Post-translational regulation of the β-secretase BACE1

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Brain Research Bulletin xxx (2016) xxx–xxx

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Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull

Review

Post-translational regulation of the ␤-secretase BACE1 Wataru Araki Department of Demyelinating Disease and Aging, National Institute of Neuroscience, NCNP, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan

a r t i c l e

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Article history: Received 2 March 2016 Received in revised form 8 April 2016 Accepted 12 April 2016 Available online xxx Keywords: Alzheimer’s disease Amyloid ␤-protein BACE1 ␤-Secretase Oligomer Trafficking

a b s t r a c t ␤-Secretase, widely known as ␤-site APP cleaving enzyme 1 (BACE1), is a membrane-associated protease that cleaves amyloid precursor protein (APP) to generate amyloid ␤-protein (A␤). As this cleavage is a pathologically relevant event in Alzheimer’s disease, BACE1 is considered a viable therapeutic target. BACE1 can be regulated at the transcriptional, post-transcriptional, translational, and post-translational levels. Elucidation of the regulatory pathways of BACE1 is critical, not only for understanding the pathological mechanisms of AD but also developing effective therapeutic strategies to inhibit activity of the protease. This mini-review focuses on the post-translational regulation of BACE1, as modulation at this level is closely associated with both physiological and pathological conditions. Current knowledge on the mechanisms underlying such BACE1 regulation and their implications for therapy are discussed. © 2016 Published by Elsevier Inc.

Contents 1. 2.

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4. 5. 6. 7.

Introduction: the functional importance of BACE1 in AD pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 BACE1 regulation via post-translational modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.1. Palmitoylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.2. Phosphorylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.3. Glycosylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.4. Acetylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 BACE1 regulation through intracellular transport and degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.1. Endocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.2. Retrograde trafficking from endosomes to the TGN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.3. Slow recycling pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.4. Sorting to lysosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 BACE1 regulation by reticulons and other proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 BACE1 and sAPP␣ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 BACE1 and amyloid ␤-protein (A␤) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction: the functional importance of BACE1 in AD pathology Basic information on the properties and functions of ␤secretase, widely known as ␤-site APP cleaving enzyme 1 (BACE1),

E-mail address: [email protected]

is provided in this section. In the molecular pathology of Alzheimer’s disease (AD), a central role of amyloid ␤-protein (A␤) has been established (Hardy and Selkoe, 2002). A␤ is a hydrophobic peptide of 40–43 amino acids highly prone to aggregation and a primary constituent of senile plaques in AD brain. A␤ is generated via two-step proteolysis of amyloid precursor protein (APP) by ␤-secretase and ␥-secretase. Specifically, ␤-secretase processing of APP generates secreted APP-␤ (sAPP␤) and a ␤-C-terminal fragment (␤-CTF), which is further cleaved by ␥-secretase to gen-

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2. BACE1 regulation via post-translational modifications 2.1. Palmitoylation

Fig. 1. Post-translational modifications of BACE1. BACE1 undergoes various modifications, including N-glycosylation (Gly), phosphorylation (P-Ser498, Thr252), acetylation (Acetyl-K299, K307, and five other sites), palmitoylation (Pal-Cys474, Cys478, Cys482, Cys485), and ubiquitination (Ub-K501). D: two aspartic proteaseactive sites; SP: signal peptide; Pro: propeptide; TM: transmembrane domain.

