Identification of a novel amyloid precursor ... - The FASEB Journal

The FASEB Journal article fj.11-200295. Published online April 6, 2012.

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Identification of a novel amyloid precursor protein processing pathway that generates secreted N-terminal fragments Laura J. Vella and Roberto Cappai1 Department of Pathology and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, Victoria, Australia ABSTRACT Alzheimer’s disease (AD) is a neurodegenerative disorder of the central nervous system. The proteolytic processing of the amyloid precursor protein (APP) into the ␤-amyloid (A␤) peptide is a central event in AD. While the pathway that generates A␤ is well described, many questions remain concerning general APP metabolism and its metabolites. It is becoming clear that the amino-terminal region of APP can be processed to release small N-terminal fragments (NTFs). The purpose of this study was to investigate the occurrence and generation of APP NTFs in vivo and in cell culture (SH-SY5Y) in order to delineate the cellular pathways implicated in their generation. We were able to detect 17- to 28-kDa APP NTFs in human and mouse brain tissue that are distinct from N-APP fragments previously reported. We show that the 17- to 28-kDa APP NTFs were highly expressed in mice from the age of 2 wk to adulthood. SH-SY5Y studies indicate the generation of APP NTFs involves a novel APP processing pathway, regulated by protein kinase C, but independent of ␣-secretase or ␤-secretase 1 (BACE) activity. These results identify a novel, developmentally regulated APP processing pathway that may play an important role in the physiological function of APP.—Vella, L. J., Cappai, R. Identification of a novel amyloid precursor protein pro-

Abbreviations: A␤, ␤-amyloid; AD, Alzheimer’s disease; APP, amyloid precursor protein; APP-CTF␣, C-terminal fragment of APP following ␣-secretase cleavage; APP-CTF␤, C-terminal fragment of APP following ␤-secretase cleavage; APPKO, amyloid precursor protein-knockout; APLP2, amyloid precursor-like protein 2; APLP2KO, amyloid precursorlike protein 2-knockout; BACE, ␤-site amyloid precursor protein-cleaving enzyme 1 (␤-secretase 1); BCA, bicinchoninic acid; CN, cognitively normal; CSF, cerebrospinal fluid; CTF, C-terminal fragment; CuBD, copper-binding domain; DR6, death receptor 6; E, embryonic day; flAPP, full-length amyloid precursor protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFD, growth factor domain; N-APP, N-terminal amyloid precursor protein; NGF, nerve growth factor; NTF, N-terminal fragment; P, postnatal day; PBS, phosphate buffered saline; PDBu, phorbol 12,13dibutyrate; PI, protease inhibitor; PKC, protein kinase C; RA, retinoic acid; sAPP, secreted amyloid precursor protein; sAPP␣, secreted amyloid precursor protein after ␣-secretase cleavage; sAPP␤, secreted amyloid precursor protein after ␤-secretase cleavage; WT, wild type 0892-6638/12/0026-0001 © FASEB

cessing pathway that generates secreted N-terminal fragments. FASEB J. 26, 000 – 000 (2012). www.fasebj.org Key Words: Alzheimer’s disease 䡠 ␣-secretase 䡠 ␤-secretase 䡠 developmental regulation Alzheimer’s disease (AD) is the most common form of dementia, characterized pathologically by the extracellular deposition of insoluble amyloid fibrils in the brain and intracellular neurofibrillary tangles. The main component of amyloid is ␤-amyloid (A␤) peptide, typically a 38- to 43-aa residue peptide produced by proteolytic cleavage from the amyloid precursor protein (APP) (1). APP is a type 1 integral membrane protein with a single membrane-spanning domain, a large extracellular amino terminus, and a short cytoplasmic carboxyl terminus (2, 3). Mature APP undergoes proteolytic cleavage by ⱖ3 proteases, termed ␣-, ␤-, and ␥-secretase, which lead to the generation of biologically active secreted APP (sAPP) species and the neurotoxic peptide A␤ (4, 5). The sAPP fragments are produced by two opposing proteolytic pathways to generate sAPP␣ or sAPP␤ (6, 7). The ␣-cleavage pathway is the main pathway under normal physiological conditions; it involves ␣-secretase cleavage within the A␤ sequence by a range of metalloproteases, with ADAM10 being the constitutive ␣-secretase (8), to release the extracellular domain, sAPP␣, and leave a membrane-bound C-terminal fragment (APP-CTF␣), which is cleaved by ␥-secretase (9) to generate a truncated species of A␤ called p3. The alternative, amyloidogenic pathway, involves cleavage by the aspartyl protease ␤-secretase 1 [␤-site APP-cleaving enzyme 1 (BACE)] to generate sAPP␤ and the membrane-bound C-terminal fragment APP-CTF␤, which is cleaved by ␥-secretase to generate A␤. sAPP␣ is present in healthy human brain and cerebrospinal fluid (CSF; ref. 10) and a number of diverse functions have been attributed to sAPP␣, including 1 Correspondence: Department of Pathology, The University of Melbourne, Victoria, Australia, 3010. E-mail: r.cappai@ unimelb.edu.au doi: 10.1096/fj.11-200295

