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. Author manuscript; available in PMC: 2012 Oct 1.
Published in final edited form as: J Neurochem. 2011 Sep 20;119(2):377–388. doi: 10.1111/j.1471-4159.2011.07419.x

FE65 Proteins Regulate NMDA Receptor Activation-induced Amyloid Precursor Protein Processing

Jaehong Suh 1, Alvin Lyckman 2, Lirong Wang 1, Elizabeth A Eckman 3,†, Suzanne Y Guénette 1
PMCID: PMC3188680  NIHMSID: NIHMS317303  PMID: 21824144

Abstract

Amyloid precursor protein (APP) family members and their proteolytic products are implicated in normal nervous system function and Alzheimer’s disease pathogenesis. APP processing and Aβ secretion are regulated by neuronal activity. Various data suggest that NMDA receptor (NMDAR) activity plays a role in both non-amyloidogenic and amyloidogenic APP processing depending on whether synaptic or extrasynaptic NMDARs are activated, respectively. The APP-interacting FE65 proteins modulate APP trafficking and processing in cell lines, but little is known about their contribution to APP trafficking and processing in neurons, either in vivo or in vitro. In this study, we examined the contribution of the FE65 protein family to APP trafficking and processing in WT and FE65/FE65L1 double knockout neurons under basal conditions and following NMDAR activation. We report that FE65 proteins facilitate neuronal Aβ secretion without affecting APP fast axonal transport to presynaptic terminals. In addition, FE65 proteins facilitate an NMDAR-dependent non-amyloidogenic APP processing pathway. Generation of high-molecular weight (HMW) species bearing an APP C-terminal epitope was also observed following NMDAR activation. These HMW species require proteasomal and calpain activities for their accumulation. Recovery of APP polypeptide fragments from electroeluted HMW species having molecular weights consistent with calpain I cleavage of APP suggests that HMW species are complexes formed from APP metabolic products. Our results indicate that the FE65 proteins contribute to physiological APP processing and accumulation of APP metabolic products resulting from NMDAR activation.

Keywords: APP, APLP2, FE65, FE65L1, NMDA, cortical neurons

Introduction

The amyloid precursor protein family consists of single transmembrane spanning proteins (APP, APLP1 and APLP2) that undergo proteolytic processing in the secretory and endocytic pathways. APP cleavage by α- or β-secretase at or near the cell surface generates the large secreted ectodomain fragments, APPsα and APPsβ, respectively, and the intracellular membrane-associated APP C-terminal fragments (APP-CTFs), C83 (α-CTF), C89 and C99 (β-CTFs). APP-CTFs are cleaved by γ-secretase giving rise to the APP intracellular domain (AICD) and Aβ peptides, the major protein component of senile plaques found in Alzheimer’s disease (AD) brain (Wolfe & Guenette 2007).

The roles of APP and its proteolytic products in synapse formation and function have been the focus of several recent studies. APP expression is detected maximally during synaptogenesis (Moya et al. 1994). Marked alterations in synaptic structure and activity are observed in neurons derived from APP family (APP, APLP1 and APLP2) knockout mice and in neurons differentiated from embryonic stem cells lacking APP and/or APLP2 genes (Wang et al. 2009, Schrenk-Siemens et al. 2008). Both, APP and APLP2 undergo fast axonal transport to presynaptic terminals (Lyckman et al. 1998). APP also localizes to post-synaptic membranes (Hoe et al. 2009). Furthermore, APP deficits at either pre- or post-synaptic membranes result in defective synapse formation in neurons lacking APLP2 (Wang et al. 2009).

NMDAR activation alters APP processing but conflicting results have been reported (Hoey et al. 2009, Marcello et al. 2007, Lesne et al. 2005, Hoe et al. 2009). These disagreements may result from activation protocols that target specific pools of NMDARs, e.g. synaptic versus extrasynaptic NMDARs. Some studies report that NMDAR activation increases APPsα secretion and reduces Aβ secretion (Hoey et al. 2009, Marcello et al. 2007), effects that have been attributed to synaptic NMDAR activation (Hoey et al. 2009). In contrast, activation of extrasynaptic NMDARs was recently reported to increase Aβ production (Bordji et al. 2010).

The FE65 family of APP-binding proteins (FE65, FE65L1 and FE65L2) modulate APP processing (King & Scott Turner 2004). FE65 or FE65L1 protein overexpression in cell lines increase Aβ production and these changes were attributed to increased trafficking of APP into the endocytic pathway (Guenette et al. 1999, Chang et al. 2003, Sabo et al. 1999). Consistent with these findings, cerebral Aβ levels were reduced in FE65/FE65L1 DKO mice (Guenette et al. 2006). Differences in neuronal APP processing between wild type (WT) and FE65/FE65L1 double knockout (DKO) mice may be responsible for the reduction in cerebral Aβ levels observed in FE65/FE65L1 DKO mice since neuron cultures established from Tg2576 mice lacking the 97KDa FE65 isoform secrete less Aβ40 and Aβ42 than control Tg2576 neurons (Wang et al. 2004).

To further assess whether and how FE65 proteins influence APP trafficking and processing in neurons, we examined the effects of FE65 protein knockout on APP transport in vivo, and on APP processing in primary cortical neuron cultures under various conditions of NMDAR activation. We find that FE65 protein deficiencies do not alter axonal transport of APP to presynaptic terminals in the CNS. However, in primary neuron cultures FE65 protein deficiencies do attenuate Aβ secretion in the absence of exogenous NMDAR activation. In addition, FE65 protein deficiencies attenuate APP processing and the accumulation of APP metabolic products following NMDAR activation.

