Much progress has been made towards deciphering some of the biological functions of APP, particularly its role in facilitating cell-cell adhesion through homo- and hetero-dimerization with the other APP family members, namely APLP1 and APLP2. However, the focus has remained mostly on its pathogenic role, as the source of the Aβ peptide in the context of Alzheimer’s disease. The identification and characterization of APP-cleaving enzymes, such as secretases (BACE1 and the γ-secretase complex) and caspases, have provided a valuable insight into the complex steps involved in the proteolytic processing of APP and the production of Aβ. Whether the Aβ peptide itself is the cause of AD remains a subject for debate. The identification of several mutations in the APP and presenilin (PS) genes in early-onset familial AD seem to support this view, as these mutations have been shown promote the production of Aβ. In late-onset AD however, the picture becomes a little more muddled. While many risk factor genes have been identified, age remains the greatest risk factor for AD. This raises several important questions: Does the cleavage process of APP change with aging or in AD, and if so what triggers these changes? Do elevated BACE1 levels in the brain play a role in AD pathogenesis, and if so what causes BACE1 to become elevated? Could dimerization of APP provide some answers?
The exact consequences of homo-dimerization of APP on the processing of APP are not fully understood. Introduction of a cysteine mutation in the juxtamembrane (JM) region of APP has been reported to enhance Aβ production through the formation of stable disulfide-linked APP dimers (Scheuermann et al., 2001
), consistent with the observation that stable Aβ dimers can be found intracellularly in vitro
and in vivo
in brains (Walsh et al., 2000
). However, other laboratories have reported the opposite effect, where enhanced dimerization of APP leads to decreased APP processing and decreased Aβ levels (Struhl, 2000
; Eggert et al., 2009
). Reconciling this dichotomy remains difficult, but it could simply be explained by differences in the manner through which APP dimerization is promoted in each model systems.
Here we show that cross-linking of endogenous APP through the use of a divalent antibody triggers the amyloidogenic pathway in cultured hippocampal neurons, resulting in a rise in the levels of intracellular Aβ. Interestingly, this increase in Aβ is observed under non-apoptotic conditions, at least within 48 hours of treatment, but is accompanied by a significant loss of dendritic spine protrusions as well as a decrease in synaptic markers, PSD-95 and Drebrin A. This is an important consideration since neuronal and non-neuronal cells undergoing apoptosis have been shown to overproduce and secrete Aβ, whether triggered by staurosporine or by trophic factor withdrawal (LeBlanc, 1995
; Barnes et al., 1998
; Galli et al., 1998
; Gervais et al., 1999
; Guo et al., 2001
; Tesco et al., 2003
; Sodhi et al., 2004
; Matrone et al., 2008a
, Matrone et al., 2008b
). This wide array of conditions and cell types raises questions as to whether the observed Aβ overproduction is a specific process or simply a general response to apoptotic stimuli. Our result supports the former view, but it is possible that both APP signaling and apoptotic stimuli share common pathways.
Whether driven by apoptosis or by APP signaling, enhanced Aβ production seems to correlate with high levels of BACE1. We show that the observed Aβ overproduction in treated neurons is due primarily to increased processing of APP by BACE1, and not by γ-secretase. This is reflected by a significant increase in the production of sAPPβ fragments correlating with elevated BACE1 protein levels. BACE1 levels rise in response to physiological stress or injury, such as oxidative stress (Tamagno et al., 2002
), traumatic brain injury (Blasko et al., 2004
), ischemia (Wen et al., 2004
), hypoxia (Zhang et al., 2007
), and energy impairment (Velliquette et al., 2005
). BACE1 is also increased in brains from late-onset and early-onset AD patients compared to cognitively normal individuals (Fukumoto et al., 2002
; Holsinger et al., 2002
; Tyler et al., 2002
; Yang et al., 2003
; Li et al., 2004
). Our results imply that in addition to age-related stress, aberrant signaling triggered by APP oligomerization may also enhance levels of BACE1 and Aβ in the brain, and drive AD pathogenesis.