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increasing synaptic density and improving memory retention (11, 12), proliferation of embryonic neural stem cells (13, 14). These studies suggest that sAPP␣ acts as a neurotrophic and neuroprotective protein in the human brain (15). The amino-terminal region of APP can be processed to release an array of small N-terminal fragments (NTFs), which can be detected in human and rodent neuronal tissues and fluids (16 –21). An N-terminal cleavage product of APP (residues 1–286, ⬃35 kDa) was reported to bind death receptor 6 (DR6) and trigger the physiological removal of synapses and axons during brain development in mice (22). In the present study, the occurrence of 17- to 28-kDa NTFs in human and mouse tissue that are distinct from those reported by Nikolaev et al. (22) were examined. We delineated the cellular pathways implicated in the generation of these fragments in the human neuronal SH-SY5Y cell line. We show that the 17- to 28-kDa NTFs are derived from a novel, developmentally regulated, APP processing pathway modulated by protein kinase C (PKC).

MATERIALS AND METHODS Reagents and antibodies Antibodies used were anti-APP antibodies 22C11 (APP 66-81) and WO2 (A␤ 5– 8) produced in house, CT20 (raised against residues 751–770 of APP 770 isoform), and amyloid precursor-like protein 2 (APLP2) from Merck Biosciences (Darmstadt, Germany); anti-sAPP␣ and anti-sAPP␤ from ImmunoBiological Laboratories Co., Ltd (Gunma, Japan); and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from Cell Signaling Technologies (Danvers, MA, USA). Chemicals all-trans-retinoic acid (RA) and phorbol 12,13-dibutyrate (PDBu) were obtained from Sigma-Aldrich (St. Louis, MO, USA); TAPI-1, G°6983, and OM99-2 were obtained from Merck Biosciences. Mouse tissue collection The APP⫺/⫺ and APLP2⫺/⫺ mice and wild-type (WT) C57BL6J ⫻ 129/Sv control mice have been described previously (23, 24). Mice at 4 mo of age were considered adult. Homogenates for biochemical analysis were prepared from brain tissue of mice that had been collected at different times: embryonic day 14 (E14), E16, postnatal day 0 (P0), P2, P8, and adult. The homogenates were prepared in 9 volumes of phosphate buffered saline (PBS) by passage through 18-, 22-, and 26-gauge needles. To ensure complete lysis of brain tissue, NET lysis buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, and protease inhibitors (PIs; Roche)], was added to the samples in a 1:1 ratio. The samples were incubated on ice for 20 min and then centrifuged for 5 min at 10,000 g, and the insoluble material was discarded. The protein concentration was quantified by bicinchoninic acid (BCA) assay (Pierce, Rockford, IL, USA) and samples analyzed by SDS-PAGE and Western blot or stored at ⫺80°C. Human tissue collection Brain tissue was collected at autopsy. The sourcing and preparation of the human brain tissue were conducted by the 2

