Function, regulation and therapeutic properties of β-secretase (BACE1)

Seminars in Cell & Developmental Biology 20 (2009) 175–182

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Review

Function, regulation and therapeutic properties of ␤-secretase (BACE1) Michael Willem ∗ , Sven Lammich ∗ , Christian Haass Center for Integrated Protein Science Munich, Adolf-Butenandt-Institute, Department of Biochemistry, Laboratory for Neurodegenerative Disease Research, Ludwig-Maximilians-University, 80336 Munich, Germany

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Article history: Available online 20 January 2009 Keywords: Alzheimer’s disease A␤ APP BACE1 Neuregulin Secretase

a b s t r a c t ␤-Secretase (␤-site amyloid precursor protein cleaving enzyme 1; BACE1) has been identified as the rate limiting enzyme for amyloid-␤-peptide (A␤) production. A␤ is the major component of amyloid plaques and vascular deposits in Alzheimer’s disease (AD) brains and believed to initiate the deadly amyloid cascade. BACE1 is the principle ␤-secretase, since its knock-out completely prevents A␤ generation. BACE1 is likely to process a number of different substrates and consequently several independent physiological functions may be exerted by BACE1. Currently the function of BACE1 in myelination is best understood. BACE1 cleaves and activates Neuregulin-1 and is thus directly involved in myelination of the peripheral nervous system during early postnatal development. However, additional physiological functions specifically within the central nervous system are so far less understood. BACE1 is upregulated in at least some AD brains. Multiple cellular mechanisms for BACE1 regulation are known including post-transcriptional regulation via its 5 -untranslated region, microRNA and non-coding anti-sense RNA. BACE1 is a primary target for A␤ lowering therapies, however the development of high affinity bio-available inhibitors has been a major challenge so far. © 2009 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4.

Maturation and cellular localization of BACE1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BACE1 substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of BACE1 expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BACE1 directed therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease worldwide. The major pathological hallmarks of AD are intracellular neurofibrillary tangles, which consist mainly of the hyperphosphorylated tau protein and extracellular amyloid plaques [1]. Amyloid plaques are composed predominantly of the amyloid-␤-peptide (A␤), a hydrophobic peptide of 39–43 amino acids [2,3]. A␤ is liberated upon endoproteolytic processing of the amyloid precursor protein (APP) by consecutive cleavages of ␤-

Abbreviations: AD, Alzheimer’s disease; ADAM, a disintegrin and metalloprotease; APP, amyloid precursor protein; A␤, amyloid-␤-peptide; BACE1, ␤-site APP cleaving enzyme 1; NRG1, Neuregulin-1; PS, presenilin. ∗ Corresponding authors. Tel.: +49 89218075462/89218075484; fax: +49 89218075415. E-mail addresses: [email protected] (M. Willem), [email protected] (S. Lammich). 1084-9521/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcdb.2009.01.003

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secretase and ␥-secretase (Fig. 1). An inherited mutation which results in the KM to NL double exchange at the ␤-secretase cleavage site in APP, the so called Swedish APP mutant (APPswe) was described to increase the efficiency of ␤-cleavage [4,5]. ␤-Secretase was discovered in 1999 independently by a number of different groups [6–10]. Therefore a confusing nomenclature exists in the literature. Accordingly, ␤-secretase has been called BACE1 (for ␤site APP cleaving enzyme 1) [6], memapsin 2 [10] or Asp 2 [7,9]. We hereafter refer to ␤-secretase as BACE1. BACE1 is a member of the pepsin-like family of aspartyl proteases. It is a type I membrane protein, which contains the characteristic dual active site motif (D-T/S-G-T/S) of aspartic proteases in its ectodomain [6,11]. Mutations in either one of the two active site motifs result in complete loss of function [7,12]. BACE1 is ubiquitously expressed with highest levels in brain and pancreas. BACE1 activity in pancreas is low due to the generation of alternatively spliced transcripts, which produce BACE1 variants with reduced proteolytic activity [13]. In

