Processing of the synaptic cell adhesion molecule neurexin-3beta by Alzheimer disease alpha- and gamma-secretases

Abstract

Neurexins (NRXNs) are synaptic cell adhesion molecules having essential roles in the assembly and maturation of synapses into fully functional units. Immunocytochemical and electrophysiological studies have shown that specific binding across the synaptic cleft of the ectodomains of presynaptic NRXNs and postsynaptic neuroligins have the potential to bidirectionally coordinate and trigger synapse formation. Moreover, in vivo studies as well as genome-wide association studies pointed out implication of NRXNs in the pathogenesis of cognitive disorders including autism spectrum disorders and different types of addictions including opioid and alcohol dependences, suggesting an important role in synaptic function. Despite extensive investigations, the mechanisms by which NRXNs modulate the properties of synapses remain largely unknown. We report here that α- and γ-secretases can sequentially process NRXN3β, leading to the formation of two final products, an ∼80-kDa N-terminal extracellular domain of Neurexin-3β (sNRXN3β) and an ∼12-kDa C-terminal intracellular NRXN3β domain (NRXN3β-ICD), both of them being potentially implicated in the regulation of NRXNs and neuroligins functions at the synapses or in yet unidentified signal transduction pathways. We further report that this processing is altered by several PS1 mutations in the catalytic subunit of the γ-secretase that cause early-onset familial Alzheimer disease.

FIGURE 1.

NRXN3β is processed by α- and γ-secretases. Shown is accumulation of NRXN3β-CTFs in HEK293T cells expressing transiently (A) or CHO cells expressing stably (B) human (h) FL-NRXN3β-FLAG exposed for 16 h to DMSO (0.5% w/v final), 10 μm γ-secretase inhibitors DAPT or CpdE, 20 μm TACE inhibitor TAPI-1, 5 μm BACE1 inhibitor GL189 in the presence or absence of 0.5 μm PMA. Whole cell lysates were immunoblotted with anti-FLAG antibodies. Non-transfected CHO cells are labeled CHO-. C, shown is accumulation of endogenous NRXN-CTFs in mouse (m) primary cortical neurons (14 DIV) treated as in (A/B). FLAG-tagged hNRXN3β proteins purified from DAPT-treated HEK293T cells expressing transiently FL-NRXN3β-FLAG were used as size controls for endogenous NRXN3β-FL and -CTFs. Linked arrowheads show full-length and glycosylated NRXN (64). The asterisks designate a minor band immunoreactive for NRXN3β. D, shown are sequence comparisons of transmembrane domains (gray) and cytosolic C-terminal domains of 16 canonical Neurexin isoforms. Identical residues in all sequences are marked by an asterisk; conserved substitutions are marked by a colon. E, shown is neuronal activity-dependent accumulation of murine NRXN-CTFs. After stimulation of PCN with either 50 μm l-glutamate or 50 mm KCl, microsomal membrane preparations were incubated with DAPT (1 μm) or DMSO at 37 °C for 4 h, and protein extracts were immunoblotted with antibodies to the C terminus of NRXNs. Densitometric analysis revealed a significant increase in CTFs when neurons were treated with l-glutamate or KCl as compared with non-treated ones. Results are expressed as the mean ± S.D. of triplicate luminescence measurements (n = 3, *, p < 0.05, t test).

FIGURE 2.

α-Secretase-mediated release of extracellular sNRXN3β. HEK293T cells transiently expressing FLAG-tagged full-length NRXN3β were maintained overnight in DMEM 10% FBS, washed, and conditioned for 16 h in serum-free DMEM medium containing 0.5 μm PMA or 20 μm TAPI-1, respectively, an activator and inhibitor of α-secretases of the ADAM family. The conditioned media were concentrated by TCA precipitation and probed by Western blotting with an anti NRXN3β antibody specific for the N-terminal ectodomain (A) or an M2 anti-FLAG antibody targeting the C terminus of full-lengthNRXN3β (B). A total cell lysate from HEK293T cells transiently expressing FL-NRXN3β-FLAG (hNRXN3β control; lane 1) and conditioned medium from untransfected HEK293T cells (−) were loaded as controls.

FIGURE 3.

Partial loss of γ-secretase activity impairs NRXN3β processing. A, Alzheimer disease-causing mutants of PS1 impair NRXN3β processing are shown. NRXN3β-CTFs were analyzed in CHO cells stably expressing FL-NRXN3β and transiently expressing increasing amounts of WT PS1 (lanes 2 and 3) or the Alzheimer disease causing mutants PS1-L166P, PS1-P436Q, and PS1-ΔE9 (lanes 4–9). FL-NRXN3β, NRXN3β-CTFs, PS1-NTF, FL-APP, and APP-CTFs were probed by Western blotting using the M2 anti-FLAG (for FL-NRXN3β/NRXN3β-CTFs), anti-hPS1, and anti-amyloid precursor protein (for FL-APP/APP-CTFs) C-terminal antibody (upper panel). Lower panel, the levels of hNRXN3β- and APP-CTFs were quantified by densitometric analysis of the bands in the Western blot. For accurate quantification, the relative levels of CTFs were normalized to these of full-length NRXN3β and APP (CTFs:FL). LV indicates the volume of lentivirus preparation used to infect 2 × 10⁶ CHO cells. β-Actin serves as a loading control. B, impaired NRXN3β processing in PS1/PS2 double knock-out MEFs is shown. Representative Western blot analysis is shown of NRXN3β-CTFs in wild-type mouse embryonic fibroblasts (MEF wt, lane 1) in PS1/PS2 double knock-out MEFs (MEF DKO, lane 2), and in PS1/PS2 double knock-out MEFs expressing hPS1 WT (MEF DKO + PS1, lane 3). *, minor band immunoreactive for NRXN3β.