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National Neural Tissue Resource Centre (Melbourne, VIC, Australia). AD pathological diagnosis was made according to standard U.S. National Institute on Aging–Reagan Institute criteria (1997). Frontal cortex was isolated from postmortem AD patients and cognitively normal (CN) aged controls. Tissue was homogenized in 1⫻ PBS (without calcium and magnesium), using an ultrasonic cell disrupter (2⫻ 30 s, 24,000 rpm; Virsonic 600; Virtis, Gardiner, NY, USA). Protein concentration was determined (BCA assay), and brain homogenates were separated into aliquots and frozen at ⫺80°C. Cell lines and drug treatments SH-SY5Y human neuroblastoma cells (American Type Culture Collection, Rockville, MD, USA) were grown in DMEM supplemented with 20% heat-inactivated fetal calf serum, 1 U/ml of penicillin, 1 ␮g/ml of streptomycin, and 2 mM glutamate (Gibco BRL; Invitrogen, Carlsbad, CA, USA) and maintained at 37°C and 5% CO2. To obtain differentiated cells, 2 ⫻ 105 cells were plated in one well of a 6-well plate and allowed to adhere for 24 h. Differentiation was started in DMEM supplemented with 1.5% FBS and 10 ␮M RA. Fresh medium, containing RA and 1.5% FBS, was applied to the cells every 1–3 d. Experiments were typically performed on cells differentiated for ⱖ14 d and displaying a differentiated neuronal phenotype, including extensive neurites and branching, as evidenced by light microscopy and expression of specific neuronal markers (MAP2B, synaptophysin, and tyrosine hydroxylase). Stock solutions of PDBu and G°6983 were dissolved in DMSO. TAPI-1 and OM99-2 were supplied in liquid form in DMSO. All drugs were subsequently diluted in DMEM. Control cultures received equivalent concentrations of DMSO in DMEM (ⱕ0.2%). For the PDBu experiments, cells differentiated for ⱖ14 d were exposed to DMSO or PDBu (0.5 ␮M) for 6 h or with PDBu (0.5 ␮M) for 15 min prior to 6 h of G°6983 treatment. For the TAPI and OM-99-2 experiments, cells differentiated for ⱖ14 d were exposed to DMSO or TAPI (80 ␮M) or OM-99-2 (5 ␮M) overnight. Western immunoblotting Equivalent amounts of brain homogenate or cells were lysed for 30 min in an equal volume of ice-cold lysis buffer (NET buffer) with PIs (Roche). After centrifugation at 3000 g for 3 min, the protein concentration was determined by BCA assay. Medium conditioned on cells for 48 h was centrifuged at 10,000 g for 3 min, and the supernatant was either TCA precipitated or used neat. All samples were then diluted in 2⫻ sample buffer (125 mM Tris-HCl, pH 6.8; 5% BME; 4% SDS; 10% glycerol; and 0.02% bromphenol blue, w/v), boiled for 5–10 min, then centrifuged for 2 min at 12,000 g, before being electrophoresed on precast 12% Bis-Tris gels (NuPage; Invitrogen). Gels were electrophoresed at 100 –150 V in MES (50 mM Tris Base, 1 mM EDTA, and 3.4 mM SDS) buffer until the bromphenol blue dye front migrated off the bottom of the gel and maximum separation had been achieved. Proteins were transferred onto nitrocellulose membrane (Bio-Rad, Gladesville, NSW, Australia); the membrane was boiled in PBS for 2 min and then allowed to cool before being blocked in 5% skim milk powder in PBST (PBS with 0.5% Tween-20) for 1 h. Membranes were probed with primary antibody diluted in PBS-T containing 2.5% skim milk powder for 1 h, then with horseradish peroxidase-conjugated secondary antibody for 1 h, and immunoreactivity was detected using enhanced chemiluminescence solutions (GE Healthcare, Piscataway, NJ, USA).

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Expression of APP NTFs is developmentally regulated in mouse brain

Figure 1. Schematic illustration of the domain organization of APP and antibody epitopes used in this study. The N-terminal growth factor domain (GFD) and copper-binding domain (CuBD) that are part of the NTF are highlighted. TM, transmembrane domain; flAPP, full-length APP. Red line indicates A␤; ␤- and ␣-secretase cleavage sites are shown. Antibodies shown are 22C11 (monoclonal antibody that binds APP, residues 66 – 81), WO2 (residues 5-8 of human A␤ sequence, which therefore binds sAPP␣ but not sAPP␤), and CT20 (residues 751–770). Statistical analysis Western blots are representative of ⱖ3 separate experiments. Quantitative analysis of Western blots was performed with ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA), by calculating the relative density of the immunoreactive bands, and expressed as a percentage of control values. Data are expressed as means ⫾ se. The statistical difference between control and treated groups were carried out using 2-way ANOVA for repeated measures. Statistical differences were determined at values of P ⬍ 0.05 and are indicated in the figure legends.