Materials and Methods

Primary neuronal cultures

Cerebral cortices were removed from brains of E15 WT or FE65/FE65L1 DKO embryos and gently triturated with a series of fire-polished pasteur pipettes of different pore sizes. Suspended dissociated cells were pooled and plated on six-well plates (~ 6 × 105 cells/well) pre-coated with poly-D-lysine (0.1 mg/ml, Sigma) and laminin (4 μg/ml, Invitrogen). Cells were plated and maintained in neuronal-selection medium consisting of Neurobasal medium, B27 supplement, 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen). Half the neuronal culture medium (1 ml) was replaced every 3–4 days and half the culture medium was replaced prior to pharmacological treatments. WT and DKO embryos obtained by mating parents of the same genotype were pooled by genotype for neuronal culture. Embryos obtained from FE65/FE65L1 DKO × FE65 +/−; FE65L1−/− crosses were individually plated and subsequently genotyped. WT neurons were similarly prepared from ICR mice (Charles River Laboratory). APP knockout mice were obtained from the Jackson Laboratory (Zheng et al. 1995). NMDA, AMPA, MK-801, CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), calpeptin, lactacystin and MG132 were purchased from Calbiochem. In our cultures, approximately 10% neuronal cell death was observed for neurons treated with 10 μM NMDA (24 hr) as assessed by lactate dehydrogenase release (data not shown).

Fast Axonal transport of APP

Metabolic labeling of neuronal proteins was performed by unilateral intraocular injection of 35S-Methionine/Cysteine (100 μCi/mouse) in nine (n=4 WT and n=5 FE65/FE65L1 DKO; 4.5–6.5 months of age) mice under avertin (0.5 mg/g i.p.) anesthesia. Animals were euthanized 4.5 hrs post-injection and brains were dissected. APP was immunoprecipitated from the superior colliculus using the A8717 APP C-terminal antibody (Sigma). Immunoprecipitates were separated on 4–12% Nupage gels with Bis-Tris running buffer; transferred to PVDF filters; and quantitated using storage phosphor imaging. All animal experiments were performed in accordance with the MGH Institutional Animal Care and Use Committee.

Semi-quantitative reverse transcription-PCR

Total RNA from primary cultured mouse neurons and brain tissue was isolated using RNAzol (Gibco). RNA integrity was confirmed by detection of the 28S and 18S ribosomal RNA bands. RNA was also confirmed to have no detectable genomic DNA contamination by PCR in the absence of reverse transcriptase. RNA (0.5 μg) was reverse transcribed into cDNA (TaqMan Reverse Transcription kit, Applied Biosystems) and each APP isoform was amplified using experimentally optimized cycles that allowed comparison between genotypes within the linear amplification range (PCR master mix, Qiagen). Oligonucleotide primers for RT-PCR of APP770, APP751, APP695, β-actin and APP exon 15 were previously reported (Lesne et al. 2005, Konig et al. 1992). PCR products were separated on 2% agarose gels and visualized with ethidium bromide staining. Relative intensities of PCR bands were analyzed using the Gel Doc 1000 video-imaging system (Bio-Rad).

Western blot analysis and antibodies

Primary neurons and embryonic brains were lysed in RIPA buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate (SDS) and protease inhibitor cocktail containing 1 mM EDTA (Roche) on ice for 15 mins. Protein samples for SDS-PAGE were prepared in NuPAGE LDS sample buffer containing β-mercaptoethanol (2.5%) and separated on 4–12% NuPAGE Novex Bis-Tris or 3–8% NuPAGE Novex Tris-Acetate gels (Invitrogen). For AICD detection, neuronal lysates were harvested directly with SDS-sample buffer containing protease inhibitors (Kimberly et al. 2005) and separated on 10–20% or 16% tricine gels (Invitrogen). Proteins were transferred to PVDF (Millipore) membranes for Western blot analyses. Both A8717 (Sigma) and C66 (Huttunen et al. 2007) were raised against the last 20 amino acid of the APP C-terminus. The epitope for CT695 (Zymed) is the last 22 amino acids of APP. 22C11 (Chemicon) recognizes amino acids 66–81 of the N-terminus of APP and APLP2. R7 (Refolo et al. 1989) and ab12269 (Abcam) are specific for KPI-APP. APP phosphorylated at the Thr668 residue (pThr668APP) was detected with a phosphoAPP (Thr668) antibody (Cell Signaling). APLP1 and APLP2 were detected with antibodies raised to the C-termini of APLP1 and APLP2 that do not cross-react with APP (Myre et al. 2009). Immunoreactive bands on Western blots were detected with enhanced chemiluminescence reagents (Amersham) on X-ray film. Band intensities were quantified by densitometric analysis using Image Gauge version 3.12 (Fuji Photo Film).

Aβ40 and Aβ42 measurements

Aβ sandwich ELISAs were performed for murine Aβ40 and Aβ42 as described previously (Suzuki et al. 1994). Endogenous murine Aβ42 in undiluted conditioned media from primary neuronal cultures was captured on ELISA plates coated with antibody MM26-2.1.3.35.86, a monoclonal antibody generated against Aβ1–42 that does not recognize Aβ ending at position 40. The bound Aβ42 was detected with horseradish peroxidase (HRP)-conjugated Aβ17–24 antibody 4G8 (Covance). Murine Aβ40 was similarly captured on ELISA plates coated with MM32.4.1, a monoclonal antibody directed against murine Aβ1–16, and was detected with HRP-conjugated Aβ40-specific antibody MM13.1.1. All monoclonal antibodies for the ELISA were generated at the Mayo Clinic. Aβ values were calculated by comparing the absorbances obtained from the samples with those obtained for synthetic Aβ1–40 and Aβ1–42 standards (Bachem).