erate A␤. Alternative processing of APP by ␣-secretase generates sAPP␣ and ␣-CTF, of which the latter is also cleaved by ␥-secretase (De Strooper et al., 2010). In 1999, a ␤-secretase designated BACE1 was identified as a transmembrane aspartyl protease abundantly expressed in neurons in the brain (Sinha et al., 1999; ,Vassar et al., 1999; Yan et al., 1999). BACE1 cleaves APP at the N-terminus of the A␤ region (␤-site), and additionally between A␤ Tyr10 and Glu11 (␤’-site) (Vassar et al., 1999). BACE2, a homolog of BACE1, is expressed ubiquitously and exerts ␣-secretase-like activity on APP (Bennett et al, 2000; Farzan et al., 2000; Yan et al., 2001; Fluhrer et al., 2002). BACE1 is an essential protease for A␤ production, as confirmed from studies on BACE1 knockout mice. Importantly, BACE1 knockout mice do not display overt abnormalities, and abrogates cerebral A␤ deposition and prevents cognitive deficits in mouse models of AD (Cai et al., 2001; Luo et al., 2001; Roberds et al., 2001; Ohno et al., 2004), supporting the feasibility of exploiting BACE1 inhibition as a therapeutic strategy for AD. BACE1, a protein of 501 amino acids, undergoes several post-translational modifications, including glycosylation, phosphorylation, and palmitoylation (Wang et al., 2013; Vassar et al., 2014), as described in the following section (Fig. 1). Earlier studies have focused on delineating the intracellular sorting mechanism of BACE1. Mature BACE1 is transported to the plasma membrane and internalized to early endosomes via the di-leucine motif at the Cterminus. BACE1 is suggested to recycle to the plasma membrane via endosomes or trans-Golgi network (TGN). BACE1 processing of APP is likely to occur in endosomal compartments with a low pH environment favorable for protease activity. ␤-Cleavage of APP is additionally supported by the Golgi/TGN environments (Zhi et al., 2011; Tan and Evin, 2012; Vassar et al., 2014). In neurons, BACE1 is distributed throughout the somatodendritic and axonal compartments (Buggia-Prévot et al., 2013; Das et al., 2013). Notably, recent studies have indicated synaptic localization of BACE1 (Kandalepas et al., 2013; Del Prete et al., 2014; Lundgren et al., 2015; Pliássova et al., 2015). As APP is also transported in axons and dendrites, it is highly likely that A␤ production occurs within these neuritic compartments (Brunholz et al., 2012). A␤ released from synaptic terminals possibly contributes to extracellular A␤ deposition (Lazarov et al., 2002; Cirrito et al., 2005). BACE1 is regulated at various levels (transcriptional, posttranscriptional, translational, and post-translational) (Rossner et al., 2006; Sun et al., 2012; Tamagno et al., 2012). This mini-review focuses on the post-translational regulation of BACE1, since modulation at this level is closely associated with both physiological and pathological conditions and previous reviews have not dealt with this particular area of research. Several mechanisms exist for this type of regulation, including post-translational modification of BACE1 itself, regulation through intracellular trafficking of BACE1, regulation by interacting molecules, such as reticulons and sAPP␣, and pathological regulation by A␤. Current knowledge on these regulatory pathways and their implications for therapy are discussed below.

Palmitoylation of BACE1 occurs at the four cysteine residues clustering in the transmembrane and C-terminal cytosolic domains, and regulates targeting to lipid rafts, which are distinct membrane domains characterized by high concentrations of cholesterol and glycosphingolipids (Benjannet et al., 2001; Vetrivel et al., 2009; Motoki et al., 2012). Protein palmitoylation exerts various effects, including protein–protein interactions, folding, trafficking, and association with lipid rafts (Charollais and Van Der Goot, 2009). For BACE1, the impact of palmitoylation on lipid raft targeting has been investigated in detail. A considerable proportion of endogenous or overexpressed BACE1 is localized to lipid rafts (Riddell et al., 2001; Ehehalt et al., 2003). The issue of whether BACE1 cleavage of APP predominantly occurs in lipid or nonlipid rafts remains controversial (Vetrivel and Thinakaran, 2010; Hicks et al., 2012; Araki and Tamaoka, 2015). Our group and that of Dr. Thinakaran independently demonstrated that lipid raft localization of BACE1 is palmitoylation-dependent, using palmitoylation-deficient mutant BACE1 with Cys to Ala substitutions at the four cysteine residues. Both wild-type and mutant BACE1 produced similar amounts of A␤ in neuroblastoma cells and primary neurons expressing APP (Vetrivel et al., 2009; Motoki et al., 2012). Furthermore, ␤-CTF was detected mainly in nonraft domains in neurons co-expressing APP and BACE1 (wild-type or mutant) as well as those expressing Swedish mutant APP only (Motoki et al., 2012). These data indicate that raft association of BACE1 does not influence ␤-cleavage of APP and A␤ generation, and support the view that BACE1 cleaves APP mainly in nonraft domains. Accordingly, we proposed a model of neuronal A␤ generation involving mobilization of ␤-CTF from nonraft to raft domains (Motoki et al., 2012). However, it should be noted that BACE1 in lipid rafts may also function in cleavage of other substrates, including neuregulin-1, ␤-subunits of voltage-gated sodium channels, and neural cell adhesion molecules L1 and CHL1 (Wong et al., 2005; Hu et al., 2016; Willem et al., 2006; Zhou et al., 2012).