To determine whether rodents express the NTFs, mouse brain homogenates from normal mice as well as APPknockout (APPKO) and APLP2-knockout (APLP2KO) mice were immunoblotted with 22C11. A set of bands of 17–28 kDa is present in WT and APLP2KO brain homogenate, but absent in APPKO (Fig. 3). These bands were not detected with CT20. This data is consistent with the 17- to 28-kDa bands being APP-derived NTFs. Since APP expression can be developmentally regulated in both animals and humans (25–28) we assessed whether the APP NTFs were developmentally regulated. Mouse brains at different time points from E14 to adult mice were analyzed by immunoblotting with 22C11. There was an increase in the 110- to 135-kDa flAPP species from E14 to P8 (Fig. 4). This result is consistent with previous studies that have analyzed the expression of APP during development, with APP expression commencing between E9.5 and E15 (26, 27), and peaking postnatally, and then depending on tissue maturation and completion of synaptic connections, decreasing between the first to third postnatal weeks (27, 29, 30). In contrast to the flAPP species, NTF expression was expressed at low levels in the embryonic to P0 stages. However, there was a clear increase in NTF expression

RESULTS APP NTFs are present in human brain tissue To determine the expression of APP NTFs in human brain, homogenates from the frontal cortex of CN control and AD brains were separated on SDS-PAGE and immunoblotted with the monoclonal antibody 22C11, which binds to residues 66 – 81 in the N-terminal growth factor domain (GFD) of APP (Fig. 1). The 22C11 recognized bands at 110 –135 kDa that represent full-length APP (flAPP) and sAPP species in both CN and AD brain. In addition, 22C11 detected a protein migrating at ⬃25 kDa in both CN and AD brains. The anti-A␤ antibody WO2 also specifically labeled the bands at 110 –135 kDa in both CN and AD samples. Moreover, the AD sample had an additional band at ⬃4 kDa corresponding to A␤. CT20, which binds the C-terminal residues of APP (Fig. 1), recognized flAPP in both CN and AD tissue. The APP CTFs were weakly recognized by CT20. The 22C11-reactive proteins migrating at ⬃25 kDa were not detected with WO2 or CT20 antibodies or the secondary antibody alone (2° alone), indicating that the ⬃25 kDa proteins are specific to 22C11 and represent a C-terminally truncated fragment of APP (Fig. 2). SECRETED APP N-TERMINAL FRAGMENTS

Figure 2. Identification of APP NTFs in CN and AD brain. Equal amounts (20 ␮g) of protein extracts from 10% gray matter homogenates were loaded per lane and separated by 12% Bis-Tris SDS-PAGE. After electroblotting, filter was probed with either 22C11, WO2, CT20, or mouse secondary antibody alone (2° alone). Representative Western blot analysis shows an N-terminal APP fragment detected by 22C11 migrating around 20 –22 kDa. WO2 and CT20 detect the ⫺COOH regions of APP. Note that the 20- to 22-kDa band detected by 22C11 was not detected by WO2 or CT20, indicating that it lacks the relative epitopes recognized by such antibodies and is an NTF of APP. GAPDH was used as a loading control. 3