Deglycosylation experiments

Cell or tissue lysates were desalted using Micro Bio-spin columns (Bio-Rad) before enzymatic reactions. Deglycosylation reactions were performed using the glycoprotein deglycosylation kit as indicated by the manufacturer (Calbiochem). The kit contains N-glycosidase F, endo-α-N-acetyl-galactosaminidase (O-glycosidase), and the exoglycosidases: 2–3,6,8,9-neuraminidase, β1,4-galactosaminidase, β-N-acetylglucosaminidase. Chondroitin sulfate conjugation to APP was analyzed by subjecting neuronal lysates to the digestion with chondroitinase ABC (Sigma) (Thinakaran et al. 1995).

Electroelution of APP and HMW species

The SDS-PAGE gel areas corresponding to full length APP (APP695) and HMW species were excised based on size markers and electroeluted using the Centrilutor micro-electroeluter (Millipore). The gel eluent obtained by applying 120 V for 3 hr was concentrated with YM-30 centricon filters (Amicon). To confirm the range of protein sizes of the electroeluted samples, silver staining was performed using the SilverSNAP stain kit (Pierce) and recovery of APP and HMW species was determined by Western blot analysis using the A8717 antibody (Sigma).

Statistical analyses

Data are expressed as the mean ± standard error of the mean (SE). Statistical comparisons using a T-test between two groups and one-way analysis of variance (ANOVA) followed by post hoc Student Neuman and Keuls (SNK) test were performed. p<0.05 was considered significant.

Results

Secreted Aβ levels are reduced in FE65/FE65L1 DKO cortical neuron cultures

To determine whether FE65 and FE65L1 protein deficiencies affect APP processing in neurons, APP695 (APP695), APP-CTFs, and AICD were examined in FE65/FE65L1 DKO and wild type (WT) primary cortical neurons. Both day in vitro 13 (DIV13) and DIV7 neurons were examined and comparable levels were found between genotypes at both time points. Levels of APP695, APP-CTFs and AICD do not differ between DIV7 WT and FE65/FE65L1 DKO neurons (Fig. 1A and Supplementary Fig. 1). In contrast, secreted murine Aβ40 (48%; 106.5±4.5 vs. 51.0 ±3.3 fmol/ml) and Aβ42 (55%; 159.9±3.4 vs. 88.3±4.9 fmol/ml) levels were significantly lower in FE65/FE65L1 DKO DIV7 neurons than in WT neurons (Fig. 1C). These data show that FE65 proteins facilitate Aβ secretion.

Figure 1. Depletion of FE65 and FE65L1 decreases secreted Aβ levels in primary cortical neurons.

Figure 1

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(a) Endogenous levels of full-length APP (APP695), APP-CTFs, and AICD in cultured cortical neurons (DIV 7) from FE65/FE65L1 DKO (DKO) and WT mouse embryos were examined by Western blot analysis using the A8717 antibody (Sigma). (b) Aβ40 and Aβ42 levels in conditioned media from WT and DKO neuronal culture (DIV7). Conditioned media was collected from neuronal cultures prepared from separate WT (n=6) and separate DKO (n=5) embryos for Aβ Sandwich ELISA. Data were normalized to the means of WT Aβ measurements which were given an arbitrary value of 100. Values are mean ± SEM. *, p < 0.05 versus WT control (t-test).

FE65/FE65L1 protein deficiencies do not impact APP fast-axonal transport

FE65/APP protein interactions alter APP subcellular sorting and APP processing in FE65 or FE65L1 overexpressing cell lines (Guenette et al. 1999, Chang et al. 2003, Sabo et al. 1999). In CNS neurons, APP is synthesized in the cell body and transported to presynaptic terminals by fast axonal transport where it co-localizes with FE65 (Sabo et al. 2003) and undergoes rapid turnover (Lyckman et al. 1998). To determine whether the FE65 proteins, FE65 and FE65L1, are required to sort nascent APP to the secretory/fast axonal transport pathway, we measured nascent, presynaptic APP in vivo in the retinal ganglion cell (RGC) terminals in the superior colliculus (SC) of WT and FE65/FE65L1 DKO mice. Nascent APP was metabolically labeled with intraocular 35S-methionine and allowed to transport to the presynaptic terminals of RGC axons in the contralateral SC. Radiolabeled APP was immunoprecipitated using A8717, separated on polyacrylamide gels, transferred to PVDF membranes, and measured by storage phosphor imaging. No difference was observed in the amounts of APP transported to the superior colliculus in WT versus FE65/FE65L1 DKO mice (Fig. 2). These data suggest that effects of FE65 protein deficiencies on Aβ secretion are unlikely to be due to changes in APP transport to presynaptic terminals.

Figure 2. Depletion of FE65 and FE65L1 has no effect on APP fast axonal transport.

Figure 2

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Storage phosphor signals for immunoprecipitated APP (A8717) from the superior colliculus of WT and FE65/FE65L1 DKO mice (WT n=4; DKO n=5) dissected 4.5 h post-radiolabeling. Raw values and mean +/− SEM are shown. T-test, p >0.05.

