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One of the main components of senile plaques in Alzheimer’s disease (AD)-affected brain is the Aβ peptide species harboring a pyroglutamate at position three pE3-Aβ. Several studies indicated that pE3-Aβ is toxic, prone to aggregation and serves as a seed of Aβ aggregation. The cyclisation of the glutamate residue is produced by glutaminyl cyclase, the pharmacological and genetic reductions of which significantly alleviate AD-related anatomical lesions and cognitive defects in mice models. The cyclisation of the glutamate in position 3 requires prior removal of the Aβ N-terminal aspartyl residue to allow subsequent biotransformation. The enzyme responsible for this rate-limiting catalytic step and its relevance as a putative trigger of AD pathology remained yet to be established. Here, we identify aminopeptidase A as the main exopeptidase involved in the N-terminal truncation of Aβ and document its key contribution to AD-related anatomical and behavioral defects. First, we show by mass spectrometry that human recombinant aminopeptidase A (APA) truncates synthetic Aβ1-40 to yield Aβ2-40. We demonstrate that the pharmacological blockade of APA with its selective inhibitor RB150 restores the density of mature spines and significantly reduced filopodia-like processes in hippocampal organotypic slices cultures virally transduced with the Swedish mutated Aβ-precursor protein (βAPP). Pharmacological reduction of APA activity and lowering of its expression by shRNA affect pE3-42Aβ- and Aβ1-42-positive plaques and expressions in 3xTg-AD mice brains. Further, we show that both APA inhibitors and shRNA partly alleviate learning and memory deficits observed in 3xTg-AD mice. Importantly, we demonstrate that, concomitantly to the occurrence of pE3-42Aβ-positive plaques, APA activity is augmented at early Braak stages in sporadic AD brains. Overall, our data indicate that APA is a key enzyme involved in Aβ N-terminal truncation and suggest the potential benefit of targeting this proteolytic activity to interfere with AD pathology. Supplementary Information The online version contains supplementary material available at 10.1007/s00401-021-02308-0.

The histopathology of Alzheimer's disease (AD) is characterized by neuronal loss, neurofibrillary tangles, and senile plaque formation. The latter results from an exacerbated production (familial AD cases) or altered degradation (sporadic cases) of 40/42-amino acid–long β-amyloid peptides (Aβ peptides) that are produced by sequential cleavages of Aβ precursor protein (βAPP) by β- and γ-secretases. The amyloid cascade hypothesis proposes a key role for the full-length Aβ42 and the Aβ40/42 ratio in AD etiology, in which soluble Aβ oligomers lead to neurotoxicity, tau hyperphosphorylation, aggregation, and, ultimately, cognitive defects. However, following this postulate, during the last decade, several clinical approaches aimed at decreasing full-length Aβ42 production or neutralizing it by immunotherapy have failed to reduce or even stabilize AD-related decline. Thus, the Aβ peptide (Aβ40/42)-centric hypothesis is probably a simplified view of a much more complex situation involving a multiplicity of APP fragments and Aβ catabolites. Indeed, biochemical analyses of AD brain deposits and fluids have unraveled an Aβ peptidome consisting of additional Aβ-related species. Such Aβ catabolites could be due to either primary enzymatic cleavages of βAPP or secondary processing of Aβ itself by exopeptidases. Here, we review the diversity of N- and C-terminally truncated Aβ peptides and their biosynthesis and outline their potential function/toxicity. We also highlight their potential as new pharmaceutical targets and biomarkers.

The amyloid cascade hypothesis, which proposes a prominent role for full-length amyloid β peptides in Alzheimer's disease, is currently being questioned. In addition to full-length amyloid β peptide, several N-terminally truncated fragments of amyloid β peptide could well contribute to Alzheimer's disease setting and/or progression. Among them, pyroGlu3-amyloid β peptide appears to be one of the main components of early anatomical lesions in Alzheimer's disease-affected brains. Little is known about the proteolytic activities that could account for the N-terminal truncations of full-length amyloid β, but they appear as the rate-limiting enzymes yielding the Glu3-amyloid β peptide sequence that undergoes subsequent cyclization by glutaminyl cyclase, thereby yielding pyroGlu3-amyloid β. Here, we investigated the contribution of dipeptidyl peptidase 4 in Glu3-amyloid β peptide formation and the functional influence of its genetic depletion or pharmacological blockade on spine maturation as well as on pyroGlu3-amyloid β peptide and amyloid β 42-positive plaques and amyloid β 42 load in the triple transgenic Alzheimer's disease mouse model. Furthermore, we examined whether reduction of dipeptidyl peptidase 4 could rescue learning and memory deficits displayed by these mice. Our data establish that dipeptidyl peptidase 4 reduction alleviates anatomical, biochemical, and behavioral Alzheimer's disease-related defects. Furthermore, we demonstrate that dipeptidyl peptidase 4 activity is increased early in sporadic Alzheimer's disease brains. Thus, our data demonstrate that dipeptidyl peptidase 4 participates in pyroGlu3-amyloid β peptide formation and that targeting this peptidase could be considered as an alternative strategy to interfere with Alzheimer's disease progression.