2.2. Phosphorylation BACE1 is phosphorylated at Ser498 in its cytoplasmic domain, which is involved in its recycling between endosomes and the cell surface (Walter et al., 2001; Pastorino et al., 2002). This serine phosphorylation appears to influence the interaction between BACE1 and GGA1, thereby modulating BACE1 intracellular trafficking (von Arnim et al., 2004; Wahle et al., 2005). However, previous experiments using a non-phosphorylated mutant form of BACE1 showed that this phosphorylation does not influence A␤ production (Walter et al., 2001; Pastorino et al., 2002). The kinase(s) mediating this phosphorylation event remain to be identified. Recently, BACE1 was shown to be phosphorylated by p25/Cdk5 at Thr252 in the extracellular (luminal) domain. Interestingly, p25/Cdk5-mediated phosphorylation increased BACE1 activity in vitro and phosphorylation-defective mutant BACE1 displayed lower activity and A␤ secretion than wild-type BACE1. Another important finding is that phospho-BACE1 is elevated in the brains of AD patients, compared to control counterparts (Song et al., 2015). This is consistent with the previous observations that Cdk5 and p25/p35 levels are significantly increased in AD brains (Tseng et al., 2002; Sadleir and Vassar, 2012). Since p25/Cdk5 is involved in BACE1 transcription (Wen et al., 2008), and is a major tau kinase (Kimura et al., 2014), p25/Cdk5 may represent a promising drug target. However, the mechanism by which Thr252 of BACE1 in the

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lumen of the organelle is phosphorylated by p25/Cdk5, which is localized in the cytosol, remains to be clarified. 2.3. Glycosylation Four potential N-glycosylation sites are encoded in the primary sequence of BACE1. BACE1 is co-translationally N-glycosylated in the endoplasmic reticulum (ER), producing an immature form. Immature BACE1 undergoes further complex glycosylation to generate mature, fully glycosylated protein in the Golgi (Capell et al., 2000; Huse et al., 2000). Recent studies showed that BACE1 is modified with bisecting N-acetylglucosamine (GlcNAc) to an increased extent in AD brains (Kizuka et al., 2015). This modification catalyzed by a GlcNAc transferase Gnt-III may play a role in stabilizing BACE1 in pathological conditions, such as oxidative stress, as its absence facilitates lysosomal degradation of the protein (Kizuka et al., 2015; Kizuka et al., 2016). However, the mechanism by which modification in the luminal region is recognized for BACE1 regulation remains to be established. 2.4. Acetylation The group of Dr. Puglielli showed that BACE1 is acetylated transiently in the ER at seven lysine residues within the N-terminal region. This modification is mediated by two acetyl CoA:lysine acetyltransferases, ATase 1 and ATase 2 (Costantini et al., 2007; Ko and Puglielli, 2009). Acetylated intermediates of the nascent protein reach the Golgi for maturation, while non-acetylated intermediates are retained and degraded in the ER/Golgi intermediate compartment. The acetylated form is assumed to be deacetylated by a Golgi-based deacetylase. Accordingly, cellular levels of BACE1 are controlled by ATase 1 and ATase 2. Interestingly, both ATases are expressed in neurons and upregulated in AD brain. The group additionally identified inhibitors of ATase 1 and ATase 2 capable of downregulating BACE expression and activity (Ding et al., 2012). These findings suggest that these ATases may be potential therapeutic targets for AD. 3. BACE1 regulation through intracellular transport and degradation The mechanisms underlying post-translational regulation of BACE1 are closely associated with its intracellular (intraneuronal) trafficking and degradation. Recent studies have disclosed that BACE1 traffics in a coordinated manner along with various regulatory molecules. BACE1 is essentially a neuronal protein abundantly localized in endosomal organelles, which are distributed throughout soma, dendrites, and axons. Thus, neuronal polarity needs to be considered in the analysis of BACE1 trafficking. As described in the Introduction, BACE1 matures through the Golgi and is transported to the plasma membrane where it is endocytosed to early endosomes. The protein is subsequently recycled to the TGN or plasma membrane or sorted to late endosomes for lysosomal degradation. Thus, BACE1 trafficking can be regulated at levels of endocytosis, recycling or sorting to lysosomes. Modulation of BACE1 trafficking and degradation occurs through four main routes: endocytosis (internalization), retrograde trafficking from endosomes to the TGN, recycling from endosomes to the cell surface, and lysosomal sorting from endosomes to lysosomes (Fig. 2). 3.1. Endocytosis As specified previously, BACE1 is internalized via the C-terminal di-leucine motif to early endosomes (Huse et al., 2000; Pastorino et al., 2002; Kang et al., 2012; Chia et al., 2013). This motif serves as