FE65 protein deficiency attenuates NMDAR activation-dependent changes in APP processing

NMDAR activation alters APP processing (Hoey et al. 2009, Lesne et al. 2005). In order to assess the contribution of FE65 proteins to NMDAR-dependent APP processing, WT and FE65/FE65L1 DKO cortical (DIV13-14) were treated with NMDA (5–15 μM) and immunoblotting of neuronal lysates was performed to determine the levels of APP and its proteolytic fragments. The increased neuronal firing with oscillatory calcium transients that results from 24h exposure to 10μM NMDA is inferred as an activation of the synaptic pool of NMDARs (Soriano et al. 2006). Using this paradigm, total APP levels did not differ significantly between NMDA-treated vs. control cultures in WT or FE65/FE65L1 DKO neurons (Fig. 3A and B). However, this paradigm significantly elevated levels of APP-CTFs, in particular C83, as compared to control treatment (Fig. 3A and C). NMDAR activation also resulted in dose-dependent accumulation of novel high molecular weight (HMW) species that migrate slower than APP695 in SDS-PAGE (Fig. 3A and D).

Figure 3. Attenuation of NMDA-mediated APP-CTF accumulation, APP Thr668 dephosphorylation and HMW species generation in FE65/FE65L1 DKO neurons.

Figure 3

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(a) Cultured cortical neurons at DIV13 from WT and FE65/FE65L1 DKO embryos were exposed to the indicated doses of NMDA for 24 hr followed by Western blot analysis using the APP C-terminal antibody A8717. A longer exposure of the membrane revealed APP-CTFs (lower panel). p-APP, pThr668-APP was detected on the same blot with a pThr668APP specific antibody and β-actin was used as a loading control. (b–d) Quantitation of APP695 (b), APP-CTFs (c), and HMW species (d) normalized toβ-actin from three independent neuronal culture preparations for each mouse genotype. An arbitrary value of 100 was given to densitometric estimates for each of these signals in WT neurons treated with 0 μM NMDA. * p < 0.05 versus WT (NMDA 0 uM); # p < 0.05 (ANOVA and Student Neuman Keuls test).

APP undergoes phosphorylation at Thr 668 and Aβ generation is reduced when Thr668 phosphorylation is abolished in cortical neurons (Lee et al. 2003). In addition to the effects of NMDA on APP processing, we found that pThr668APP levels were reduced with increasing NMDA concentrations (Fig. 3A). These data suggest that NMDA receptor activation either leads to pThr668APP dephosphorylation or reduced phosphorylation of APP at this residue.

The effects of NMDAR activation on C83, HMW species and pThr668APP levels observed in WT cultures upon NMDAR activation were significantly attenuated in FE65/FE65L1 DKO neurons (Fig. 3A, C, and D). Thus, APP/FE65 interactions appear to facilitate NMDAR-dependent APP processing.

Higher levels of NMDA (≥ 20 μM) are reported to produce linear Ca++ increases that peak within 1 h and that result in maximal neuronal cell death by 24 hrs (Sattler et al. 1998). Therefore, we examined the contribution of FE65 proteins to NMDAR-dependent APP processing in neurons using bath applications of 100μM NMDA. Both WT and DKO neurons showed prominent cell body swelling observed by light microscopy within 1 hr suggesting rapid activation of pathways leading to excitotoxic cell death (Choi 1987). APP-CTF levels were dramatically increased within 0.5 h of NMDA treatment and remained elevated at 1 hr (Fig. 4A). In contrast, no change in AICD levels was observed (Supplementary Fig. 2). The increase in APP-CTF levels was transient since longer incubations with NMDA (100 μM, 2.5 hrs) resulted in APP-CTF levels that are comparable to those of control neurons (Fig. 4A). Shorter incubations (10 mins.) with NMDA (100 μM) did not produce a notable increase in APP-CTFs in our neuronal cultures (Supplementary Fig. 3). HMW species were only faintly detectable in neurons exposed to NMDA (100 μM) for 1h (Fig. 4A), revealing temporal uncoupling between the accumulation of HMW species and APP-CTF generation. In contrast, reduced levels of APP695 were found to coincide with accumulation of HMW species in neurons treated with high concentrations of NMDA (100 μM). FE65-dependent differences in APP-CTF and HMW species accumulation were no longer apparent at high concentrations of NMDA (100 μM, 3 hrs), but are comparable in neurons treated with a wide range of NMDA concentrations (10, 30 and 50 μM; 3 hrs) (Fig. 4A and B). Collectively, these data suggest that the FE65 proteins facilitate NMDA-dependent changes in APP processing, but that cellular events resulting from excessive NMDAR activation can mask or override this effect.

Figure 4. Cytotoxic NMDA levels abrogate the differential effect of FE65 protein deficiencies on NMDAR-activation dependent APP processing.

Figure 4

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(a) WT and FE65/FE65L1 DKO cortical neurons (DIV 13) were exposed to NMDA (100 μM) for the indicated times. MK801, an NMDA antagonist, was co-incubated with NMDA for the 2.5 hr treatment group. (b) WT or DKO neurons (DIV13) were exposed to the indicated doses of NMDA for 3 hr. (c) WT neurons (DIV 13) were exposed to NMDA (100 μM) or AMPA (10 μM) alone, or in combination with MK801 (10 μM) or CNQX (10 μM) for 3 hr. APP695, APP-CTFs and HMW species were detected by Western blot analysis using the APP C-terminal antibody (A8717). p-APP, pThr668-APP was detected on the same blot with a pThr668APP specific antibody and β-actin was used as a loading control.

A reduction in FE65 protein levels in NMDA (100 μM) treated WT neurons may account for the lack of difference in APP-CTF accumulation between WT and FE65/FE65L1 DKO neurons. Reduced FE65 protein levels were observed with continued NMDA treatment (Supplementary Fig. 4), but does not account for the lack of difference in APP-CTF accumulation at high NMDA (100 μM) doses because both FE65 protein and APP-CTF levels were comparable between control and NMDA treated (100 μM, 0.5 hr) WT neurons (Supplementary Fig. 4).

