulation of EGFRs in enlarged early endosomes and partially blocked their delivery to lysosomes where they are normally degraded. These studies indicate that GGA3 is involved in the delivery of endosomal cargoes to lysosomes. Recently, He et al. (2005)
showed that RNAi silencing of GGAs significantly increases the levels of BACE in endosomes. They also proposed that GGAs are necessary for BACE and MPRs to be transported back to TGN. However, BACE turnover was not assessed in that study, and the accumulation of BACE in endosomes could also be due to decreased degradation resembling the effect of GGA3 downregulation on EGFR degradation (Puertollano and Bonifacino, 2004
). The mechanism by which GGA3 targets some cargo (e.g. EGFR) to lysosomes has been shown to be ubiquitin-dependent (Puertollano and Bonifacino, 2004
). While there is some evidence that BACE is ubiquitinated (Qing et al., 2004
), future studies will be required to determine whether GGA3-dependent degradation of BACE requires ubiquitination, or whether it occurs via an alternate mechanism (e.g. the binding to GGA3-VHS domain).
We have shown that caspase-mediated degradation of GGA3 occurs following cerebral ischemia and propose that it may explain our and others' observations of elevated β-secretase levels (and activity) following cerebral ischemia. As shown in the Nun Study (Kalaria, 2000
; Nolan et al., 1998; Snowdon et al., 1997
), and recently confirmed in additional prospective autopsy studies (Petrovitch et al., 2005; Riekse et al., 2004), individuals with AD and cerebrovascular pathologies show greater cognitive impairment than those exhibiting either pathology alone. These studies indicate that there is an additive or synergistic interaction between AD and cerebrovascular pathologies. Furthermore, evidence is accumulating that stroke and transient ischemic attacks significantly increase the risk of AD in elderly individuals (Honig et al., 2003
; Zlokovic, 2002
). A recent family-based study has shown that stroke increases the risk of AD to a similar extent as the presence of an APOE-ε4 allele in Latinos (Rippon et al., 2006
). Thus, stroke may represent either a precipitating or a triggering event in AD.
While there is an increasing body of knowledge indicating a strong association between cerebrovascular disease and AD (Honig et al., 2003
; Rippon et al., 2006
), the potential role of apoptosis and cerebral ischemia in AD pathogenesis has remained unclear. Apoptosis has been firmly established to enhance Aβ production in neuronal and non-neuronal cells (Barnes et al., 1998; Galli et al., 1998; Gervais et al., 1999
; Guo et al., 2001; LeBlanc, 1995
; Sodhi et al., 2004; Tesco et al., 2003
). Thus, apoptotic events in the brain, e.g. induced by stroke and ischemia could increase risk for, or trigger AD by driving cerebral Aβ accumulation. Several studies have shown cerebral ischemia to upregulate APP messages containing the Kunitz-type protease inhibitor domain, between 1 and 21 days after reperfusion (Abe et al., 1991
; Kim et al., 1998; Koistinaho et al., 1996; Shi et al., 2000). Additionally, APP protein levels were increased between 1 and 10 weeks after reperfusion (Banati et al., 1995; Kalaria et al., 1993
; Stephenson et al., 1992; Wakita et al., 1992). BACE protein levels and β-secretase activity have also been shown to be increased in animal models of traumatic brain injury, including cerebral ischemia (Wen et al., 2004
) and head injury, which is also a risk factor for AD (Blasko et al., 2004
; Chen et al., 2004). More recently, caspase inhibition therapy has been shown to prevent brain trauma-induced increases in Aβ peptide (Abrahamson et al., 2006). Collectively, these findings, taken together with our current data, suggest that accumulative insults to the brain over one's lifetime would progressively increase risk for AD by elevating cerebral Aβ accumulation via BACE stabilization owing to caspase-mediated depletion of GGA3. Furthermore, the effect of BACE stabilization on Aβ levels could also be amplified by other events (e.g. upregulation of APP levels at much later time points, e.g. several days after the ischemic event).
We have also shown that GGA3 levels are reduced both in the temporal cortex and cerebellum of AD patients (versus controls). The decrease in GGA3 levels was more pronounced in the temporal cortex versus cerebellum, which is relatively spared of AD pathology. Importantly, decreased levels of GGA3 were inversely correlated with increased levels of BACE only in the temporal cortex, which is strongly impacted by AD pathology. In contrast, BACE levels were not significantly increased in the cerebellum of AD patients as compared to control subjects. These findings suggest that some subjects have lower levels of GGA3 independently of AD pathology, e.g. in cerebellum. Subjects with lower levels of GGA3 may be at risk of developing AD given that conditions associated with caspase activation e.g stroke, which is a risk factor for AD, may further decrease GGA3 levels triggering or precipitating AD pathology.
The contribution of apoptosis to the etiology and pathogenesis of AD remains unclear largely due to the difficulties involved in identifying classic apoptotic markers in vivo
(for review see (Cribbs et al., 2004; LeBlanc, 2005
). This is most likely due to the long duration of AD and the very rapid clearance of apoptotic cells. Contradictory results could be at least partially due to the use of post-mortem tissues. Many factors (e.g. length of agonal state, collection of tissue at the end point of the disease and time interval before freezing the tissue) may significantly affect the analysis of enzymatic activities. It is also possible that many senile plaques, which can take many years to form, are no longer surrounded by apoptotic neurons by the time of autopsy. On the other hand genetic evidence for sporadic AD, such as the disease associations with DAPK1
(Li et al., 2006), GAPD
(Li et al., 2004b) and LOC439999
(Grupe et al., 2006) variants, also point to apoptosis as a disease-relevant process. While there is increasing evidence for caspase activation in AD brain (for review see (Cribbs et al., 2004; LeBlanc, 2005
), we cannot rule out the possibility that genetic factors and/or other post-translational mechanisms (e.g. other proteases) may contribute to GGA3 depletion in AD brain.
In summary, our studies suggest that elevated BACE protein levels found in AD patients and animal models of traumatic brain injury including ischemia and acute head trauma, may be at least partly due to impaired degradation and stabilization of BACE. This would then lead to increased production of the Aβ peptide, thereby contributing to AD pathogenesis. Since Aβ has also been reported to induce apoptosis, this could result in a vicious cycle that autopotentiates Aβ generation and cell death. Finally, our in vivo and in vitro data implicate GGA3 as the key player in regulating degradation of BACE in its capacity as a trafficking molecule that delivers BACE to the endosomal-lysosomal system.