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a sorting signal, often through binding to clathrin adaptor proteins, such as AP-1 and AP-2. Recent studies have indicated that AP-2 is positively involved in BACE1 endocytosis. The DDISLL sequence of BACE1 fits the [DE]XXXL[LI] consensus AP binding motif. SiRNAmediated knockdown of AP-2 increased localization of BACE1 to the plasma membrane, while its overexpression promoted formation of the BACE1/AP2/clathrin complex and BACE1 internalization, clearly suggesting that BACE1 internalization is AP-2/clathrindependent (Prabhu et al., 2012; Chia et al., 2013; Maesako et al., 2015). However, another study reported that BACE1 internalization is mediated by Arf6, and independent of clathrin and AP-2 (Sannerud et al., 2011). The concept that BACE1 internalization is a factor controlling A␤ production is supported by the recent finding that dynamin 1, a GTPase that plays a critical role in endocytic vesicle fission, regulates A␤ generation through modulation of BACE1 (Zhu et al., 2012). It is also suggested that high fat diet promotes BACE1 cleavage of APP by increasing AP-2 levels (Maesako et al., 2015). BACE1 endocytosis is additionally negatively regulated by SNX12, a member of the sorting nexin family comprising a diverse group of cellular trafficking proteins unified by the presence of a phospholipid-binding motif. Specifically, SNX12 interacts with BACE1, and its downregulation accelerates BACE1 endocytosis and decreases the steady-state level of cell surface BACE1 (Zhao et al., 2012a). 3.2. Retrograde trafficking from endosomes to the TGN The mechanisms governing retrograde trafficking of BACE1 from endosomes to the TGN are complex, but this pathway also appears important for the maintenance and regulation of BACE1. The molecules initially identified in this pathway are GGAs (Golgi-localized ␥-ear-containing, ADP ribosylation factor-binding proteins), such as GGA1. Interactions of BACE1 with GGAs may regulate retrieval of BACE1 from endosomes to the TGN (Wahle et al., 2005; He et al., 2005). The DDISLL sequence of BACE1 is a dualspecificity signal that can alternatively interact with GGAs at the TGN and endosomes. Another study has implicated flotillin-1 and flotillin-2 in the endosomal sorting of BACE1 (John et al., 2014). Several reports support the view that the retromer complex plays a role in retrograde trafficking of BACE1 from endosomes to the TGN. For example, sortilin, a VPS10p domain receptor, interacts with BACE1 through the cytoplasmic domain and mediates retrograde trafficking (Finan et al., 2011). Another study showed that SNX6, a putative component of the retromer, associates with BACE1 and negatively modulates its retrograde transport (Okada et al., 2010). In addition, Vps35, a major component of the retromer complex, is reported to regulate BACE1 trafficking. Suppression of Vps35 expression leads to increased BACE1 localization in endosomes (Wen et al., 2011; Wang et al., 2012). Thus, retromermediated retrograde trafficking may exert an inhibitory effect on the A␤-producing activity of BACE1. Considering the important roles of the retromer in APP trafficking, retromer dysfunction is likely to be linked to AD pathogenesis (Small and Petsko, 2015). Retromer is considered as a tractable therapeutic target, as a recent study has reported that pharmacological chaperones that stabilize retromer levels can reduce amyloidogenic APP processing in neurons (Mecozzi et al., 2014; Small and Petsko, 2015). 3.3. Slow recycling pathway Two types of recycling pathways exist: a rapid recycling pathway (mediated by Rab4) and slower recycling endosome pathways (mediated by Rab11a and Rab8a) (Goldenring, 2015). The recycling pathway also appears to play an important role in regulating BACE1, especially in differentiated neurons. A recent RNAi screen of human