The exact mechanism of this up-regulation is not fully understood and hypotheses vary from transcriptional, post-transcriptional, translational and post-translational modifications of BACE1 (Holsinger et al., 2002
; Zhao et al., 2007
; Faghihi et al., 2008
; Hébert et al., 2008
; Wen et al., 2008
). Our results indicate that the BACE1 increase may be due, at least in part, to enhanced protein stabilization and accumulation. This is reflected by the fact that no significant changes are observed in BACE1 mRNA in treated hippocampal neurons. Alternatively, BACE1 half-life is significantly prolonged in treated neurons, persisting to near control levels after 18 hours of cycloheximide treatment. These results suggest that oligomerization of APP may trigger a signaling cascade that directly interferes with the normal degradation of BACE1 protein, allowing BACE1 to accumulate in treated neurons. Our data shows that activation of this pathway may lead to the loss of function of the GGA3 protein. GGA family proteins are known to be involved in the trafficking of proteins, such as BACE1, which contain the DXXLL signal between different compartments, e.g. Golgi complex, endosomes and lysosomes (reviewed in Bonifacino, 2004
). We demonstrated that activation of caspase-3 in treated neurons promotes cleavage of GGA3, perhaps generating increased amounts of the dominant negative N-terminal fragment (Tesco et al., 2007
). The accumulation of BACE1 in early endosomal compartments in treated neurons was consistent with a loss of function of the GGA3 protein. Furthermore, downregulation of caspase-3 by siRNA prevented the accumulation of BACE1 in endosomes as well as an increase in intracellular Aβ. A similar effect was observed in cells over-expressing human GGA3, which did not exhibit an accumulation of BACE1 or overproduction of Aβ.
The mechanism by which GGA3 targets its cargo to lysosomes has been shown to be ubiquitin-dependent (Puertollano, 2004
). While there is some evidence that BACE1 is ubiquitinated (Qing et al., 2004
), future studies will be required to determine whether GGA3-dependent degradation of BACE1 requires ubiquitination or whether it occurs via
an alternate mechanism (e.g. binding the VHS domain of GGA3). Additionally, RNAi silencing of GGA1 and GGA2 has also been shown to lead to the accumulation of BACE1 in endosomes. However, unlike GGA3, which shuttles BACE1 from endosomes to lysosomes, GGA1 and GGA2 appear to regulate retrograde transport of BACE1 from endosomes to the TGN (Wahle et al., 2005
). Further studies will be required to determine whether loss of function or depletion of GGA1 and GGA2 contribute to BACE1 accumulation in our model. Finally, phosphorylation of BACE1 at Serine498
facilitates its binding to GGA proteins (He et al., 2002; He et al., 2003
; Shiba et al., 2004
; von Arnim et al., 2004
). However, we did not observe any changes in the phosphorylation status of BACE1 in treated neurons (data not shown).
In summary, our results argue for a well-defined mechanism through which aberrant APP signaling can trigger amyloidogenic processing of APP, without affecting neuronal survival, as depicted in . As we discussed earlier, alterations in synaptic density occur early in AD and strongly correlate with the cognitive decline observed in the disease (reviewed in Scheff, 2006
). Our results suggest that aberrant signaling through APP oligomerization is sufficient to drive synaptic dysfunction as well as promote Aβ production in hippocampal neurons. Whether, these effects are dependent on each other remains unclear. It is interesting to note however, that in our model system, while Aβ production is dependent on caspase-3, downregulation of caspase-3 did not protect neurons against the synaptotoxic effects of 22C11 suggesting on the surface that the effects we observed are independent of Aβ production. While somewhat puzzling, a simple explanation could be that signaling through APP results in the activation of a parallel pathway, which itself drives synaptic dysfunction and perhaps even neuronal death. Indeed, another consequence of APP cross-linking is the production of another toxic 31 amino acid cytosolic fragment, termed C31 (Bertrand et al., 2001
; Lu et al. 2003
; Shaked et al., 2006
), which exerts its effects independently of Aβ (Park et al., 2009
). This raises the interesting possibility that abnormal oligomerization of APP initiates a positive feedback loop in an affected neuronal population, resulting in local synaptic dysfunction while simultaneously raising the levels of Aβ, which can spread and “infect” healthy neurons in a manner similar to Prion’s disease. This may also suggest that the development of drugs aimed at disrupting APP dimerization may be a viable therapeutic approach worth exploring.
Schematic illustration of 22C11-induced BACE1 accumulation and Aβ production in neurons