Axonal BACE1 dynamics and targeting in hippocampal neurons: a role for Rab11 GTPase

A transmembrane aspartyl protease, termed β-site APP cleaving enzyme 1 (BACE1), initiates Alzheimer's disease (AD) β-amyloid (Aβ) production by sequential proteolysis of amyloid precursor protein (APP) [1‐5]. The release of Aβ from its precursor involves initial processing by BACE1 to release the APP ectodomain, followed by intramembrane proteolysis by γ-secretase [6]. Aβ is the major component of cerebral amyloid plaques in brains of aged individuals and those with AD. Several lines of evidence suggest that Aβ plays a central role in AD pathogenesis. For example, familial AD-linked mutations near the amino terminus of the Aβ region in APP, found in two Swedish families, cause AD by significantly increasing Aβ production due to enhanced BACE1 cleavage of APP [7‐9]. Moreover, a single amino acid substitution adjacent to the BACE1 cleavage site of APP, which significantly reduces BACE1 cleavage and Aβ peptide generation in cultured cells, has been recently found to protect against disease onset as well as cognitive decline in the elderly without AD [10].

APP and BACE1 are type I transmembrane proteins that undergo secretory and endocytic trafficking. However, in cultured cell lines and primary neurons, only a subset of full-length APP is processed to generate Aβ. This implies either BACE1 cleavage of APP is rather inefficient or that BACE1 has limited access to APP due to their distinct intracellular itineraries and/or spatially restricted localization in intracellular organelles. Over the years, non-neuronal cells were used as experimental systems to characterize the cellular organelles and sorting pathways involved in amyloidogenic processing of APP. Most studies recognize the importance of endocytic trafficking of APP for Aβ production [reviewed in [6, 11, 12]. This notion is in agreement with the predominant steady-state localization of BACE1 in endocytic organelles, and its trafficking and recycling between the plasma membrane and endosomes [1, 13, 14]. Moreover, BACE1 activity has a low pH optimum in vitro[1], supporting a model where APP cleavage by BACE1 occurs in acidic intracellular organelles such as the endosomes. Indeed, siRNA knockdown of Golgi-associated, γ-adaptin homologous, ADP-ribosylation factor-interacting proteins enhance BACE1 localization in early endosomes and a concomitant increase in Aβ secretion [15‐17]. Finally, membrane targeting of a BACE1 transition-state inhibitor to endosomal organelles by linking it to a sterol moiety efficiently reduced BACE1 activity as compared with the free inhibitor [18].

In the mouse brain, ~70% of Aβ released into the interstitial fluid requires ongoing endocytosis, and synaptic activity regulates the vast majority of this endocytosis-dependent Aβ production [19, 20]. The underlying mechanism likely involves activity-dependent regulation of endocytic trafficking of APP as well as its secretases [21]. Neuronal endosomes are not only found in the soma but are distributed throughout the dendrites and axons where they undergo bidirectional transport, adding to the complexity of the trafficking mechanisms [22]. Indeed, APP undergoes BACE1-mediated cleavage during anterograde axonal transport, and Aβ can be generated and released at or near presynaptic sites in vivo[19, 23‐27]. BACE1 has been reported to localize in dendrites and axons in cultured neurons and in the brain [21, 28‐32]. Axonal BACE1 localization is significant because abnormal accumulation of BACE1 in axon terminals has been documented in the brains of individuals afflicted with AD. This later finding raises the possibility that local elevation in BACE1 processing could contribute to amyloid burden in AD [30, 33]. However, the molecular mechanisms responsible for axonal sorting of BACE1 have not been fully explored.

Here, we used live-cell imaging to characterize dynamic BACE1 transport in hippocampal neurons in vitro and in brain slices in situ. We report BACE1 colocalization and dynamic transport in recycling endosomes within the dendrites and axons of cultured hippocampal neurons. Interestingly, our results show that efficient axonal sorting of BACE1 requires Rab11 activity. Together, these findings provide the first demonstration of dynamic BACE1 transport in hippocampal neurons in situ and the involvement of neuronal recycling endosomes in axonal sorting of BACE1.