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Fig. 2. Intracellular trafficking of BACE1. Mature BACE1 (B) is transported from the TGN to the cell surface, and endocytosed to early endosomes (Pathway 1). BACE1 in endosomes is retrogradely trafficked to the TGN (Pathway 2), recycled via recycling endosomes (Pathway 3) or sorted to late endosomes and lysosomes for degradation (Pathway 4).

Rab proteins revealed that Rab11a regulates BACE1 cleavage of APP as well as A␤ generation (Udayar et al., 2013). BACE1 is colocalized with Rab11-positive recycling endosomes and expression of dominant-negative mutant Rab11 affects recycling of BACE1 (Chia et al., 2013; Udayar et al., 2013). Another study presented evidence of BACE1 and Rab11 colocalization in neurons (Buggia-Prévot et al., 2014). Thus, the Rab11-dependent slow recycling pathway appears to be significantly involved in BACE1 trafficking as well as A␤ generation. However, the roles mediated by recycling endosomes are yet to be established. The pool of BACE1 is possibly replenished by recycling, and BACE1 cleavage of APP may occur in recycling as well as early endosomes. Importantly, a recent study suggests that BACE1-APP interactions occur in recycling endosomes throughout neuronal processes of hippocampal neurons (Das et al., 2016). It is also noteworthy that internalized APP is predominantly routed to late endosomes and lysosomes, escaping BACE1 cleavage (Chia et al., 2013; Das et al., 2016). Interestingly, exome sequencing revealed a significant genetic association of Rab11A with late-onset AD (Udayar et al., 2013). 3.4. Sorting to lysosomes The main degradation pathway of BACE1 is thought to be the lysosomal pathway (Koh et al., 2005; Lefort et al., 2012; Kandalepas et al., 2013), although the proteasomal pathway appears to play a minor role (Qing et al., 2004). For example, treatment with lysosomal inhibitors has been shown to induce augmentation of BACE1 levels. Therefore, it is reasonable to assume that BACE1 can be regulated via modulation of trafficking to the lysosomal pathway. Several recent studies have investigated this aspect of BACE1 regulation. The group of Dr. Tesco showed that GGA3, one of the GGA proteins involved in transport of cargo proteins from the TGN to endosomes, plays a positive role in BACE1 sorting from endosomes to lysosomes. Interestingly, decrease in GGA3 mediated by caspase cleavage led to stabilization of BACE1. Moreover, GGA3 protein levels were significantly decreased and inversely correlated with increased BACE1 in AD brain (Tesco et al., 2007; Walker et al., 2012). However, the mechanisms underlying GGA3 involvement in BACE1 regulation in neurons, in particular, dendrites and axons, remain to be established.