Neuronal network dysfunction and cognitive decline constitute the most prominent features of Alzheimer’s disease (AD), although mechanisms causing such impairments are yet to be determined. Here we report that virus-mediated delivery of the active spliced transcription factor X-Box binding protein 1s (XBP1s) in the hippocampus rescued spine density, synaptic plasticity and memory function in a mouse model of AD. XBP1s transcriptionally activated Kalirin-7 (Kal7), a protein that controls synaptic plasticity. In addition, we found reduced levels of Kal7 in primary neurons exposed to Aβ oligomers, transgenic mouse models and human AD brains. Short hairpin RNA-mediated knockdown of Kal7 altered synaptic plasticity and memory formation in naive mice. Further, reduction of endogenous Kal7 compromised the beneficial effects of XBP1s in Alzheimer’s model. Hence, our findings reveal that XBP1s is neuroprotective through a mechanism that engages Kal7 pathway with therapeutic implications in AD pathology.

The aspartyl protease β-site APP cleaving enzyme, BACE1, is the rate-limiting enzyme involved in the production of amyloid-β peptide, which accumulates in both sporadic and familial cases of Alzheimer’s disease and is at the center of gravity of the amyloid cascade hypothesis. In this context, unravelling the molecular mechanisms controlling BACE1 expression and activity in both physiological and pathological conditions remains of major importance. We previously demonstrated that Aβ controlled BACE1 transcription in an NFκB-dependent manner. Here, we delineate an additional cellular pathway by which natural and synthetic Aβ42 oligomers enhance active X-box binding protein XBP-1s. XBP-1s lowers BACE1 expression and activity indirectly, via the up-regulation of the ubiquitin-ligase HRD1 that acts as an endogenous down-regulator of BACE1. Thus, we delineate a novel pathway by which cells could compensate for Aβ42 oligomers production and thus, associated toxicity, by triggering a compensatory mechanism aimed at lowering BACE-1-mediated Aβ production by a molecular cascade involving XBP-1s and HRD1. It thus identifies HRD1 as a potential target for a novel Aβ-centered therapeutic strategy.

Endoplasmic reticulum (ER) is the cellular compartment where secreted and integral membrane proteins are folded and matured. The accumulation of unfolded or misfolded proteins triggers a stress that is physiologically controlled by an adaptative protective response called Unfolded Protein Response (UPR). UPR is primordial to induce a quality control response and to restore ER homeostasis. When this adaptative response is defective, protein aggregates overwhelm cells and affect, among other mechanisms, synaptic function, signaling transduction and cell survival. Such dysfunction likely contributes to several neurodegenerative diseases that are indeed characterized by exacerbated protein aggregation, protein folding impairment, increased ER stress and UPR activation. This review briefly documents various aspects of the biology of the transcription factor XBP-1 (X-box Binding Protein-1) and summarizes recent findings concerning its putative contribution to the altered UPR response observed in various neurodegenerative disorders including Parkinson's and Alzheimer's diseases.