In human brain, BACE1 can be observed by immunostaining in dendrites of CA1 neurons [34]. Interestingly, in mouse brain, endogenous BACE1 is highly enriched in hippocampal mossy fiber terminals, and only low levels of BACE1 can be detected in the neuronal soma and dendrites (Figure 1) [33, 34]. These apparent differences likely represent the balance between the biosynthetic level and the efficiency of the transport machinery responsible for BACE1 trafficking. Thus, overexpression of BACE1-YFP in cultured hippocampal neurons allows us to appreciate dynamic sorting of BACE1 between dendrites and axons, which is not possible to discern from steady-state analysis of endogenous proteins that have reached their preferred final destination. Indeed, our results on BACE1 localization are analogous to what has been documented for the localization of kainate-type glutamate receptor subunits GluK2 and GluK3. When analyzed by immunohistochemistry, the distribution of GluK2 and GluK3 is prominent at hippocampal mossy fiber synapses in mouse brain [44]. However, detailed analysis of these receptors by transfection in cultured hippocampal neurons reveals a prominent dendritic localization and demonstrates that the steady-state localization of kainate receptors is achieved by dynamic activity-dependent polarized endocytic sorting from the dendrites through recycling or degradative pathways [45, 46]. The progressive increase in BACE1 levels in axons concomitant with its decrease in dendrites over time following transfection suggest the existence of saturable mechanisms that mediate polarized axonal sorting of BACE1 (Figure 3). Axonal sorting and presynaptic localization of BACE1 are highly relevant because of the recent identification of several presynaptic proteins such as L1, CHL1, LRRN1, brain EGF-repeat containing transmembrane protein, and neurexin-1a as neuronal BACE1 substrates [47, 48]. Moreover, loss of BACE1 expression results in axon guidance defects in the hippocampus and the olfactory bulb [36, 44, 48].

Although the machinery and the molecular mechanisms governing polarized sorting of proteins in neurons needs to be further explored, the involvement of three pathways for the axonal targeting of transmembrane proteins has been suggested: 1) direct polarized delivery of nascent proteins from the secretory pathway to the dendrites or axons in TGN-derived vesicles, 2) non-polarized delivery to dendrites and axons followed by selective retrieval and retention involving endocytosis and degradation pathways, 3) indirect polarized delivery via endosomes, also called transcytosis, whereby nascent proteins are first delivered to the somato-dendritic cell surface, and then following endocytosis get routed to the axons [22]. The extensive localization of BACE1 in endosomes and the highly polarized axonal localization of BACE1 in hippocampal mossy fibers ([34], Figures 1 and 5) suggest that the direct polarized delivery is unlikely to play a major role in BACE1 axonal targeting. The results detailed in this study suggest that BACE1 sorting to Rab11-positive recycling endosomes plays an important role for further transport to the axons and presynaptic terminals. First, BACE1 is efficiently sorted in endosomes positive for three known markers of recycling endosomes: Rab11, syntaxin 13, and TfR [21, 34]. Second, the dynamic characteristics of BACE1 transport in hippocampal neurons, characterized by live-cell imaging, are consistent with protein trafficking in recycling endosomes [37], a conclusion supported by two-color imaging of BACE1 co-transport with Rab11 in dendrites and axons (Figure 5, Additional file 4). Third, the impairment of Rab11 activity by dominant-negative mutant expression caused internalized BACE1 accumulation in the soma with a concomitant decrease in axons. The accumulation of internalized BACE1 in the soma and unperturbed relative subcellular distribution in endosomes and lysosomes (Figure 7) suggest that BACE1-containing vesicles are not rerouted for lysosomal degradation when a block in recycling itinerary is imposed by DN Rab11 expression. Because we have not found convincing evidence for the involvement of degradative pathways to explain the paucity of BACE1 in soma and dendrites, we hypothesize that proper BACE1 sorting to axons and presynaptic terminals requires its transcytosis in a Rab11-dependent manner rather than aspecific sorting followed by selective retention/retrieval mechanisms. Interestingly, BACE1 shows only partial colocalization with Rab11 in axons, when observed by immunofluorescence staining or dual-color live imaging (Figure 5). These findings suggest that in axons a subset of BACE1 is sorted independent of Rab11-positive endosomes or Rab11 detaches from a subset of BACE1-positive vesicles. It will be of interest to identify other trafficking adaptors that regulate dynamic BACE1 transport in axons. In this regard, we recently reported that loss of Eps15-homology-domain containing 1 and 3 protein function in hippocampal neurons compromises dynamic axonal transport and overall BACE1 levels in axons [34].