During normal glutamatergic neurotransmission, NMDAR activation typically follows activation of postsynaptic AMPA receptors. Therefore, we further examined the relative contributions of NMDA- versus AMPA-glutamate receptor activation to APP processing in WT versus DKO cortical neurons. First, neurons were treated with NMDA alone, or in combination with either the NMDAR antagonist MK801 or the AMPA antagonist CNQX. All NMDA-induced APP changes in WT and DKO neurons were inhibited by MK801 treatment and not affected by CNQX, suggesting that NMDAR activation is responsible for these changes (Fig. 4A and 4C). Second, AMPA treatment of neuronal cultures also resulted in HMW species accumulation (Fig. 4C) and this was blocked by incubation with CNQX (Fig. 4C). Third, MK801 treatment blocked the AMPA-induced accumulation of HMW species, suggesting this accumulation depends on secondary activation of NMDARs (Fig. 4C). Taken together, these data suggest that postsynaptic processing of APP is critically dependent on NMDAR activity.

NMDAR activation lowers secreted Aβ

NMDA treatment resulted in a significant reduction of secreted Aβ40 and Aβ42 levels (Fig. 5). A dramatic decrease in Aβ40 (controls; 25.9±4.4 fmol/ml vs NMDA-treated; n.d.) and Aβ42 (controls; 20.9±3.3 vs NMDA-treated; 3.7±0.5 fmol/ml) levels was observed for neurons treated with 10 μM NMDA for 24 h (Fig. 5A). Both Aβ40 and Aβ42 measurements were at the limit of detection of our assay for neurons treated with 10 μM NMDA. A less dramatic decrease in Aβ40 (controls; 30.2±7.1 vs NMDA-treated; 18.2±5.5) and Aβ42 (controls; 25.4±5.6 vs NMDA-treated; 17.4±4.3 fmol/ml) was observed for neurons treated with 100 μM NMDA for 3 h (Fig. 5B).

Figure 5. NMDAR activation reduces Aβ secretion.

Figure 5

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Aβ40 and Aβ42 levels in conditioned media from WT neuronal cultures (DIV13), untreated or treated with NMDA (n=6 samples for each condition). (a) 10 μM NMDA for 24 h. (b) 100 μM NMDA for 3 h. Data were normalized to the mean of the untreated Aβ measurements, which were given an arbitrary value of 100. Values are mean ± SEM. T-tests: *, p < 0.05; **, p<0.01 versus untreated control. N.d., not detected in undiluted media.

HMW species accumulation resulting from NMDAR activation is not due to alternative splicing of APP transcripts

The predominant APP species in neurons is APP695, but minor APP isoforms with reduced gel mobility are generated by alternative splicing (Sandbrink et al. 1993, Pangalos et al. 1995). We examined whether alternative splicing of APP contributes to HMW species accumulation upon NMDAR activation. Semi-quantitative RT-PCR analyses of APP transcript levels in control and NMDA-treated (100 μM, 2.5 h) WT and FE65/FE65L1 DKO neurons were performed using primers specific for Kunitz protease inhibitor (KPI) domain containing mRNAs, APP751 and APP770, and APP mRNAs lacking exon 15. Exclusion of APP exon 15 was examined because it generates a chondroitin sulfate conjugation site that produces APP isoforms of 140 to 250 kDa in SDS-PAGE that are expressed in neurons (Pangalos et al. 1995, Lyckman et al. 1998). Although APP751, APP770 and to a lesser extent mRNAs lacking exon 15 were detected in neuronal cultures and in brain, their expression levels were far lower than APP695 (Fig. 6A). No differences in the levels of these alternatively spliced APP mRNAs were found between WT and FE65/FE65L1 DKO neurons (Fig. 6A). Accordingly, we could not detect the induction of KPI-APP proteins by NMDAR activation in Western blot analyses using KPI-APP specific (Fig. 6B), or chondroitin sulfate specific antibodies (Fig. 8B). HMW species detected with the A8717 antibody (Fig. 3) also cross-react with two additional APP antibodies raised against the APP C-terminal 20–22 amino acid residues (C66 and CT695) (Supplementary Fig. 5). Collectively, these results indicate that HMW species while containing APP epitopes are not the product of KPI- or chondroitin sulfate modified APP isoforms.

Figure 6. HMW species contain neither a KPI domain nor a chondroitin sulfate conjugation site produced by exon 15 splicing.

Figure 6

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(a) Cortical neurons (DIV13) were exposed to sham control or 100 μM NMDA for 2.5 hr and the expression patterns of APP mRNA isoforms were examined by semi-quantitative RT-PCR. Endogenous levels of each APP mRNA isoform were also analyzed from 1-month-old brains of WT and DKO mice. Arrowhead indicates APP mRNA missing exon 15. PCR cycle number is shown in parentheses. (b) Cortical neurons (DIV13) were exposed to sham control or 100 μM NMDA for 2.5 hr and Western blot analysis for HMW species using KPI-APP specific (ab12268, upper panel) and APP-CTF (A8717, lower panel) antibodies. A lysate of H4 neuroglioma cells overexpressing APP751 was used as a positive control. Longer film exposures did not reveal KPI-APP in neuronal lysates. A similar result was obtained with the R7 KPI-APP specific antibody (data not shown).

Figure 8. N-glycan chains are predominantly responsible for the reduced gel mobility of HMW species.