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ity [29,31,32], therefore in the following we will focus on BACE1 only. 1. Maturation and cellular localization of BACE1

Fig. 1. Processing of APP. Proteolytic processing of APP occurs by two alternative pathways: BACE1 dependent cleavage results in the release of soluble APP (␤-APPs). The C-terminal membrane-bound stub is subjected to intramembraneous proteolysis by ␥-secretase in a stepwise manner at the ␧- and ␥-site which liberates the intracellular domain of APP (AICD) into the cytoplasm and A␤ into the lumen. Alternatively the smaller fragment p3 can be released in a similar way by concerted ␣and ␥-secretase mediated proteolysis. The anti-amyloidogenic pathway is initiated by the ␣-secretases ADAM9, ADAM10 or ADAM17.

contrast high BACE1 enzymatic activity was found in human brain extracts. This is consistent with the finding that neurons produce the highest levels of A␤ [8,14]. BACE1 cleaves APP into two fragments. The N-terminal soluble ␤-APPs is released upon shedding by BACE1. A C-terminal fragment (C99) is retained in the membrane, which is the immediate precursor for A␤ generation (Fig. 1). ␥-Secretase, a protein complex consisting of nicastrin, APH-1 (anterior pharynx-defective phenotype-1), PEN-2 (PS enhancer 2) and presenilin (PS), cleaves C99 in a stepwise manner within its transmembrane domain at the ␧-, ␨- and ␥-site [3,15]. These cleavage events generate the APP intracellular domain (AICD) and several species of A␤. If AICD serves as a transcriptional regulator like other ␥-secretase cleavage products is currently under debate [16–20]. The liberated hydrophobic A␤ peptide forms toxic oligomers, which induce the amyloid cascade and probably cause cognitive impairment and neuronal loss. A␤ accumulation is counterbalanced by its proteolytic degradation and drainage from the brain [21–23]. APP can not only be shed by BACE1 but it is also cleaved in the middle of the A␤ domain by ␣-secretase, thereby precluding the formation of A␤ (Fig. 1). Three members of the ADAM (a disintegrin and metalloprotease) family of metalloproteases are described to have ␣-secretase activity, namely ADAM9, ADAM10 and ADAM17 [24–26]. The processing of APP by ␣-secretase generates the soluble ␣-APPs ectodomain, which may have neuroprotective and neurotrophic properties [27] and a membrane-bound C-terminal fragment (C83). Whether the truncated ␤-APPs lacks this function is currently unclear. C83 is further cleaved by ␥-secretase, producing the non-amyloidogenic p3 as well as AICD. Together with BACE1, another membrane-bound aspartyl protease with substantial sequence homology to BACE1, termed BACE2 (memapsin 1 or Asp 1) was discovered [28–31]. However, BACE2 seems to function primarily as an ␣-secretase-like activity [29,31,32] and is preferentially expressed in non-neuronal cells [28]. BACE2 is clearly not involved in A␤ generation as an alternative ␤-secretase activ-

