η-Secretase processing of APP inhibits neuronal activity in the hippocampus

Abstract

Alzheimer disease (AD) is characterized by the accumulation of amyloid plaques, which are predominantly composed of amyloid-β peptide. Two principal physiological pathways either prevent or promote amyloid-β generation from its precursor, β-amyloid precursor protein (APP), in a competitive manner. Although APP processing has been studied in great detail, unknown proteolytic events seem to hinder stoichiometric analyses of APP metabolism in vivo. Here we describe a new physiological APP processing pathway, which generates proteolytic fragments capable of inhibiting neuronal activity within the hippocampus. We identify higher molecular mass carboxy-terminal fragments (CTFs) of APP, termed CTF-η, in addition to the long-known CTF-α and CTF-β fragments generated by the α- and β-secretases ADAM10 (a disintegrin and metalloproteinase 10) and BACE1 (β-site APP cleaving enzyme 1), respectively. CTF-η generation is mediated in part by membrane-bound matrix metalloproteinases such as MT5-MMP, referred to as η-secretase activity. η-Secretase cleavage occurs primarily at amino acids 504-505 of APP695, releasing a truncated ectodomain. After shedding of this ectodomain, CTF-η is further processed by ADAM10 and BACE1 to release long and short Aη peptides (termed Aη-α and Aη-β). CTFs produced by η-secretase are enriched in dystrophic neurites in an AD mouse model and in human AD brains. Genetic and pharmacological inhibition of BACE1 activity results in robust accumulation of CTF-η and Aη-α. In mice treated with a potent BACE1 inhibitor, hippocampal long-term potentiation was reduced. Notably, when recombinant or synthetic Aη-α was applied on hippocampal slices ex vivo, long-term potentiation was lowered. Furthermore, in vivo single-cell two-photon calcium imaging showed that hippocampal neuronal activity was attenuated by Aη-α. These findings not only demonstrate a major functionally relevant APP processing pathway, but may also indicate potential translational relevance for therapeutic strategies targeting APP processing.

Figure 1. A novel proteolytic processing pathway of APP.

a, A 30 kDa N-terminally elongated APP-CTF-η fragment is detected in membrane fractions obtained from brains of adult (22 month) and postnatal day 10 (P10) mice using antibody Y188 directed against the C-terminus of APP. CTF-η is specifically found in young and old wild type (WT) mice but absent in APPKO. In addition to this novel fragment, Y188 is detecting CTF-β and CTF-α. Full-length APP (APP-FL) was detected with antibody 22C11. β-Actin served as loading control. b, Aη was identified as several closely spaced peptides detected in the soluble fraction of adult and P10 mice by antibody M3.2. A similar pattern is detected by antibody 9478D that is specifically recognizing an N-terminal part of the Aη peptide (antibody 9478D may not be sensitive enough to detect the lower Aη levels in adult brain). sAPP-α and sAPP-β are shown as additional controls. APPKO brains were used as controls for antibody specificities. β-Actin served as a loading control. c, Higher levels of CTF-η are observed in RIPA lysates of APPPS1-21 mouse brains (long exposure) as compared to WT. Full-length APP (APP-FL) was detected with antibody 22C11. β-Actin served as a loading control. d, Soluble extracts of APPPS1-21 mouse brains contained Aη species detected by 2E9. Aη-β(swe) was selectively detected by antibody 192swe in addition to sAPP-β(swe). While 2D8 antibody detected robust levels of sAPP-α, only low levels of Aη-α could be detected in APPPS1-21 brain lysates due to the overexpression of APPswe transgene. e, Aη and Aβ were readily detectable in 10 μl of human CSF by antibody 2D8. Antibody 2E9 allowed the selective detection of Aη in the same samples, while 192swe specifically detected BACE1 cleaved Aη-β(swe) in the mutation carriers, but not in controls. f, Mass spectrometry analysis of peptides isolated by immunoprecipitation with antibodies 2E9, 2D8, 9478D and 9476M (supplementary Fig. S3 a and b). Peptide intensities were summed per amino acid residue and plotted in relation to each other. We detected peptides from the complete Aη-α sequence (see also supplementary Fig. S3c). The fragmentation spectrum of the N-terminal Aη peptide (APP505-5013) shows good coverage of the b- and y-ion series and an Andromeda score of 88.5 [Doi 10.1021/Pr101065j].