Notably, BACE1 is ubiquitinated at Lys-501 and mainly undergoes mono- and Lys-63-linked polyubiquitination. This modification appears to play a positive role in sorting BACE1 to lysosomes for degradation (Kang et al., 2010, 2012). Dr. Cai and colleagues presented evidence that snapin is involved in the retrograde transport of BACE1 and its trafficking from endosomes to lysosomes (Ye and Cai, 2014). Snapin is a dynein motor adaptor that coordinates retrograde transport and late endsomal-lysosomal trafficking (Cai and Sheng, 2011). The group showed that BACE1 retrograde trafficking and lysosomal targeting are reduced in snapin-deficient neurons, compared to control. Moreover, increased levels of BACE1 and APP ␤-CTF were observed in brains of snapin-deficient mice, and snapin overexpression reduced amyloidogenic APP processing by suppressing the BACE1 level. These findings suggest that snapin-mediated retrograde trafficking facilitates BACE1 translocation to lysosomes for degradation. Our group recently demonstrated that low-density lipoprotein receptor-related protein 1 (LRP1), a multi-functional receptor (Kanekiyo and Bu, 2014), has an inhibitory effect on BACE1 protein expression in primary neurons as well as non-neuronal cells. We showed that LRP1 specifically downregulates BACE1 protein expression by facilitating its intracellular trafficking from early to late endosomes through protein–protein interactions, thereby promoting lysosomal degradation (Tanokashira et al., 2015). Several studies have revealed a critical role of LRP1 in brain-to-blood and cellular clearance of A␤ (Kanekiyo and Bu, 2014). Therefore, LRP1 appears to have a beneficial function against A␤ accumulation by promoting clearance and reducing production. Taking this aspect into consideration, any agent that is capable of upregulating LRP1 in the brain may be of therapeutic potential. A recent report demonstrated that inhibition of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) enhances BACE1 levels while its overexpression accelerates its degradation. Increased BACE1 and ␤-CTF levels were detected in brains of gad mice with disruption of the UCH-L1 gene (Zhang et al., 2012). Additionally, UCH-L1 appears to accelerate lysosomal degradation of APP (Zhang et al., 2014). A recent report showed that A␤42 induces upregulation of BACE1 and downregulation of UCH-L1 in neuronal cells. The latter process seems to interfere with BACE1 lysosomal degradation (Guglielmotto et al., 2012). Thus, UCH-L1 is proposed to

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play a positive role in lysosomal degradation of BACE1, although the underlying mechanism remains to be clarified. Potentiation of UCH-L1 could be a disease-modifying strategy for AD. Trafficking from endosomes to lysosomes may be involved in aberrant upregulation of BACE1 under pathological conditions, as described below.

4. BACE1 regulation by reticulons and other proteins BACE1 is additionally regulated by its interacting proteins, among which the reticulon (RTN) protein family has been investigated most rigorously. RTNs are membrane proteins with two long hydrophobic regions separated by a long hydrophilic loop. Although the most well-known aspect of RTNs is their role in shaping and morphogenesis of the ER, they have other distinct functions (Chiurchiù et al., 2014). From 2004 to 2006, our group and that of Dr. Yan independently used proteomic approaches to show that BACE1 interacts with RTN3 and RTN4-B/C (Nogo-B/C) (He et al., 2004; Murayama et al., 2006). Importantly, RTNs appear to exert an inhibitory effect on BACE1-induced generation of A␤, as revealed by the finding that RTN overexpression led to reduced A␤ production through modulation of BACE1 processing of APP (He et al., 2004; Murayama et al., 2006). Conversely, siRNA-mediated suppression of RTN3 led to increased ␤-CTF levels and A␤ secretion (He et al., 2004). Owing to neuronal expression of RTN3 in the brain (Moreira et al., 1999), studies on the regulation of BACE1 by RTNs have mainly focused on RTN3. The inhibitory effects of RTN3 on BACE1 have been verified in mouse models of AD. Notably, overexpression of RTN3 reduced A␤ deposition in a transgenic mouse model of AD (Shi et al., 2009; Araki et al., 2013), whereas RTN3 deficiency led to increased A␤ deposition (Shi et al., 2014). The mechanisms by which RTNs inhibit BACE1 are only partly understood at present. Using various deletion constructs of RTN3 and RTN4-C, we showed that the tertiary structure of RTNs with two hydrophobic domains is essential for their inhibitory function (Kume et al., 2009b). At the cellular level, BACE1 and RTN3 colocalize at the same subcellular sites (He et al., 2004; Kume et al., 2009a; Shi et al., 2009). Studies by Dr. Yan and co-workers disclosed that RTN3 overexpression alters the intracellular localization of BACE1 to increase retention in the ER and reduce levels on the cell surface (Shi et al., 2009). The group additionally suggested that RTN3 expression leads to reduced axonal transport of BACE1 in cultured neurons (Deng et al., 2013). Although the mechanisms underlying RTN3 regulation in the brain are currently unclear, agents that induce RTN3 expression may have therapeutic efficacy against AD. A recent study reported that the heavy component of CUTA, the mammalian CutA divalent cation tolerance homolog (Escherichia coli), modulates amyloidogenic APP processing and A␤ production through interactions with BACE1 (Zhao et al., 2012b). The data showed that downregulation of CUTA decelerates BACE1 transport from the Golgi/TGN to the plasma membrane and reduces the steady-state levels of cell surface BACE1, resulting in increased A␤ generation. BRI2, a protein mutated in familial British and Danish Dementias, has been shown to interact with APP and affect its processing (Fotinopoulou et al., 2005). Recently, a related study reported that BRI2 physically interacts with BACE1 and reduces its cellular levels by promoting lysosomal degradation and reducing mRNA expression (Tsachaki et al., 2013). Another group of investigators reported that Fbx2, a neuronspecific F-box protein, interacts with BACE1 and promotes its degradation via the ubiquitin-proteasome system (Gong et al., 2010).