The identification of the aspartyl protease BACE1 as ␤-secretase allowed the rapid characterization of this enzyme. Purified BACE1 has an optimal enzymatic activity at an acidic pH of approximately 4.5, which reflects its primary site of action inside the cell, i.e. in acidified endosomes [6,14,33]. However, APP containing the Swedish mutation is, due to the better cleavage site, already cleaved within the Golgi apparatus, thus allowing BACE1 to outcompete anti-amyloidogenic processing by ␣-secretase activities [34,35]. If BACE1 principally exists as a monomeric enzyme or as a dimer is not well understood. However, in brain tissue of mice and humans BACE1 forms homodimers. Interestingly, such homodimers exert a higher enzymatic activity than monomeric BACE1 [36,37]. BACE1 is synthesized in the endoplasmatic reticulum (ER) as an immature proenzyme with a molecular weight of 60 kDa. BACE1 then rapidly matures to the 70 kDa form. Maturation involves disulfide bridge formation, N-glycosylation, carbohydrate sulfation, propeptide removal by prohormone protein convertases like Furin and palmitoylation at its junction of the trans-membrane and cytoplasmic domain [38–43]. While deletion of the glycosylation sites has a significant impact on BACE1 activity [44], removal of the prodomain does not diminish the enzymatic activity of BACE1 towards its substrate APP to a great extent [43,45]. After its maturation in the ER and the Golgi apparatus BACE1 is transported to the plasma membrane. It is discussed that A␤ generation is increased in lipid rafts because of colocalization of BACE1 and its substrate APP [46–49]. However, a recent study suggests that the activity of BACE1 is independent of its palmitoylation and thereby can occur in non-raft subdomains of the plasma membrane [50]. Similar to APP, BACE1 is internalized from the plasma membrane to early endosomes, the main compartment of A␤ generation [33]. The internalization is driven primarily by the di-leucine sorting signal within the Cterminus (DISLLK) [39,51]. BACE1 is recycled from early endosomal compartments to the trans-Golgi-network (TGN). The phosphorylation of a single serine residue (S498) in the C-terminus near to the di-leucine motif of BACE1 is required for this event. Wild type BACE1 and a S498D carrying BACE1 mutant which mimics phosphorylated BACE1 are efficiently retrieved from early endosomes to the TGN, whereas the non-phosphorylatable mutant BACE1 S498A is retained in early endosomes [52]. The recycling step depends on Golgi-localized ␥-ear containing ADP (GGA) ribosylation factor binding proteins [53–55]. GGA1 mediated rerouting from endosomes to the TGN and recycling to the plasma membrane lead to a longer half-life of BACE1. In addition, it was reported that phosphorylated BACE1 could be transported in a GGA3 dependent manner from endosomes to lysosomes where BACE1 can be degraded [56,57]. Finally, BACE1 undergoes proteolytic degradation by the proteasome [58] although the physiological relevance of BACE1 processing by the proteasome is unclear. In neurons BACE1 is transported to axonal membrane surfaces and is most likely localized to pre-synaptic terminals in vivo [59,60]. Thus, axonally derived A␤ is eventually released from axon terminals [60]. In support of this Kamenetz et al. described an upregulation of BACE1 by synaptic activity and they put forward a role for A␤, which in a feedback loop might regulate BACE1 levels via modulation of synaptic activity [61]. 2. BACE1 substrates BACE1 knock-out mice fail to produce any A␤, thus BACE1 is the sole enzyme with a bonafide ␤-secretase activity. These mice

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Fig. 2. Cleavage of NRG1 by BACE1. BACE1 and ADAM17 cleavage sites are found close to the EGF-like domain in type III NRG1 (red box). For activation of type III NRG1 we propose a model of a sequential dual cleavage in which ADAM17 and/or BACE1 cleave first within the juxtamembrane region close to the C-terminus of the EGF-like domain and second release the EGF-like domain potentially by an additional BACE1 dependent cleavage. The C-terminal membrane-bound NRG1 stub is a substrate of the ␥-secretase complex.