Triple-transgenic mice (3xTgAD) overexpressing Swedish-mutated β-amyloiḍprecursoṛprotein (βAPPswe), P310L-Tau (TauP301L) and physiological levels of M146V-presenilin-1 (PS1M146V) display extracellular amyloid-β peptides (Aβ) deposits and Tau tangles. More disputed is the observation that these mice accumulate intraneuronal Aβ that has been linked to synaptic dysfunction and cognitive deficits. Here, we provide immunohistological, genetic and pharmacological evidences for early, age-dependent and hippocampus-specific accumulation of the β-secretase-derived βAPP fragment C99 that is observed in 3 month-old mice and enhanced by pharmacological blockade of γ-secretase. Notably, intracellular Aβ is only detectable several months later and appears, as is the case for C99, in enlarged cathepsin B-positive structures, while extracellular Aβ deposits are detected around 12 months of age and beyond. Early C99 production occurs mainly in the CA1/subicular interchange area of the hippocampus corresponding to the first region exhibiting plaques and tangles in old mice. The examination of two other mice models harboring mutated βAPP but endogenous wild type PS1 and Tau protein (TgCRND8 or Tg2576) indicate that C99 levels are largely higher in all animal models than in their respective control mice. Furthermore, the comparison of 3xTgAD mice with double transgenic mice bearing the βAPPswe and TauP301L mutations but expressing endogenous PS1 (2xTgAD) demonstrate that C99 accumulation could not be accounted for by a loss of function triggered by PS1 mutation that would have prevented C99 secondary cleavage by γ-secretase. Altogether, our work identifies C99 as the earliest βAPP catabolite and main contributor to the intracellular βAPP-related immunoreactivity in 3xTgAD mice, suggesting its implication as an initiator of the neurodegenerative process and cognitive alterations taking place in this mice model.

Activation of EphB receptors by ephrinB (efnB) ligands on neuronal cell surface regulates important functions, including neurite outgrowth, axonal guidance, and synaptic plasticity. Here, we show that efnB rescues primary cortical neuronal cultures from necrotic cell death induced by glutamate excitotoxicity and that this function depends on EphB receptors. Importantly, the neuroprotective function of the efnB/EphB system depends on presenilin 1 (PS1), a protein that plays crucial roles in Alzheimer's disease (AD) neurodegeneration. Furthermore, absence of one PS1 allele results in significantly decreased neuroprotection, indicating that both PS1 alleles are necessary for full expression of the neuroprotective activity of the efnB/EphB system. We also show that the ability of brain-derived neurotrophic factor (BDNF) to protect neuronal cultures from glutamate-induced cell death depends on PS1. Neuroprotective functions of both efnB and BDNF, however, were independent of γ-secretase activity. Absence of PS1 decreases cell surface expression of neuronal TrkB and EphB2 without affecting total cellular levels of the receptors. Furthermore, PS1-knockout neurons show defective ligand-dependent internalization and decreased ligand-induced degradation of TrkB and Eph receptors. Our data show that PS1 mediates the neuroprotective activities of efnB and BDNF against excitotoxicity and regulates surface expression and ligand-induced metabolism of their cognate receptors. Together, our observations indicate that PS1 promotes neuronal survival by regulating neuroprotective functions of ligand-receptor systems.

International audience; The senile plaques found in the brains of patients with Alzheimer's disease are mainly due to the accumulation of amyloid beta-peptides (A beta) that are liberated by gamma-secretase, a high molecular weight complex including presenilins, PEN-2, APH-1 and nicastrin. The depletion of each of these proteins disrupts the complex assembly into a functional protease. Here, we describe another level of regulation of this multimeric protease. The depletion of both presenilins drastically reduces Pen2 mRNA levels and its promoter transactivation. Furthermore, overexpression of presenilin-1 lowers Pen2 promoter transactivation, a phenotype abolished by a double mutation known to prevent presenilin-dependent gamma-secretase activity. PEN-2 expression is decreased by depletion of beta-amyloid precursor protein (APP) and increased by the APP intracellular domain (AICD). We show that AICD and APP complement for Pen2 mRNA levels in APP/APLP1-2 knockout fibroblasts. Interestingly, overexpression of presenilin-2 greatly increases Pen2 promoter transactivation. The opposite effect triggered by both presenilins was reminiscent of our previous study, which showed that these two proteins elicit antagonistic effects on p53. Therefore, we examined the contribution of p53 on Pen2 transcription. Pen2 promoter transactivation, and Pen2 mRNA and protein levels were drastically reduced in p53(-/-) fibroblasts. Furthermore, PEN-2 expression could be rescued by p53 complementation in p53- and APP-deficient cells. Interestingly, PEN-2 expression was also reduced in p53-deficient mouse brain. Overall, our study describes a p53-dependent regulation of PEN-2 expression by other members of the gamma-secretase complex, namely presenilins.