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5. BACE1 and sAPP␣ sAPP␣, a metabolite of non-amyloidogenic APP processing, has both neurotrophic and neuroprotective properties (Kögel et al., 2012). Recent studies by Dr. Tan and colleagues showed that sAPP␣ is capable of inhibiting BACE1 though physical interactions (Obregon et al., 2012). The group showed that sAPP␣ inhibits ␤-cleavage of APP and interacts with BACE1 in cultured cells. Furthermore, experiments with transgenic mice expressing sAPP␣ demonstrated that sAPP␣ inhibits amyloidogenic APP processing and reduces A␤ pathology in a mouse model of AD. Conversely, immunoneutralization of sAPP␣ enhances A␤ generation in the AD mice. However, sAPP␣ also ameliorates A␤ pathology via a mechanism unrelated to BACE1 inhibition (Fol et al., 2016). Another recent report disclosed that sAPP␣ acts as an endogenous direct inhibitor of BACE1 via an allosteric mechanism, but not sAPP␤ (Peters-Libeu et al., 2015). These studies suggest a novel mechanism of BACE1 regulation whereby BACE1 activity and A␤ generation are regulated via sAPP␣. Further studies on the BACE1 inhibitory effects of sAPP␣ may contribute to the development of novel approaches for BACE1 suppression. For example, sAPP␣ enhancing agents or sAPP␣ mimetics may have therapeutic value to AD.

6. BACE1 and amyloid ␤-protein (A␤) Soluble A␤ oligomers in the brain are key pathogenic structures in AD. A␤ oligomers specifically bind neurons, evoking neurotoxicity and synapse deterioration, and are thought to be the initiators of AD pathology (Viola and Klein, 2015). BACE1 protein levels are elevated in AD brain (Hampel and Shen, 2009). Consistently, increased BACE1 levels have been detected in the brains of AD mice (Zhao et al., 2007; Zhang et al., 2009; Devi and Ohno, 2010). Previous pathological studies indicate accumulation of BACE1 in dystrophic neurites surrounding amyloid plaques in brains of AD patients and AD model mice (Kandalepas et al., 2013). One critical question is whether a direct relationship exists between aberrant BACE1 expression and A␤ accumulation. While a number of previous reports have documented A␤induced transcriptional elevation of BACE1 in non-neuronal and neuronal cells (Chami and Checler, 2012; Tamagno et al., 2012), Dr. Vassar and colleagues showed that A␤42 oligomers trigger BACE1 elevation via a post-transcriptional mechanism in mouse primary neurons (Sadleir and Vassar, 2012). However, the issue of whether A␤ modulates BACE1 at the translational or post-translational level remains to be established. In this regard, the group suggested that A␤-associated BACE1 elevation is not mediated by translational control via eIF2␣ phosphorylation (O’Connor et al., 2008; Sadleir et al., 2014). We independently investigated the relationship between A␤ and BACE1 using a neuronal model system in which rat primary cultured neurons were treated with relatively low concentrations of A␤42 oligomers or fibrils for 2-3 days. Our results indicated that A␤ oligomers induce significant upregulation of both endogenous and exogenous BACE1. As exogenous BACE1 was independent of translational control via eIF2␣, and BACE1 mRNA levels were unaffected by A␤ oligomer treatment, we inferred that A␤ oligomers influence BACE1 expression at the posttranslational level. Further analyses revealed that A␤ oligomers increase BACE1 immunoreactivity in neurites (both axons and dendrites), but not soma of neurons. The most likely possibility is that A␤ oligomers modulate BACE1 via a post-translational mechanism involving altered subcellular localization, which may result from impairment of BACE1 trafficking and degradation (Mamada et al., 2015). Activation of calpain is suggested to be involved in BACE1 upregulation induced by aggregated A␤ (Liang et al., 2010). Consistent