during development, we also analyzed sAPP␣ and CTF␣ levels. Both sAPP␣ and CTF␣ had a similar expression profile to flAPP, with levels decreasing postnatally from P8 to adulthood. Densitometric analysis showed significant increases in NTF levels relative to P0 in P14 (P⬍0.05) to P18 (P⬍0.05) and adult brains (P⬍0.01) (Fig. 4B). NTF levels were 87, 162, and 169% higher, respectively, and contrasted with the rapid decrease in flAPP, sAPP␣, and CTF␣ levels from P14 to adult. Our data indicates that APP NTFs are largely generated postnatally, with expression highest in the adult mouse brain. This would suggest that N-terminal cleavage of APP might occur in mature neuronal cells. APP NTFs are generated on neuronal differentiation Figure 3. Identification of APP NTFs in mouse brain. Equal amounts (20 ␮g) of protein extracts from 10% homogenates were loaded per lane and separated by 12% Bis-Tris SDSPAGE. Membranes were probed with either 22C11 or CT20. Incubation with 22C11 followed by anti-mouse horseradish peroxidase-linked secondary antibody showed a set of APPspecifc bands of 18 –28 kDa for WT and APLP2KO homogenates. In contrast, brain homogenates from APP-KO mice showed neither APP-specific bands of flAPP or sAPP size nor any predominant shorter polypeptides. CT20 antibody-immunoblotted membranes did not show a set of APP-specific bands of 17–28 kDa for WT or APLP2KO homogenates, illustrating that the bands detected by 22C11 are specific for the N-terminal region of APP.

following birth, and this continued through to adulthood. To understand how NTF expression correlates with the expression of other APP processing products

To address whether N-terminal cleavage of APP occurs in mature neuronal cells, we assessed APP NTF generation during neuronal maturation. The human neuroblastoma cell line SH-SY5Y can be terminally differentiated on treatment with RA, recapitulating many of phenotypes seen in mature neurons (31, 32). After 12 d of RA treatment, the SY-SY5Y cell line acquired a differentiated neuronal phenotype, with extensive neurites and branching, as evidenced by light microscopy and expression of specific neuronal markers (MAP2B, synaptophysin, and tyrosine hydroxylase; data not shown). RA-induced differentiation of SH-SY5Y cells is associated with significant increases in sAPP␣ levels in conditioned media of differentiated vs. undifferentiated cells (33–35); suggesting that differentiation shifts APP processing toward the nonamyloidogenic pathway (36, 37). In our study, induction of differentiation

Figure 4. A) Mouse brains were collected at different prenatal and postnatal times, homogenized, and immunoblotted for APP and its processing products. Representative immunoblots are shown. flAPP and CTF␣ were detected with antibody CT20, sAPP␣ was detected with an sAPP␣-specific antibody, and APP NTFs were detected with 22C11. GAPDH was used as a loading control. B) Densitometric analysis, expressed as mean ⫾ se values of 3 independent experiments. Values were normalized using GAPDH as an internal control, and the percentage difference was calculated relative to P0. *P ⬍ 0.05, **P ⬍ 0.01; ANOVA. 4

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Figure 5. Modulation of APP NTF formation by neuronal differentiation. SH-SY5Y cells endogenously expressing APP were exposed to retinoic acid for up to 15 d. Media were changed 48 h prior to collection of conditioned media and cells. A) Cell lysate was probed with synaptophysin or WO2 or, to show equal loading, GAPDH. Neat media were analyzed for sAPP␣; TCA-precipitated media were analyzed for APP NTFs with 22C11. B) Densitometric analysis, expressed as mean ⫾ se values of 3 independent experiments. Values were normalized using GAPDH as an internal control, and the percentage difference was calculated relative to control. *P ⬍ 0.05, **P ⬍ 0.01; ANOVA.

increased synaptopysin, APP, and sAPP␣ levels, as well as APP NTFs (Fig. 5). The APP NTFs were detected in precipitated conditioned media. We were unable to detect NTFs in cell lysate or bands of appropriate molecular weight to the corresponding APP-CTF in cell lysate or medium. Differentiation caused a time-dependent increase in cell-associated APP and sAPP␣ levels, with a 3- and 7-fold increase by d 15 for cell-associated APP and sAPP␣, respectively (Fig. 5B). The NTFs also increased in a time-dependent manner, and showed the greatest increase with a 10-fold increase by d 15, compared to undifferentiated cells. GAPDH levels remained unaltered. This suggests that NTF formation is linked to the

maturation and differentiation state of the neuron and may be associated with nonamyloidogenic processing. PKC is involved in APP NTF generation To determine whether there is a relationship between NTF formation and the nonamyloidogenic pathway, differentiated SH SY-5Y cells were treated with PDBu, a phorbol ester that activates PKC. PKC mediates the action of many proteins, regulating several signal transduction pathways through protein phosphorylation (38). It is well established that PKC activation stimulates nonamyloidogenic processing and increases sAPP␣ levels (39 – 41), with a concomitant inhibition of the