Figure 2. Inhibition of BACE1 results in elevated levels of CTF-η and of Aη-α.

a, Conditioned media of CHO cells expressing human APP_V717F without or with BACE1 inhibition (BI; 2 μM Merck IV) were compared to synthetic peptides of Aη-β and Aη-α. Increased Aη-α peptide levels were observed upon BACE1 inhibitor treatment. The peptide with the lowest molecular weight, co-migrating with the synthetic peptide Aη-β, was diminished upon BACE1 inhibition. b-c, After overnight incubation without or with a BACE inhibitor (BI; 2 μM Merck IV), supernatants (sup.) (b) and lysates (lys.) (c) of DIV16 primary hippocampal neurons were analyzed by Western blotting. A strong increase of endogenous Aη-α was observed upon BACE inhibition. (b). Total levels of secreted APP (22C11) were unchanged while sAPP-α levels increased. The absence of sAPP-β and Aβ proves the effective blockade of BACE1 (b). In cell lysates CTF-β was undetectable when the BACE inhibitor was applied and CTF-η was strongly increased (c). While APP-FL levels were accumulating, CTF-α levels remained unchanged. BACE1 levels were similar in all samples. β-Actin served as loading control (c). d-g, Similarly, human neurons differentiated from H9 embryonic stem cells were incubated for 48 h with a BACE1 inhibitor (BI; 1 μM LY2886721). d, In cell lysates enriched CTF-η levels were detected, while CTF-β levels were diminished. APP-FL and BACE1 levels were similar in all samples. β-Actin served as loading control. e-g, Supernatants were analyzed by Western blotting. While sAPP-β levels dropped to undetectable levels upon BACE inhibition, Aη-α levels strongly increased as indicated by antibody 2D8 and 2E9 (g, ImageG quantified intensities for 2D8 signal in e; 64,8% increase upon BACE1 inhibition, n=8; p < 0.001; Student’s t-test). With antibody 2E9 we additionally detected a faster migrating band disappearing when BACE was blocked, demonstrating the selective reduction of Aη-β. In supernatants of hippocampal neurons and of H9 induced neurons Aβ could be detected only upon longer exposure, while for the detection of Aη-α much shorter exposures were sufficient. h, BACE1 inhibition in vivo resulted in enhanced production of Aη-α species in APP_V717I mice. BACE1 inhibitor RO5508887 treated mice and vehicle treated controls were sacrificed and analyzed after 5, 8 or 24 h. BACE inhibition reduced sAPP-β and CTF-β and increased levels of Aη-α at 5 and 8 h after treatment. 24 h after the treatment these changes were normalized due to the clearance of the inhibitor. Background bands obtained with 2D8 and Y188 are indicated by asterisks. i, Western blot analysis of soluble extracts of P10 BACE1-/- mouse brains revealed a significant increase in Aη-α peptides as compared to controls. Total levels of secreted APP (22C11) were unchanged while sAPP-α levels increased. CTF-η levels were increased in membrane lysates of the BACE1-/- mouse brain. As expected after an efficient BACE1 block, CTF-β and sAPP-β were severely reduced.