Thus far, only a few proteins have been characterized to undergo neuronal somaodendritic-to-axonal transcytosis; notable examples are the axonal cell adhesion molecule L1/NgCAM [49], tropomyosin-related kinase Trk receptors [41], and Contactin-associated protein 2, Caspr2 [50]. In each case, it is clear that endosomal sorting is required for axonal targeting, but the machinery and the molecular players involved in achieving polarized sorting are not fully understood. Also, differences could be already found between the steady-state localization of these proteins suggesting the existence of different regulatory mechanisms in the transcytotic pathway. For example, L1/NgCAM axonal surface expression is prominent and highly polarized in transfected cultured neurons [49], which is not the case for BACE1. In addition, L1/NgCAM is sorted mainly in NEEP21-positive vesicles in soma/dendrites before transcytosis, and shows only low levels of colocalization with transferrin-positive recycling endosomes. In contrast, a large fraction of BACE1 is found in recycling endosomes positive for TfR and Rab11, with only a relatively smaller fraction present in EEA1 or NEEP21-positive early endosomes (Figure 5, [34]). Thus, it appears that L1/NgCAM and BACE1 are subject to different the endosomal sorting steps prior to transcytosis. BACE1 sorting in recycling endosomes and dynamics share similarities with Trk receptors. Similar to BACE1 trafficking in hippocampal neurons, TrkA is also dynamically co-transported in axons in Rab11-positive vesicles and its trafficking in sympathetic neurons also requires Rab11-GTPase activity for efficient transcytosis [41]. It has been hypothesized that transcytosis serves as a means to facilitate efficient axonal delivery of receptors in response to signals often involving ligand:receptor interaction in presynaptic terminals as it is shown for TrkA transcytosis mediated by nerve growth factor signaling [41]. Since BACE1 is the first reported enzyme to undergo transcytosis, exploring potential signals that regulate BACE1 transcytosis would be of great interest especially for a better understanding of the mechanism underlying its abnormal accumulation in swollen presynaptic terminals in Alzheimer’s disease. Since synaptic activity regulates BACE1 trafficking and APP processing [19‐21] it is possible that synaptic activity impairment observed in Alzheimer’s disease could affect BACE1 transcytosis in a manner that promotes its accumulation in dystrophic neurites, thus contributing to local Aβ production near the presynaptic terminals.

Abnormalities of the endosomal system such as the fusion of early and recycling endosomes have long been implicated in Alzheimer’s disease pathogenesis (reviewed in [11]). Interestingly, Rab11 was recently identified as one of the two major regulators of Aβ production, in an unbiased RNAi screen of human Rab GTPases and Rab GTPase-activating proteins by Rajendran and colleagues [51]. Overexpression of dominant-negative mutants of Rab11a or Rab11b also reduced Aβ and sAPPβ levels significantly. Moreover, siRNA knockdown of Rab11 expression in primary neurons significantly reduce sAPPβ and Aβ levels demonstrating that Rab11 function is crucial for β-cleavage and Aβ generation [51]. Our characterization of Rab11 as a novel regulator of BACE1 axonal sorting in neurons, along with the identification of Rab11 as a modulator of Aβ production, raises the possibility that dysfunction of Rab11 may underlie pathogenesis in a subset of sporadic Alzheimer’s disease cases. Indeed, aberrant Rab11 trafficking has been reported in Huntington's disease and contributes to oxidative stress and neuronal cell death [52]. Future studies aimed at manipulating of polarized neuronal BACE1 trafficking to assess potential modulation of Aβ production in vivo would be necessary to evaluate if trafficking modulation of BACE1 could serve as a therapeutic strategy in Alzheimer’s disease to reduce cerebral amyloid burden.