Figure 6. Differentiated SH-SY5Y cells (differentiated for ⬃14 d) were incubated with DMSO (control) or PDBu (0.5 ␮M) for 6 h. A) Cell lysate was probed with CT20 to illustrate flAPP and CTF␣ or GAPDH to show equal loading. Neat media were analyzed for sAPP␣; TCA-precipitated media were analyzed for APP NTFs with 22C11. B) Densitometric analysis, expressed as mean ⫾ se values of 3 independent experiments. Values were normalized using GAPDH as an internal control, and the percentage difference was calculated relative to control. PBDu stimulated the release of sAPP␣ and APP NTFs. *P ⬍ 0.05; ANOVA. SECRETED APP N-TERMINAL FRAGMENTS

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Figure 7. Differentiated SH-SY5Y cells (differentiated for ⬃14 d) were incubated with DMSO (control) or PDBu (0.5 ␮M) for 6 h or with PDBu (0.5 ␮M) for 15 min prior to 6 h of G°6983 treatment. A) Neat media were analyzed for sAPP␣; TCA-precipitated media were analyzed for APP NTFs with 22C11. Cell lysate was probed with GAPDH to show equal loading. B) Densitometric analysis, expressed as mean ⫾ se values of 3 independent experiments. Values were normalized using GAPDH as an internal control, and the percentage difference was calculated relative to control. *P ⬍ 0.05; ANOVA.

amyloidogenic pathway (42– 44). In accordance with previous studies, 0.5 ␮M PDBu stimulated ␣-secretase cleavage of APP in SH-SY-5Y cells, resulting in 2.5-fold increase in extracellular sAPP␣ levels in neat conditioned cell medium and a 2-fold increase in intracellular CTF␣ levels (Fig. 6). Western blot analysis of TCA-precipitated medium from the differentiated PDBu-treated cells showed that PDBu caused an ⬃2fold significant increase in APP NTF levels as compared to untreated controls (Fig. 6B), comparable to the increases in sAPP␣ and CTF␣ levels. To verify that PKC modulates NTF generation, we used a broad PKC inhibitor, G°6983, to test whether PDBu-stimulated NTF generation could be prevented. Treatment of PDBu-stimulated differentiated SH-SY-5Y

cells with G°6983 caused sAPP␣ levels to decrease to almost control levels, supporting PKC inhibition (Fig. 7). G°6983 treatment also inhibited PDBu-stimulated generation of the NTFs to unstimulated levels, providing strong evidence for the direct involvement of PKC in APP NTF generation. APP NTF generation does not involve ␣-secretase or BACE PKC activates ␣-secretase, and since PKC is involved in NTF formation, we tested whether ␣-secretase mediates cleavage at the APP N terminus to generate the NTFs. Endogenous ␣-secretase activity can be inhibited with the metalloprotease inhibitor TAPI-1, thereby reducing

Figure 8. APP NTFs are generated in a pathway distinct from the ␣-secretase pathway. To determine whether ␣-secretase plays a role in NTF generation, SY5Y cells differentiated for 14 d were treated with 80 ␮M TAPI overnight. A) Neat media were analyzed for sAPP␣; TCAprecipitated media were analyzed for APP NTFs with 22C11. Cell lysate was probed with GAPDH to show equal loading. B) Densitometric analysis, expressed as mean ⫾ se values of 4 independent experiments. Values were normalized using GAPDH as an internal control, and the percentage difference was calculated relative to control. *P ⬍ 0.05; paired Student’s t test.