Figure 3. Aη-α impairs hippocampal LTP.

a, In membrane lysates of brains obtained from BACE inhibitor (BI, 100mg/kg SCH1682496) treated animals an increase in CTF-η was observed, which was paralleled by a strong reduction of CTF-β, while CTF-α was unchanged (left panel). APP-FL and BACE1 signals remained unchanged (asterisk indicates background band). Calnexin served as a loading control (Left panel). In the soluble fraction BACE inhibition resulted in enhanced production of Aη-α species which was detected by antibody M3.2 (right panel). Reduced sAPP-β levels indicated efficient BACE1 inhibition. revealed a 95,4% increase upon BACE1 inhibition, n=3; p < 0.01, Student`s t-test). b-c, Pharmacological inhibition of BACE lowers hippocampal LTP. Three hours after a single gavage of SCH1682496 (100 mg/kg) or vehicle, hippocampal slices were cut and baseline transmission and LTP measured. Note, that compared to vehicle treated controls in slices from inhibitor treated mice LTP was notably reduced. c, Representative fEPSPs recorded in CA1 area prior and 45 min after tetanization of Schaffer collaterals (top) with summary plots of the effects of the inhibitor and vehicle on fEPSP slopes in all examined groups. d, Soluble Aη-α and Aη-β peptides were expressed in CHO cells. Conditioned media were analyzed with antibodies 2D8, 2E9 and 9478D for the presence of the larger Aη-α and the smaller Aη-β peptides. e-h, SEC fractions containing Aη were diluted (1:15) in ACSF for the treatment of hippocampal slices and LTP measurements. Aη-α or Aη-β and control SEC fractions (obtained from CHO cells transfected with the empty vector) were perfused over mouse hippocampal slices for 20 min after obtaining a stable baseline of a fEPSP at the CA3-CA1 synapse. After 20 minutes a high-frequency stimulation protocol was applied (HFS; 2x (100 Hz, 1 s) at 20 second inter-stimulus interval) to induce long-term potentiation (LTP). e, Supernatants from CHO cells expressing Aη-α significantly inhibited LTP; f, Supernatants from CHO cells expressing Aη-β did not alter LTP. g, Supernatants from untransfected CHO cells did not alter LTP when compared to the control condition (ACSF only); h, summary graph of LTP magnitudes (as % of baseline) calculated 45-60 minutes post-HFS from graphs in e-g with statistical analysis (*p < 0.05); error bars represent s.e.m. n = number of fields. For each condition, sample fEPSP traces pre-LTP (black) and 45-60 min post-LTP (grey) induction are shown.

Figure 4. Aη-α reduces neuronal activity in vivo.

a-d (left panels), In vivo two-photon images of CA1 hippocampal neurons labeled with the fluorescent calcium indicator fluo-8 AM. (middle and right panels), Calcium transients of 5 representative neurons, marked in the corresponding left panels a-d, before and during bath-application of Aη peptides (panels b-d) and CHO conditioned media (panel a). e, Calcium transients in hippocampal neurons before, during and after local application of synthetic Aη-α. f, Summary results of the changes in the average rates of calcium transients (error bars represent s.e.m; p < 0.001 for CHO-Aη-α (12.36 transients/min ± 0.43 vs. 6.91 ± 0.44, n = 206 cells in three mice), bath-applied synthetic Aη-α (16.19 ± 0.56 vs. 7.06 ± 0.53, n = 163 cells in three mice) and locally applied Aη-α (14.30 ± 0.5 vs. 8.05 ± 0.44, n = 198 cells in four mice); p > 0.05 for CHO (10.65 ± 0.51 vs. 10.09 ± 0.52, n = 188 cells in three mice), control peptide (12.62 ± 0.44 vs. 13.17 ± 0.40, n = 212 cells in three mice) and CHO-Aη-β (11.23 ± 0.46 vs. 11.72 ± 0.55, n = 186 cells in three mice); Students T-test). g, Summary results, displayed as bar graphs, of the changes in the fractions of silent neurons (p < 0.001 for CHO-Aη-α (1.94 % vs. 17.48), bath-applied synthetic Aη-α (0.62 vs. 12.27), locally applied Aη-α (0 vs. 15.15); p > 0.05 for CHO (1.06 vs. 0.53), control peptide (0.94 vs. 1.90) and CHO-Aη-β (2.15 vs. 4.30); Fisher´s exact test)