Figure 8

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(a) Immunoblot (A8717) of NMDA-treated or untreated neuronal extracts after overnight deglycosylation. The glycosidases used were N-glycosidase F (N-glyc), a mixture of o-glycosidase and exoglycosidases to remove O-linked glycans (O-glyc) and neuraminidase, which removes sialic acid residues. Thick and narrow arrows indicate HMW species before and after deglycosylation, respectively. (b) Immunoblot (A8717) after chondroitinase ABC treatment of NMDA-treated or untreated neuronal lysates. (c) Immunoblot (A8717) after overnight deglycosylation similar to (a), but a 3–8% Tris-acetate gel was used for improved resolution of the deglycosylated proteins. (d) Immunoblot (A8717) of an N-glycosidase F time-course preformed on NMDA-treated neuronal lysates (3–8% Tris-acetate gel). Arrowheads, mature and immature forms of APP695; thin arrows, partially deglycosylated HMW species; large arrows, HMW species. -, no enzyme; N-glyc, N-glycosidase F; O-glyc, a mixture of O-glycosidase and exoglycosidases (see methods).

Calpain and proteasomal activities are necessary for HMW species accumulation

APP can be degraded by several proteolytic systems, including calpain-1 (Siman et al. 1990), the proteasome (Nunan et al. 2001), and various caspases (Gervais et al. 1999). Furthermore, these proteolytic systems can be triggered by NMDAR activation (Wang 2000, Yi & Ehlers 2005). We examined whether these activities contributed to HMW species accumulation and APP processing following NMDAR activation. HMW species were not present after pharmacological inhibition of calpain activity with calpeptin (Fig. 7A and B). Proteasome inhibitors, MG132 and lactacystin, also inhibited HMW species accumulation, with MG132 being more effective than lactacystin (Fig. 7A and B). The latter observation may be due to partial inhibition of calpain by MG132 (Lee & Goldberg 1998). In contrast, ZVAD, a broad-spectrum caspase inhibitor, had no effect on HMW species accumulation (Fig. 7A and B). Inhibition of α-, β- or γ-secretases with TAPI-1, C3, or DAPT, respectively, failed to attenuate HMW species induction (Supplementary Fig. 6). These data suggest that HMW species generated by NMDAR activation arise from proteolytic processing of APP, but not from APP-secretase activities.

Figure 7. Calpain and proteasome inhibition block NMDAR activation-mediated HMW species induction.

Figure 7

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(a) Cortical neurons (DIV13) were treated with sham control or NMDA (50 μM), alone or concurrently with zVAD (100 μM), calpeptin (10 μM), MG132 (10 μM) or lactacystin (10μM) for 3 hr. Western blot analysis was performed with the A8717 antibody. (b) Densitometric analysis of HMW species, APP695, and APP-CTFs in A. An arbitrary value of 100 was given to the mean for sham controls. -, no treatment; lactacys, lactacystin. * p < 0.05 versus NMDA alone, n=3 (ANOVA and Student-Neuman-Keuls test).

APP-CTF levels were dramatically increased in neurons treated with calpeptin or MG132 in addition to NMDA (Fig 7A and B). However, neurons treated with MG132 or calpeptin under basal conditions also accumulated APP-CTFs (Fig. 7A). A previous study showed that inhibition of calpain activity in cultured cell lines increases APP-CTF generation as a result of a partial redistribution of APP to the cell surface (Mathews et al. 2002). Similar increases in APP-CTFs were observed in cultured cell lines treated with proteasome inhibitors (Nunan et al. 2001, Myre et al. 2009). The differential effect of calpain inhibition on HMW species and APP-CTF accumulation provides additional support for our conclusion that HMW species accumulation does not depend on the generation of secretase-derived APP-CTFs.

N-glycan chains make a significant contribution to the reduced gel mobility of HMW species

The generation of HMW species from APP proteolysis is paradoxical. However, the apparent gel mobility of APP is also a function of glycosylation. Mature APP is N- and O-glycosylated and immature APP is N-glycosylated (Weidemann et al. 1989). The gel mobility of NMDA-induced HMW species in SDS-PAGE is dependent on the choice of extraction and sample loading buffers suggesting that HMW species tertiary structures are more resistant to denaturation than APP695 (Supplementary Fig. 7A). To examine whether differential glycosylation may be contributing to the mobility of HMW species in SDS-PAGE, we incubated neuronal cell lysates with a mixture of glycosidases. Mature and immature APP695 showed the expected increase in mobility when digested with a mixture of N-glycosidase F, three exoglycosidases and an O-glycosidase (see methods) (Fig. 8A). The gel mobility of HMW species was robustly increased by digestion with the same enzyme cocktail. However, digestion with N-glycosidase F alone achieved a similar shift in gel mobility indicating that N-glycan chains make a major contribution to the gel mobility of HMW species (Fig. 8A). O-glycosylation and sialylation, which were found to contribute to the molecular weight shift of APP in PS1-depleted N2A cells (Leem et al. 2002), did not produce a notable HMW species gel mobility shift (Fig. 8A and C). A time course of N-glycan chain removal revealed a stepwise shift in the mobility of HMW species suggesting the presence of two N-glycan chains in HMW species (Fig. 8D). However, extensive enzymatic digestion to remove glycosyl residues failed to fully abolish the difference between deglycosylated HMW-APP and APP695 (Fig. 8A and D). This may be due to the presence of less common glycan chains that are refractory to digestion with the enzymes used, other post-translational modifications that contribute to the retarded mobility of deglycosylated HMW species or the existence of several A8717-positive polypeptides in HMW species. In support of the latter possibility, two distinct bands are visible after a short digest with N-glycosidase F (Fig. 8D, 0.2 h).