develop normally, are healthy, fertile and appear to have no obvious morphological phenotype [62–66]. This finding suggests that inhibition of BACE1 could be a useful therapeutic approach for the treatment of AD, since a blockade of BACE1 activity should not interfere too much with physiological mechanisms exerted by BACE1 cleavage products. However, behavioral assays revealed memory impairment and changes in spontaneous activity [65,67,68]. Moreover, close analysis of BACE1 knock-out mice revealed an important morphological phenotype, which is related to a major neuronally expressed substrate of BACE1, namely Neuregulin-1 (NRG1) [69,70]. Beside many other functions NRG1 signaling via ErbB receptors is important for the early phase of Schwann cell myelination in the peripheral nervous system (PNS) [71]. During this developmental phase strong BACE1 expression is found in dorsal root ganglia (DRGs) and in motorneurons of the spinal cord [69]. BACE1 knock-out mice show severe hypomyelination of the PNS similar to NRG1 heterozygous mice. Since under these conditions, uncleaved full-length NRG1 accumulates, hypomyelination appears to be a consequence of the lack of NRG1 processing in these animals [69,70]. A contribution of BACE1 mediated NRG1 cleavage in CNS oligodendrocyte myelination is also claimed [70] but this was not observed by others [69]. Moreover, CNS myelination by oligodendrocytes was found to be independent of NRG1 and its receptors ErbB3 and ErbB4 [72]. The proteolytic activation of NRG1 signaling could occur by a single cleavage of type III NRG1 by ADAM17 or BACE1 (Fig. 2). Thereafter the NRG1 EGF-like domain is still retained in the membrane and could activate ErbB receptors on the target cell (Fig. 2). Moreover, an activation of ErbB4 receptors by soluble NRG1 is expected, since ErbB4 internalization is necessary for NRG1 signaling [73]. The soluble EGF-like domain of type III NRG1 may thus be liberated by a dual cleavage to allow for paracrine signaling as shown for type I NRG1 (Fig. 2) [74–76]. The NRG1 intracellular domain (NRG1-ICD), released by ␥-secretase, might exert intracellular functions via nuclear signaling mechanisms [77,78]. NRG1 is an important candidate gene for Schizophrenia and NRG1-ErbB4 signaling has a function in adult neuronal plasticity, since NRG1 modulates synaptic transmission of GABAergic and glutamatergic synapses [76,79–81]. Suppression of NMDA receptor signaling is an important mechanism in Schizophrenia [82–84]. Preventing NRG1-ErbB4 signaling destabilizes synaptic AMPA receptors and leads to loss of synaptic NMDA currents and spines [84]. Recently, Savonenko et al. demonstrate that BACE1 knock-out mice show, beside an impaired NRG1 processing, a vari-