International audience; Nicastrin (NCT) is a component of the presenilin (PS)-dependent gamma-secretase complexes that liberate amyloid beta-peptides from the beta-Amyloid Precursor Protein. Several lines of evidence indicate that the members of these complexes could also contribute to the control of cell death. Here we show that over-expression of NCT increases the viability of human embryonic kidney (HEK293) cells and decreases staurosporine (STS)- and thapsigargin (TPS)-induced caspase-3 activation in various cell lines from human and neuronal origins by Akt-dependent pathway. NCT lowers p53 expression, transcriptional activity and promoter transactivation and reduces p53 phosphorylation. NCT-associated protection against STS-stimulated cell death was completely abolished by p53 deficiency. Conversely, the depletion of NCT drastically enhances STS-induced caspase-3 activation and p53 pathway and favored p53 nuclear translocation. We examined whether NCT protective function depends on PS-dependent gamma-secretase activity. First, a 29-amino acid deletion known to reduce NCT-dependent amyloid beta-peptide production did not affect NCT-associated protective phenotype. Second, NCT still reduces STS-induced caspase-3 activation in fibroblasts lacking PS1 and PS2. Third, the gamma-secretase inhibitor DFK167 did not affect NCT-mediated reduction of p53 activity. Altogether, our study indicates that NCT controls cell death via phosphoinositide 3-kinase/Akt and p53-dependent pathways and that this function remains independent of the activity and molecular integrity of the gamma-secretase complexes.

We recently established that neprilysin (NEP), one of the putative amyloid β-peptide (Aβ)-degrading enzymes, is transcriptionally upregulated by AICDs, the C-terminal fragments of the β-amyloid precursor protein (βAPP/APLP) that are generated by presenilin (PS)-dependent γ- and ɛ-secretase activities (Pardossi-Piquard et al., 2005). Chen and Selkoe now report lack of evidence of NEP upregulation by PS-dependent γ-secretase. To explore the reasons for this difference, we have repeated several experiments, conducted new experiments, and carefully examined the data presented by Chen and Selkoe.

The presenilin-dependent gamma-secretase activity, which is responsible for the generation of amyloid beta-peptide, is a high molecular weight complex composed of at least four components, namely, presenilin-1 (or presenilin-2), nicastrin, Aph-1, and Pen-2. Previous data indicated that presenilins, which are thought to harbor the catalytic core of the complex, also control p53-dependent cell death. Whether the other components of the gamma-secretase complex could also modulate the cell death process in mammalian neurons remained to be established. Here, we examined the putative contribution of Aph-1 and Pen-2 in the control of apoptosis in TSM1 cells from a neuronal origin. We show by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and DNA fragmentation analyses that the overexpression of Aph-1a, Aph-1b, or Pen-2 drastically lowered staurosporine-induced cellular toxicity. In support of an apoptosis rather than necrosis process, Aph-1 and Pen-2 also lower staurosporine- and etoposide-induced caspase-3 expression and diminished caspase-3 activity and poly(ADP-ribose) polymerase inactivation. The Aph-1 and Pen-2 anti-apoptotic phenotype was associated with a drastic reduction of p53 expression and activity and lowered p53 mRNA transcription. Furthermore, the Aph-1- and Pen-2-associated reduction of staurosporine-induced caspase-3 activation was fully abolished by p53 deficiency. Conversely, Aph-1a, Aph-1b, and Pen-2 gene inactivation increases both caspase-3 activity and p53 mRNA levels. Finally, we show that Aph-1 and Pen-2 did not trigger an anti-apoptotic response in cells devoid of presenilins or nicastrin, whereas the protective response was still observed in fibroblasts devoid of beta-amyloid precursor protein and amyloid precursor protein like-protein 2. Furthermore, Aph-1- and Pen-2-associated protection against staurosporine-induced caspase-3 activation was not affected by the gamma-secretase inhibitors N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester and difluoromethylketone. Altogether, our study indicates that Aph-1 and Pen-2 trigger an anti-apoptotic response by lowering p53-dependent control of caspase-3. Our work also demonstrates that this phenotype is strictly dependent on the molecular integrity of the gamma-secretase complex but remains independent of the gamma-secretase catalytic activity.