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APP shedding and decreasing sAPP␣ generation (44 – 46). Overnight treatment of differentiated SH-SY5Y cells with TAPI-1 significantly decreased sAPP␣ levels by ⬃25%; however, there was no significant change in APP NTF levels, suggesting that ␣-secretase does mediate the cleavage of APP to form sAPP␣ but not NTFs (Fig. 8). We investigated whether the APP NTFs identified in our study were derived from sAPP␤. Treatment of differentiated SH-SY5Y cells with the BACE inhibitor OM-99 decreased sAPP␤ levels by 40%, as anticipated; however, it had no effect on APP NTF levels (Fig. 9). This indicates that neither ␣-secretase nor BACE is responsible for NTF generation. No effect of RA withdrawal on APP NTF generation Recently, a candidate pathway for axonal degeneration following nerve growth factor (NGF) withdrawal was identified (22). In this model, the removal of NGF initiates a cascade of events whereby axonal APP is cleaved by BACE to yield sAPP␤, which undergoes further cleavage to yield a 55-kDa CTF and a 35-kDa NTF (termed N-APP; ref. 22). The shed N-APP fragment activates the tumor necrosis factor receptor DR6, mediating axon pruning and degeneration (22). We investigated whether the APP NTFs identified in our study were generated in response to RA deprivation. Differentiated SH-SY5Y cells were incubated in medium without RA for 72 h. sAPP␤ levels increased ⬃5-fold following RA withdrawal; however, no significant change in APP NTFs was observed (Fig. 10).

DISCUSSION In addition to the proteolytic cleavage of APP around the A␤ region, APP is also processed to generate other

metabolites. It is clear that the amino-terminal region of APP/sAPP is processed to release small NTFs ranging in size from 12 to 30 kDa on immunoblots from human peripheral blood (21), human CSF (17), rat brain (20), mouse and human brain (18, 47), human tear fluid (48), cultured rat cortical neurons (16), N2a cells (19), and differentiated SH-SY5Y cells (16). Our current study identified 17- to 28-kDa APP NTFs in human and mouse brains and showed they are developmentally regulated in mice, with high expression from the second postnatal week through to adulthood. The generation of the APP NTFs involves a novel APP processing pathway that is regulated by PKC, but is independent of ␣-secretase or BACE activity. The 17- to 28-kDa APP NTFs are not regulated by NGF withdrawal or derived from sAPP␤, suggesting that they are a different species from the larger, 35-kDa N-APP fragment described by Nikolaev et al. (22), who reported that a 35-kDa fragment is generated by cleavage of sAPP␤ in sensory axons of the peripheral nervous system. We did not detect a 35-kDa N-APP fragment in our studies, presumably due to differences between model systems. It is possible that the 35-kDa N-APP fragment is generated from sAPP␤ in the peripheral nervous system, while smaller NTFs are generated in the central nervous system. N-terminal cleavage of APP is modulated by PKC, since NTF generation was prevented by inhibitors of PKC and stimulated by activation of PKC. Further evidence for the role of PKC in NTF generation was provided by the neuronal differentiation experiments, whereby RA treatment increased NTF levels. RA is known to increase both PKC protein levels and activity (49, 50), with binding of RA to tyrosine receptor kinase B (TrkB) or TrkA, activating the PKC signaling cascade. The phosphorylation of PKC substrates that act as intermediate molecules may regulate APP cleavage.

Figure 9. APP NTF generation is not affected by BACE inhibition. SY5Y cells differentiated for 14 d were treated with 5 ␮M OM99-2 overnight. A) TCA-precipitated media were analyzed for either sAPP␣ or APP NTFs. As expected, sAPP␤ levels decreased following BACE inhibition; conversely, APP NTF levels were unchanged. Cell lysate was probed with WO2 and GAPDH as a control. B) Densitometric analysis, expressed as mean ⫾ se values of 4 independent experiments. Values were normalized using GAPDH as an internal control, and the percentage difference was calculated relative to control. ***P ⬍ 0.005; paired Student’s t test. SECRETED APP N-TERMINAL FRAGMENTS

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Figure 10. APP NTF generation is not affected by RA withdrawal. SH-SY5Y cells differentiated for 14 d were incubated in medium without RA for 72 h [RA withdrawal (RA WD)]. A) Neat media were analyzed for sAPP␣; TCA-precipitated media were analyzed for either sAPP␣ or APP NTFs. sAPP␤ levels increased following 72 h of RA WD; conversely, APP NTF levels were unchanged Cell lysate was probed with WO2 and GAPDH as a control. B) Densitometric analysis, expressed as mean ⫾ se values of 4 independent experiments. Values were normalized using GAPDH as an internal control, and the percentage difference was calculated relative to control. *P ⬍ 0.05, ***P ⬍ 0.005; ANOVA.