FIGURE 4.

γ-Secretase-dependent processing of NRXN3β in a cell-based luciferase reporter assay. A, a schematic representation of the NRXN3β-specific luciferase reporter assay is shown. Full-length NRXN3β fused to the Gal4 DNA binding domain and to the VP16 activation domain (NRXN3β-FL-GV) was expressed under the control of the tetracycline operator (TO) in T-Rex HeLa cells constitutively expressing the tetracycline repressor (tetR) protein transfected with pGL4.31, a plasmid encoding a Gal4-driven luciferase reporter gene. In the absence of tetracycline (−Tet), tetR binds to the tetracycline operator and represses the transcription of NRXN3β-FL-GV, resulting in low expression of luciferase reporter. Added tetracycline (+Tet) results in tetR release from the tetracycline operator and consequently to the expression of the chimeric proteins NRXN3β-FL-GV and high expression of luciferase reporter. In this system, sequential processing by α- and γ-secretase of NRXN3β-FL-GV leads to the formation of NRXN3β-ICD-GV, which gets released from the membrane and translocated into the nucleus, where it activates transcription of the luciferase reporter gene. Inhibition of α- or γ-secretase is, thus, expected to prevent expression of the luciferase reporter gene. B, the γ-secretase inhibitor DAPT blocks the proteolysis of NRXN3β-CTF-GV or APP-C99-GV and prevents the expression of the luciferase reporter gene. Left, shown is luminescence (counts per second) emitted by cells treated with tetracycline (1 μg/ml) in the presence or absence of DAPT (10 μm). Results are expressed as the means ± S.D. of triplicate luminescence measurements (n = 3, *, p < 0.05, t test). Right, a representative reaction was Western-blotted for NRXN3β-GV and APP-C99-GV by using an anti VP16 antibody. Light (top) and dark (bottom) exposures are shown.

FIGURE 5.

γ-Secretase-dependent processing of NRXN3β-CTFs and NRXN3β-ICD production. A, γ-secretase activity assays performed with membranes isolated from CHO cells stably expressing full-length NRXN3β-FLAG are shown. Cell membranes enriched in FL-NRXN3β-FLAG were incubated for 3 h at 37 °C in the presence or absence of 1 μm DAPT or 1 μm Compound E (CpdE) (lanes 1–5). Alternatively, membranes prepared from cells incubated for 16 h in the presence or absence of DAPT (10 μm) were incubated for 2 h at 37 °C (lanes 6 and 7). B, γ-secretase activity assays were performed with purified NRXN3β-based substrates and purified γ-secretase. FLAG-tagged NRXN3β-CTFs purified from HEK293T cells transiently expressing FL-NRXN3β-FLAG were incubated with purified γ-secretase for 4 h at 37 °C with 0.1% PC, 0.025% PE, and in the presence or absence of 1 μm DAPT or CpdE. NRXN3β-ICDs in the representative Western blot were quantified by densitometry (right panel) and normalized to the NRXN3β-ICDs produced in the absence of γ-secretase inhibitor (100%). C, γ-secretase activity assays were performed with purified recombinant (r) NRXN3β-based substrates and purified γ-secretase. Left panel, shown is a schematic representation of the membrane anchored full-length NRXN3β and the recombinant NRXN3β-CTF substrate consisting of the C-terminal amino acids 551–630 plus a methionine residue at the N terminus and a FLAG tag at the C terminus. The gray box (amino acid residues 564–584) represents the predicted transmembrane domain. Middle panel, γ-secretase from solubilized S20 membranes diluted in 0.25% CHAPSO-HEPES, pH 7.5, was incubated at 37 °C for 4 h with 1 μm APP-C100FLAG (lanes 1–4) or 1 μm rNRXN3β-CTF-551 (lanes 5–8), 0.1% PC, 0.025% PE in the presence or absence of 1 μm DAPT or CpdE. The reactions were Western-blotted for APP intracellular domain (AICD)-FLAG and NRXN3β-ICD-FLAG by using the anti FLAG M2 antibody. Recombinant NRXN3β-ICDs and APP intracellular domains in the representative Western blots were quantified by densitometry (right panel) and normalized to the ICDs produced in the absence of γ-secretase inhibitor (100%).

FIGURE 6.

Model for NRXN3β processing by α- and γ-secretases and hypothetical implications on synaptic activity and plasticity. Full-length NRXN3β (FL-NRXN3β) is synthesized as a type I transmembrane protein forming trans-synaptic complexes with NLGNs required for synapse maturation and function (A). FL-NRXN3β can undergo cleavage at the α-secretase sites to release large ectodomains designated sNRXN3β and leave membrane-embedded fragments NRXN3β-CTFs (B). Proteolysis of these CTFs by γ-secretase releases intracellular domains NRXN3β-ICDs and extracellular NRXN3β peptides (NRXN3β-Ps). Considering the essential roles of NRXNs in synaptic activity and plasticity, one can speculate that the processing of FL-NRXN3β by α-secretase (1) and/or the release of soluble sNRXN3β into the synaptic cleft (2) can modulate NLGNs and NRXN-dependent activities. The intracellular release of NRXN3β-ICD (3) might trigger signaling pathways and/or affect neurotransmitter vesicle clustering and release.