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with this theory, a novel calpain inhibitor led to downregulation of BACE1 in a mouse model of AD (Medeiros et al., 2012). Accordingly, a vicious cycle appears to exist whereby soluble A␤ oligomers augment A␤ production by promoting amyloidogenic processing of APP through BACE1 modulation. The direct association between A␤ oligomers and BACE1 has important implications for AD. It is plausible that the cycle is closely associated with the pathogenetic mechanism of AD, and therapeutic interventions to disrupt the cycle may therefore aid in preventing disease progression. Further elucidation of the precise mechanisms underlying A␤ oligomer-induced BACE1 upregulation should facilitate the identification of new therapeutic targets for AD. 7. Conclusions BACE1 is modulated at the post-translational level by several factors, which is associated with both physiological and pathological functions. The significance of BACE1 modulation can be highlighted from two viewpoints: the pathogenetic mechanism of AD, and development of therapeutic strategies. It is proposed that A␤ oligomers induce BACE1 augmentation in neurons via a post-translational mechanism, generating a vicious cycle of A␤ production (Mamada et al., 2015). However, further research is needed to elucidate the precise mechanisms responsible for A␤ oligomer-induced BACE1 upregulation. Moreover, genetic risk may be associated with BACE1 modulation. For instance, Rab11A is genetically associated with late-onset AD (Udayar et al., 2013). BIN1, a genetic risk factor of AD (Tan et al., 2013), is also potentially involved in BACE1 trafficking (Miyagawa et al., 2014). As BACE1 is a promising therapeutic target, many smallmolecule inhibitors have been developed, and clinical trials are in progress (Vassar, 2014). Recent research on post-translational modulation of BACE1 has also highlighted the possibility of BACE1 modification with agents other than inhibitors. In particular, elucidation of the molecular mechanisms underlying the inhibitory effects of BACE1-associated molecules, such as sAPP␣, should provide important insights that facilitate the development of novel strategies to disrupt the pathological progression of AD. Conflict of interest The author declares no competing interests. Acknowledgment This work was supported, in part, by a Grant-in-Aid for Scientific Research Grant Number 22590951 from JSPS, Japan, and an Intramural Research Grant [27-9] for Neurological and Psychiatric Disorders of the National Center of Neurology and Psychiatry. Please note that citation of many relevant and contributory references was not possible within this concise review. References Araki, W., Tamaoka, A., 2015. Amyloid beta-protein and lipid rafts: focused on biogenesis and catabolism. Front. Biosci. (Landmark Ed.) 20, 314–324. Araki, W., Oda, A., Motoki, K., Hattori, K., Itoh, M., Yuasa, S., Konishi, Y., Shin, R.W., Tamaoka, A., Ogino, K., 2013. Reduction of ␤−amyloid accumulation by reticulon 3 in transgenic mice. Curr. Alzheimer Res. 10, 135–142. Benjannet, S., Elagoz, A., Wickham, L., Mamarbachi, M., Munzer, J.S., Basak, A., Lazure, C., Cromlish, J.A., Sisodia, S., Checler, F., Chrétien, M., Seidah, N.G., 2001. Post-translational processing of beta-secretase (beta-amyloid-converting enzyme) and its ectodomain shedding. The pro- and transmembrane/cytosolic domains affect its cellular activity and amyloid-beta production. J. Biol. Chem. 276, 10879–10887. Bennett, B.D., Babu-Khan, S., Loeloff, R., Louis, J.C., Curran, E., Citron, M., Vassar, R., 2000. Expression analysis of BACE2 in brain and peripheral tissues. J. Biol. Chem. 275, 20647–20651.

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Please cite this article in press as: Araki, W., Post-translational regulation of the ␤-secretase BACE1. Brain Res. Bull. (2016), http://dx.doi.org/10.1016/j.brainresbull.2016.04.009