PKC-dependent processing of APP is mediated by phosphorylation of the cytoskeletal protein myosin II-B (51) or sorting protein-related receptor (sorLA; ref. 52), leading to enhanced delivery of APP to cellular compartments or the plasma membrane for cleavage to generate APP NTFs. A functional link between PKC and APP NTF generation may be to regulate APP-mediated modulation of synaptic currents. The application of the APP residues 16 –290 to Xenopus cultures containing developing neuromuscular synapses increased the amplitude of evoked synaptic currents (ESCs; ref. 53). This effect by APP residues 16 –290, a region that would contain the NTFs, on ESC amplitude was mediated by PKC, highlighting a potential functional role for the PKC-modulated NTFs we have identified. In the developing embryo, flAPP expression correlates with differentiation, maturation, and synaptogenesis (28, 54, 55). Postnatally, APP expression generally peaks and then, depending on tissue maturation and completion of synaptic connections, decreases between postnatal weeks 1–3 (30). Similarly, in our study, a continuous increase in flAPP, sAPP␣, and CTF␣ was observed from E12 to P8, followed by decreased levels from P14 to adulthood. Expression of the APP NTFs, on the other hand, was significantly different. APP NTFs were up-regulated from P14 to adulthood, relative to sAPP␣ and CTF␣, suggesting that there is a developmental increase in APP N-terminal cleavage in the mature mouse brain. This distinct expression pattern for APP N-terminal cleavage indicates that APP NTFs may play a functional role in the maturing and/or adult brain and supports their processing via a distinct APP pathway. Neurogenesis occurs mainly during prenatal development, as well in as some regions in the 8

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adult brain, including the lateral subventricular (SVZ) and subgranular zones (SGZ) of the dentate gyrus (56). sAPP␣ can regulate SVZ progenitor proliferation in the adult central nervous system (14, 57, 58) with cell proliferation being partly dependent on EGF-induced release of sAPP into the medium. As the domain within sAPP responsible for this action has not been identified, it is interesting to speculate that APP NTFs perform this function within sAPP, thus implicating the N-terminal cysteine-rich region as the active site. Similarly, the role of APP in synaptogenesis has been largely attributed to sAPP (55, 59). High concentrations of sAPP are present at synaptic sites and neuromuscular junctions of human and rat brain (60), and in the adult olfactory bulb, where continuous synaptogenesis occurs in the adult animal (30). It is possible that NTFs, rather than sAPP, play a role in synaptogenesis in adults. On the basis of the fragment sizes and immunoreactivity to 22C11, whose epitope is residues 66 – 81, the NTFs are expected to encompass the cysteine-rich N-terminal region, which encodes the GFD and copperbinding domain (CuBD) and would terminate just prior to the acidic domain. The CuBD can bind Cu(II), reducing it to Cu(I) in vitro and is presumed to regulate copper homeostasis and copper-mediated toxicity (61– 64). The N-terminal cysteine-rich region of APP is well conserved between its homologues and other species (65). The crystal structure of the GFD (residues 28 – 123) revealed that it has similarities with other cysteinerich growth factors (66). The GFD is capable of binding to heparin (67, 68), fibulin (an extracellular matrix protein; ref. 69), and reelin (70), as well as to itself or other APP-family members (71–74). We have recently

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shown that the GFD portion of the NTF is neuroprotective in a traumatic brain injury model (75). It remains to be determined how the GFD and CuBD are relevant to the function of the NTFs identified in this study and how the physiological functions attributed to sAPP␣ relate to NTFs. The authors thank Giuseppe Ciccotosto and Andrea Connor (Department of Pathology, University of Melbourne) for assistance in providing the mouse and human brain tissue respectively. We thank the National Neural Tissue Resource Centre for the human brain tissues. L.J.V. is an Alzheimer’s Australia Research Viertel Fellowship recipient. R.C. is an Australia National Health and Medical Research Council (NHMRC) Senior Research Fellow. Funding was provided by the NHMRC and the Australian Research Council to R.C.

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