PS (presenilin)-dependent γ-secretase occurs as a high-molecular-mass complex composed of either PS1 or PS2 associated with Nct (nicastrin), PEN2 (presenilin enhancer 2 homologue) and APH1 (anterior pharynx defective 1 homologue). Numerous reports have documented the very complicated physical and functional cross-talk between these proteins that ultimately governs the biological activity of the γ-secretase, but very few studies examined the fate of the components of the complex. We show that, in both HEK-293 cells and the TSM1 neuronal cell line, the immunoreactivities of overexpressed myc-tagged-APH1a and -PEN2 were enhanced by the proteasome inhibitors ZIE and lactacystin, whereas a broad range of protease inhibitors had no effect. By contrast, proteasome inhibitors were totally unable to affect the cellular expression of endogenous APH1aL and PEN2 in HEK-293 cells, TSM1 and primary cultured cortical neurons. To explain this apparent discrepancy, we examined the degradation of myc-tagged-APH1a and -PEN2, in vitro, by cell extracts containing endogenous proteasome and by purified 20S proteasome. Strikingly, myc-tagged-APH1a and -PEN2 resist proteolysis by endogenous proteasome and purified 20S proteasome. We also show that endogenous PEN2 expression was drastically higher in wild-type than in PS- and Nct-deficient fibroblasts and was enhanced by proteasome inhibitors only in the two deficient cell systems. However, here again, purified 20S proteasome appeared unable to cleave endogenous PEN2 present in PS-deficient fibroblasts. The levels of endogenous APH1aL-like immunoreactivity were not modified by proteasome inhibitors and were unaffected by PS deficiency. Altogether, our results indicate that endogenous PEN2 and APH1aL do not undergo proteasomal degradation under physiological conditions in HEK-293 cells, TSM1 cells and fibroblasts and that the clearance of PEN2 in PS- and Nct-deficient fibroblasts is not mediated by 20S proteasome. Whether the 26S proteasome participates to PEN2 proteolysis in deficient fibroblasts remains to be established.

We established previously that alpha-synuclein displayed a protective anti-apoptotic phenotype in neurons, mainly by down-regulating p53-dependent caspase-3 activation (Alves da Costa, C., Ancolio, K., and Checler, F. (2000) J. Biol. Chem. 275, 24065-24069; Alves da Costa, C., Paitel, E., Vincent, B., and Checler, F. (2002) J. Biol. Chem. 277, 50980-50984). This function was abolished by Parkinson disease-linked pathogenic mutations and by the dopaminergic toxin, 6-hydroxydopamine (6OH-DOPA) (Alves da Costa, C., Paitel, E., Vincent, B., and Checler, F. (2002) J. Biol. Chem. 277, 50980-50984). However, the mechanisms by which 6OH-DOPA interfered with alpha-synuclein function remained unclear. Here we showed that 6OH-DOPA prevents alpha-synuclein-mediated anti-apoptotic function by altering its degradation. Thus, 6OH-DOPA treatment of TSM1 neurons and SH-SY5Y neuroblastoma cells enhances endogenous alpha-synuclein-like immunoreactivity and inhibits the catabolism of endogenous and recombinant alpha-synucleins by purified 20 S proteasome. Furthermore, we demonstrated that 6OH-DOPA directly inhibits endogenous proteasomal activity in TSM1 and SH-SY5Y cells and also blocks purified proteasome activity in vitro. This inhibitory effect can be prevented by the anti-oxidant phenyl-N-butylnitrone. We also established that 6OH-DOPA triggers the aggregation of recombinant alpha-synuclein in vitro. Therefore, we conclude that 6OH-DOPA abolishes alpha-synuclein anti-apoptotic phenotype by inhibiting its proteasomal degradation, thereby increasing its intracellular concentration and potential propensity to aggregation, the latter phenomenon being directly exacerbated by 6OH-DOPA itself. Interestingly, 1-methyl-4-phenylpyridinium (MPP(+)), another toxin inducer of Parkinson disease-like pathology, does not affect alpha-synuclein protective function and fails to trigger aggregation of recombinant alpha-synuclein. Furthermore, MPP(+) does not alter cellular proteasomal activity, and only high concentrations of the toxin affect purified 20 S proteasome by a mechanism that remains insensitive to phenyl-N-butylnitrone. The drastically distinct effects of 6OH-DOPA and MPP(+) on alpha-synuclein function are discussed with respect to Parkinson disease pathology and animal models mimicking this pathology.