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ety of behavioral abnormalities reminiscent of those identified in Schizophrenia, including prepulse inhibition impairments, novelty induced hyperactivity, alterations in social recognition and cognitive deficits [85]. Therefore, a complete inhibition of BACE1 as a therapeutic approach for AD treatment could cause side effects. BACE1, as any other protease, may process many substrates and may thus exert multiple physiological functions. Indeed, it was recently shown that BACE1 is also involved in the regulation of voltage dependent sodium channels and neuronal activity [86,87]. Neuronal action potentials are dependent on the voltage dependent sodium channel Nav 1.1. Interestingly, it was found that sodium current densities are markedly reduced in adult hippocampal neurons from BACE1 overexpressing transgenic mice [87]. Consistent with this finding, BACE1 was found to be involved in the turnover of sodium channels by cleaving the ␤2-subunit of Nav 1.1 [87]. In analogy to APP processing, a ␥-secretase mediated cleavage is following BACE1 dependent shedding of the ␤2-subunit. The ICD of the Nav 1 ␤2-subunit apparently regulates in a feedback mechanism the transcription of the ␣-subunit [87], thereby leading to the accumulation of ␣-subunits inside the cell and a decrease of functional sodium channels at the cell surface. However, whether sodium current densities are compromised in brains of BACE1 knock-out mice remains to be shown. Type II ␣-2,6-sialyltransferase (ST6Gal-1) was also identified as a BACE1 substrate [88]. ST6Gal-1, a Golgi apparatus resident protein, is secreted upon BACE1 cleavage in BACE1 transgenic mice and this cleavage event is decreased in BACE1 knock-out mice [89]. Moreover, the authors observed higher plasma levels of ST6Gal-1 due to increased BACE1 expression in LEC rats, a model of Wilson’s disease. These animals accumulate copper in their livers, due to a defect of a Golgi apparatus resident copper transporter, arguing that copper induced stress in the liver might lead to elevated BACE1 transcription. Finally, platelet selectin glycoprotein ligand1 (PSGL-1) and the interleukin receptor type II were also shown to be cleaved by BACE1 [90,91], but a deficiency in the immune response was not demonstrated in BACE1 knock-out mice so far [66]. 3. Regulation of BACE1 expression The regulation of BACE1 at the transcriptional level was studied mainly in vitro so far [92–97]. Several promoter elements important for the transcriptional regulation of BACE1, e.g. transcription factor binding sites for signal transducer and activator of transcription (STAT1/3 and STAT6) have been identified in the BACE1 promoter [94,98]. Recently it was found that overexpression of p25, the activator of cyclin dependent kinase 5 (CDK5), results in a 2fold induction of A␤ levels [98]. In this study, p25 overexpression in transgenic mice lead to hyperphosphorylation of STAT3 which affects BACE1 promoter activity. As a consequence increased BACE1 mRNA and protein levels could be observed in these transgenic animals. These findings seem to be particularly interesting, since tangles, a pathological hallmark of AD, are composed of hyperphosphorylated tau, which is phosphorylated by glycogen synthase kinase 3␤ (GSK3-␤) and CDK5. Thus, CDK5 seems to be involved in both neuropathological aspects of AD, the buildup of amyloid plaques and in tangle formation. Transcriptional and translational upregulation of BACE1 can also be triggered by cellular and especially oxidative stress [99]. 4Hydroxy-nonenal (HNE), a product of lipid peroxidation, is a likely mediator of this effect [100,101]. Furthermore, activated microglial cells producing TNF␣ and INF␥ could mediate BACE1 upregulation [102]. Additionally, it was reported that acute hypoxia leads to elevated BACE1 mRNA levels and subsequently elevated BACE1 protein levels [103]. This effect seems to be mediated by binding of hypoxia-inducible factor 1␣ to the promoter region of BACE1.

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Fig. 3. Translational regulation of BACE1. (A) BACE1 expression is regulated via multiple post-transcriptional pathways. Capped BACE1 (yellow ellipsoid) mRNA contains a long GC-rich 5 UTR with short uORFs (red boxes) able to form complex secondary structures. Unknown proteins (blue boxes) might bind to the 5 UTR, as well as to the 3 UTR and thereby orchestrate the translational efficiency of BACE1. Moreover, it was shown that BACE1 translation was regulated by specific miRNAs (green rectangles). Translational control is further mediated via a short natural anti-sense transcript which forms heteroduplexes with the ORF of the BACE1 mRNA. (B) The 5 UTR of BACE1 contains 3 uORFs. Initiation of translation normally occurs at the first AUG within the 5 UTR of BACE1. After translation of the first uORF, the 60S ribosomal subunit dissociates off the mRNA, however few 40S ribosomal subunits might stay attached to the mRNA. Under normal conditions, high levels of ternary complex exist in the cell, allowing a rapid recharging of these scanning 40S ribosomal subunits. As a consequence reinitiation at uORF2 might occur, which decreases the probability of reinitiation at the BACE1 start codon leading to low levels of BACE1 protein. However under stress conditions, low levels of ternary complex exist in the cell, due to phosphorylation of eIF2␣. Therefore the 40S ribosomal subunit might spend more time scanning the 5 UTR of BACE1. This increases the probability that the scanning 40S ribosomal bypasses uORFs and secondary structure elements within the 5 UTR, before the recharging step with ternary complex occurs. As a result, increased reinitiation at the BACE1 start codon might lead to increased BACE1 protein.