APP and APLP2 fragments are components of NMDA-induced HMW species

We compared the properties of HMW species to APP695 by enriching HMW species (140–170 kDa) and APP695 (80–100 kDa in our gel system) from acrylamide gels by electroelution because HMW species were not recovered in A8717 immunoprecipitates (Supplementary Fig. 7B). Silver staining of the eluted polypeptides showed recovery of proteins that co-migrate with HMW species or APP695 and fainter bands in the 40–60 kDa range in both isolates (Fig. 9A). The 40–60 KDa bands were differentially recognized by A8717, C66, an APP C-terminal antibody, and 22C11, an APP N-terminal antibody (Fig. 9A). The enrichment process itself does not lead to proteolytic degradation since intact APP695 polypeptides were recovered following electroelution. Detection of APP fragments from electroeluted HMW species suggests that HMW species may be aggregates of APP fragments. This is consistent with our data showing that calpain and proteosome activities are necessary for the generation of HMW species following NMDAR activation. Surprisingly, a 60 kDa APLP2 immunoreactive fragment was observed using a C-terminal APLP2 antibody (Fig. 9B). In contrast, an APLP1 C-terminal antibody failed to recognize fragments resulting from electroelution of HMW species (data not shown). HMW species are eluted from anion exchange resin at the same high salt concentration as APP695 and like APP can subsequently be enriched from copper-loaded chelating sepharose (data not shown) (Hesse et al. 1994). These data indicate that HMW species share biochemical properties that are characteristic of APP and APLP2.

Figure 9. HMW species contain APP and APLP2 fragments.

Figure 9

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(a) Lysates obtained from NMDA-treated (100 μM, 3 hr) neurons (DIV13) were separated on SDS-PAGE and gel regions corresponding to APP695 (FL) and HMW species (H) were excised. After electroelution (Millipore) of the gel slices, eluents from the gel regions containing APP695 and HMW species were subjected to silver staining or Western blot analysis with (a) APP antibodies (A8717, C66, 22C11) or (b) an APLP2 C-terminal antibody. Results are representative of more than three independent gel elution experiments. (c) Primary cortical neurons (DIV 8) from APP Het (+/−) or KO (−/−) embryos were exposed to sham control or 100 μM NMDA for 3 hr. * indicates a cross-reacting band that is detected in the cytosolic fraction of neuronal lysates.

To further examine the possibility that HMW species are not exclusively derived from APP, cortical neuron cultures were established from APP KO embryos and treated with NMDA. Accumulation of a HMW A8717-positive band with an apparent molecular weight identical to HMW species was observed in NMDA-treated APP KO neurons (Fig. 9C). The 20 amino acid peptide used to generate the A8717 antibody contains a sequence of eight consecutive amino acid residues that are identifcal between APP and APLP2 that may serve as a common epitope. Given that we found an APLP2 fragment in the electroeluted HMW species, these data suggest that APLP2 fragments are components of HMW species.

Discussion

In the present study, we examined the impact of FE65/FE65L1 deficiency on APP trafficking and processing in neurons. We report that FE65 proteins contribute to APP processing in primary cortical neuron cultures, but do not impact APP fast axonal transport. The FE65 proteins facilitate NMDAR-dependent α-secretase processing of APP and accumulation of secreted Aβ in the absence of exogenous neuronal activation. In addition, prolonged NMDAR activation resulted in accumulation of HMW species that consist of APP and/or APLP2 proteolytic products requiring calpain and proteasome activities for their generation. These data show that FE65 proteins facilitate endogenous APP processing and metabolism in primary neurons in the presence and absence of NMDAR activation.

Aβ secretion correlates with neuronal activity in vitro and in vivo (Kamenetz et al. 2003, Cirrito et al. 2005, Tampellini et al. 2009). Both altered APP processing and stimulation of Aβ secretion are implicated in neuronal activity-dependent Aβ secretion (Kamenetz et al. 2003, Cirrito et al. 2005). We find that FE65 protein deficiencies attenuate secreted Aβ40 and Aβ42 levels in cortical neuron cultures. Our data are in agreement with a previous study showing decreased levels of secreted Aβ40 and Aβ42 in cortical neuron cultures derived from FE65 isoform (97 kDa)- specific knockout/APP transgenic (Tg2576) hybrid mice when compared to Tg2576 controls (Wang et al. 2004). The similar decrease in neuronal Aβ secretion observed in these two FE65 knockout mouse lines suggest that the p97 isoform of FE65 is largely responsible for these effects. These data are also consistent with the reduced cerebral Aβ levels observed in FE65/FE65L1 DKO mice when compared to WT controls (Guenette et al. 2006).

The impact of NMDAR activation on APP processing has previously been examined with conflicting results (Lesne et al. 2005, Hoey et al. 2009, Marcello et al. 2007, Hoe et al. 2009). This may be due to treatment conditions that produced different intracellular Ca++ loading. Chronic extrasynaptic NMDAR activation results in a sustained accumulation of Ca++ at low levels, while synaptic NMDAR activation produces transient intracellular Ca++ increases (Bordji et al. 2010, Soriano et al. 2006). Neuronal culture and treatment conditions used in our study closely resemble those of Hoey et al. (2009) and our observation that increased C83 levels and reduced Aβ levels are generated upon NMDAR activation are consistent with their results. They are also in agreement with a report showing that physiologic NMDAR activation does not contribute to Aβ secretion (Wei et al. 2010). Our data showing an initial increase in APP-CTF levels and subsequent normalization over 3 hrs of NMDA (100 μM) treatment suggests a shift away from non-amyloidogenic APP processing with continued exposure to high NMDA concentrations. Given that α-secretase processing of APP precludes Aβ generation, the time-dependent attenuation of α-secretase processing in neurons treated with 100 μM NMDA may at least in part be responsible for higher secreted Aβ levels in this paradigm than in neuronal cultures treated with 10 μM NMDA (24 hrs). Decreased Aβ levels were also accompanied by a reduction in pThr668APP in NMDA-treated neurons. pThr668APP was previously shown to contribute to Aβ production in primary cortical neurons (Lee et al. 2003). The decrease in pThr668APP observed upon NMDAR activation may also be partly responsible for lowered secreted Aβ levels in cultures treated with NMDA (10 μM) for 24 hrs. Phosphorylation of APP at Thr668 has been shown to play a key role in trafficking APP to neurites and the plasma membrane (Muresan & Muresan 2005, Santos et al. 2011). Therefore, conditions that sustain low levels of pThr668APP for extended periods of time, may result in much lower levels of secreted Aβ.