Increased BACE1 protein levels were observed in some brain samples of sporadic AD patients without a parallel increase in mRNA expression [104–109]. This prompted several researchers to uncover the regulatory mechanism behind this phenomenon. Multiple post-transcriptional regulatory mechanisms are currently discussed (Fig. 3A) [96,99,110]. In general, complex secondary structures, the presence of upstream open reading frames (uORF) and the binding of transactivating factors to the 5 UTR (untranslated region) and/or the 3 UTR are shown to influence translational efficiency. In addition, microRNA (miRNA) mediated translational control can occur via binding to the 3 UTR [111]. Several researchers independently demonstrated that BACE1 expression is restricted via its 5 UTR [112–116]. In cells transfected with BACE1 cDNA containing or lacking the 5 UTR of BACE1, large differences in BACE1 protein expression were observed. In the absence of the 5 UTR, BACE1 protein levels were significantly increased, whereas mRNA levels were not affected. Thus, the 5 UTR represses translation of BACE1. Extensive mutagenesis predicted that the GC-rich region of the 5 UTR forms a constitutive translation barrier, which may prevent the ribosome from efficiently initiating translation of the BACE1 mRNA [113]. Additionally, it was

proposed that after the translation of a uORF within the 5 UTR of BACE1, scanning ribosomes will dissociate off the BACE1 mRNA, thereby contributing to the translational repression effect of the highly structured 5 UTR [115,116]. Interestingly, an increase of BACE1 protein level in mouse brain was detected after energy starvation obtained by insulin-induced hypoglycaemia or administration of 2-deoxy-glucose, kainic acid and 3-nitropropionic acid [117]. No alterations of BACE1 transcript levels were observed, supporting the assumption that BACE1 expression might be translationally controlled in vivo. One possible explanation for this finding is that stress induces global translational inhibition and a subsequent shift to selective translation of stress-response transcripts (Fig. 3B) [111,118]. Depending on the stress condition, eIF2␣ is phosphorylated by one of four different kinases which results in the inhibition of global translation. The phosphorylation event reduces the dissociation of eIF2B from eIF2␣-GDP, which catalyses the GDP-GTP exchange on eIF2␣, thereby preventing the formation of the ternary complex consisting of the methionyl-initiator-tRNA bound to eIF2␣-GTP. However, specific mRNAs, which are important for cell survival, are selectively translated under stress conditions. The best understood

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examples for such a mechanism are yeast GCN4 and mammalian ATF4 [111,118]. Both transcripts contain several uORFs. The translation of these transcripts depends on the availability of active ternary complexes. Initiation of translation occurs in general at the first AUG. Under normal conditions, where high concentrations of the active ternary complexes exist in the cell, few 40S ribosomal subunits are able to reinitiate translation at a downstream uORF. Afterwards ribosomes dissociate off the mRNA. As a consequence, efficient translation at the ORF of both mRNAs is strongly reduced. However, under stress conditions the 40S ribosomal subunits scan through the following uORF(s) along the 5 UTR and the probability for reinitiation at the start codon of the mRNAs increases, which leads to elevated levels of protein expression. In accordance with this model, increased BACE1 and phosphorylated eIF2␣ protein levels were observed in vitro and in vivo after energy deprivation arguing that under this condition the 40S ribosomal subunits might bypass uORFs and secondary structure elements within the 5 UTR of BACE1, thereby increasing the probability of efficient reinitiation on the start codon of BACE1 (Fig. 3B) [119]. Recently, miRNA mediated downregulation of BACE1 translation via the 3 UTR of BACE1 was described for different miRNAs [120,121]. Decreased expression levels of miRNA-29a and of miRNA29b were observed in brains of AD patients with abnormally high BACE1 expression. However, the functional role of miRNAs in the developmental regulation of BACE1 or in pathological deregulation in AD needs to be addressed in more detail, because currently it is still unclear whether local BACE1 translational upregulation is a result of downregulation of the corresponding miRNAs in the same neurons. Since the binding site in the BACE1 3 UTR is highly conserved for miRNA-29b, which is upregulated in the first weeks after birth, this miRNA may be a good candidate as a key regulator of the postnatal downregulation of BACE1 protein levels [69,120,122]. In addition to miRNA mediated BACE1 regulation, a non-coding anti-sense RNA against BACE1 was found to be elevated in the brain of AD patients. Surprisingly, in that case an upregulation of BACE1 expression was demonstrated to result from the stabilization of BACE1 mRNA anti-sense duplexes [123]. This was described as a concordant regulatory mechanism in contrast to the discordant mechanism of anti-sense mRNA upregulation, which leads to the degradation of the corresponding sense mRNA[124]. The authors propose that the elevation of the BACE1 anti-sense RNA, resulting from the actions of AD related cell stressors, forms the basis for deleterious feed-forward cycle of AD progression [123].