The reduction in Aβ secretion and NMDAR activation-dependent C83 generation in FE65/FE65L1 DKO neurons suggests that FE65/APP interactions contribute to both pro- and non-amyloidogenic APP processing in neurons with the outcome possibly depending on the cellular signaling events elicited. Thus, effects of altering FE65 levels on APP processing could result in altered trafficking of APP and C99 to subcellular sites of α- and γ-secretase cleavage, respectively. In support of this hypothesis, previous studies have shown that surface localization, APPsα secretion and Aβ generation were increased when FE65 or FE65L1 were overexpressed in cell lines (Guenette et al. 1999, Chang et al. 2003, Sabo et al. 1999). However, not all APP sorting is altered by FE65 protein deficiencies. Our data show no effect on fast axonal transport of APP to presynaptic terminals. These data are consistent with the observation that recombinant C-terminal truncated APP undergoes fast axonal transport in the same vesicles as full-length APP (Szodorai et al. 2009). Since FE65 proteins bind APP, it remains possible that FE65 proteins not only contribute to physiological APP processing resulting from NMDAR activation but also to APP trafficking and processing in other neuronal compartments.

We showed previously that FE65 protein binding to APP is required for its effects on APP processing (Chang et al. 2003). The contribution of FE65 proteins to APP processing in NMDA treated neurons was no longer detectable at high NMDA (100 μM) concentrations. Although a dose-dependent reduction in FE65 protein levels was observed upon NMDAR treatment, it does not account for this observation. However, it remains possible that FE65/APP interactions are attenuated at high NMDA doses.

Excess FE65 was previously shown to stabilize AICD (Kimberly et al. 2001). In our study, AICD levels between WT in FE65/FE65L1 DKO neurons were no different under basal conditions or following NMDAR activation. Our results do not rule out the possibility that regulated increases in FE65 protein levels stabilize AICD, but physiological events leading to FE65 upregulation and AICD stabilization have not yet been reported.

Despite the existence of two potential N-glycosylation sites on APP695, Asn467 and Asn496, APP695 translated in vitro or expressed in CHO cells is only glycosylated at Asn467 (Pahlsson et al. 1992). Therefore, the detection of two N-glycan chains in HMW species and two bands for extensively deglycosylated HMW species suggests that HMW species consist of a complex of discrete polypeptides that share the A8717 APP C-terminal epitope.

We report that activated calpain and the proteasome play a role in HMW species accumulation. These cytosolic enzymes would normally have limited access to the intraluminal ectodomain of APP. However, NMDAR activation results in the unfolded protein response (Uehara et al. 2006). Furthermore, APP undergoes retrotranslocation from the endoplasmic reticulum to the cytosol where it accumulates when proteasomal activity is inhibited (Huttunen et al. 2007). Thus, direct degradation of APP by calpain and the proteasome may be occurring following NMDAR activation. Three major APP calpain I cleavage products have been identified in vitro (Siman et al. 1990). A calpain I cleavage site predicted to reside between the acidic domain and the N-glycosylation sites of APP produces a doublet migrating at 60 KDa in SDS-PAGE (Siman et al. 1990). It is possible that the 60 kDa APP and APLP2 fragments identified as polypeptides present in HMW species arise from NMDA-dependent calpain I activation. Our current study does not formally exclude the possibility that polypeptides unrelated to APP are found in HMW species. However, our data support that HMW species contain APP and APLP2 polypeptides for the following reasons: HMW species are recognized by several APP C-terminal antibodies, APP and APLP2 immunoreactive fragments with the expected molecular weights for calpain I cleavage are recovered from electroeluted HMW species and HMW species bind copper-chelating sepharose, a property of APP and APLP2 that is found in less than 1% of the proteome (Andreini et al. 2009).

Synaptic loss correlates with AD-related cognitive deficits (Terry et al. 1991). Functional and morphological impairments mediated by Aβ at synapses involve NMDAR-dependent calcium dysregulation (Wei et al. 2010, Bezprozvanny & Mattson 2008). Little is known about the metabolic fate of APP following synapse loss in AD. The HMW APP species identified in our study have not been reported in AD brains but may represent a form of APP that is present in vulnerable neurons.

Collectively, our results suggest that FE65 proteins facilitate Aβ generation and post-synaptic APP processing and metabolism resulting from NMDA receptor-dependent neuronal activation.

Supplementary Material

Supp Fig S1-S7

Acknowledgments

We thank Dr. Nicolaos Robakis for providing R7 antibody; Dr. W. Wasco for providing the APLP2 and APLP1 C-terminal antibodies and Dr. D.M. Kovacs for providing the C66 APP C-terminal antibody. We also thank Drs. Doo Yeon Kim, Henri Huttunen and Robert Moir for their scientific expertise. Sources of funding for this study include NIH AG15903 (S.Y.G. and J.S.) and the Korea Research Foundation fellowship (KRF-2006-214-E00024. J.S.).

Footnotes

Authors report no conflict of interest.

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Supplementary Materials

Supp Fig S1-S7
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