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By hooking up to fatty acid moieties, inhibitors could also become enriched in lipid raft environments where they eventually reach higher concentrations at the site of BACE activity [131]. However, as described above the functional relevance of lipid raft associated BACE1 is under debate [50]. At present potent active site inhibitors are developed, which are useful to study BACE1 function in cellular assays and after direct administration to the brain of AD-mouse models for a short period. However, the major problem of the available BACE1 inhibitors is their ability to cross the blood–brain barrier and block BACE1 activity in the brain. Since a BACE1 inhibitor must inevitably exhibit activity intracranially, it is imperative that it is not only able to cross the blood–brain barrier, but also to achieve sustainably high drug levels. Many of the established BACE1 inhibitors are substrates of the P-glycoprotein (Pgp) transporter and efficiently pumped out of the brain [132,133]. When BACE1 inhibitors were applied together with Pgp inhibitors the BACE1 inhibitors remained in the brains at much higher levels and A␤ synthesis could be lowered significantly even after oral administration in rodents and rhesus monkeys [132–135]. Nevertheless, one should be aware that due to the identification of physiological relevant BACE1 substrates, a complete block of BACE1 activity by BACE1 inhibitors may also cause side effects. Whether myelination may be affected upon treatment of adults is unknown, however major side effects are rather unlikely, since re-myelination after nerve crush is obviously independent of NRG1 mediated ErbB2 signaling [136]. Beside a direct pharmaceutical inhibition of BACE1, alternative approaches, which address the normalization of the deregulated BACE1 expression level, could be employed in the future. However, since a pharmacological interference with regulatory mechanisms is not only very difficult to achieve but may also cause rather pleiotropic effects, it is still much too early to speculate about the potential of a therapeutic control of BACE1 levels. Acknowledgements The authors thank Dr. Richard Page for comments on the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (DFG), the Leibniz Award (to C.H.), the SFB596 (Project A9; to C.H., S. L. and M.W.), the National Genome Research Network (NGFNplus; to C.H.), the Helmholtz Alliance for Mental Health and Ageing (HELMA; to C.H. and M.W.) and the Virtual Institute Neurodegeneration & Ageing (to C.H.). C.H. is supported by a Forschungsprofessur of the Ludwig-MaximiliansUniversity.

4. BACE1 directed therapy References Both amyloidogenic secretases, BACE1 and ␥-secretase, are attractive and obvious targets for the therapeutic treatment of AD. It was shown that BACE1 seems to prefer more bulky residues at P1, like in APPswe [125], therefore wild type APP is rather inefficiently cleaved by BACE1 [126,127]. While the KM to NL exchange at the ␤-secretase cleavage site in APPswe results in an increased affinity for BACE1, a M to V substitution at the position P1 reduced the affinity significantly [125]. From the BACE1 substrate cleavage sites described so far, no convincing common substrate recognition signature for BACE1 cleavage could be determined. The elucidation of the BACE1 crystal structure in the presence of an inhibitor forced the development of highly selective peptidomimetic inhibitors [11,126,128–130]. However, it remains an open question whether BACE1 inhibitors designed to the BACE1 homodimer, for which a higher substrate affinity could be demonstrated [36,37], rather than to the monomer would result in compounds with higher affinity.

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