Ectodomain shedding of PLA2R1 is mediated by the metalloproteases ADAM10 and ADAM17.

Phospholipase A2 receptor 1 (PLA2R1) is a 180-kDa transmembrane protein that plays a role in inflammation and cancer, and is the major autoantigen in membranous nephropathy (MN), a rare but severe autoimmune kidney disease. A soluble form of PLA2R1 has been detected in mouse and human serum. It is likely produced by proteolytic shedding of membrane-bound PLA2R1 but the mechanism is unknown. Here, we show that human PLA2R1 is cleaved by A Disintegrin And Metalloprotease 10 (ADAM10) and ADAM17 in HEK293 cells, mouse embryonic fibroblasts and human podocytes. By combining site-directed mutagenesis and sequencing, we determined the exact cleavage site within the extracellular juxtamembrane stalk of human PLA2R1. Orthologs and paralogs of PLA2R1 are also shed. By using pharmacological inhibitors and genetic approaches with RNA interference and knock-out cellular models, we identified a major role of ADAM10 in the constitutive shedding of PLA2R1, and a dual role of ADAM10 and ADAM17 in the stimulated shedding. We did not observe evidence for cleavage by ß- or γ-secretase, suggesting that PLA2R1 may not be a substrate for Regulated Intramembrane Proteolysis. PLA2R1 shedding occurs constitutively and can be triggered by the calcium ionophore ionomycin, the protein kinase C inducer PMA, cytokines and lipopolysaccharides, in vitro and in vivo. Altogether, our results show that PLA2R1 is a novel substrate for ADAM10 and ADAM17, producing a soluble form that is increased in inflammatory conditions and likely exerts various functions in physiological and pathophysiological conditions including inflammation, cancer and MN.

The η-secretase-derived APP fragment ηCTF is localized in Golgi, endosomes and extracellular vesicles and contributes to Aß production.

The processing of the amyloid precursor protein (APP) is one of the key events contributing to Alzheimer's disease (AD) etiology. Canonical cleavages by ß- and γ-secretases lead to Aß production which accumulate in amyloid plaques. Recently, the matrix metalloprotease MT5-MMP, referred to as η-secretase, has been identified as a novel APP cleaving enzyme producing a transmembrane fragment, ηCTF that undergoes subsequent cleavages by α- and ß-secretases yielding the Aηα and AÎ·ß peptides, respectively. The functions and contributions of ηCTF and its related fragments to AD pathology are poorly understood. In this study, we designed a novel immunological probe referred to as ηCTF-NTer antibody that specifically interacts with the N-terminal part of ηCTF targeting ηCTF, Aηα, AÎ·ß but not C99, C83 and Aß. We examined the fate and localization of ηCTF fragment in various cell models and in mice. We found that overexpressed ηCTF undergoes degradation in the proteasomal and autophagic pathways and accumulates mainly in the Golgi and in endosomes. Moreover, we observed the presence of ηCTF in small extracellular vesicles purified from neuroblastoma cells or from mouse brains expressing ηCTF. Importantly, the expression of ηCTF in fibroblasts devoid on APP leads to Aß production demonstrating its contribution to the amyloidogenic pathway. Finally, we observed an ηCTF-like immunoreactivity around amyloid plaques and an age-dependent accumulation of ηCTF in the triple-transgenic mouse AD model. Thus, our study suggests that the ηCTF fragment likely contributes to AD pathology by its exosomal spreading and involvement in Aß production.

Is γ-secretase a beneficial inactivating enzyme of the toxic APP C-terminal fragment C99?

Genetic, biochemical, and anatomical grounds led to the proposal of the amyloid cascade hypothesis centered on the accumulation of amyloid beta peptides (Aß) to explain Alzheimer's disease (AD) etiology. In this context, a bulk of efforts have aimed at developing therapeutic strategies seeking to reduce Aß levels, either by blocking its production (γ- and ß-secretase inhibitors) or by neutralizing it once formed (Aß-directed immunotherapies). However, so far the vast majority of, if not all, clinical trials based on these strategies have failed, since they have not been able to restore cognitive function in AD patients, and even in many cases, they have worsened the clinical picture. We here propose that AD could be more complex than a simple Aß-linked pathology and discuss the possibility that a way to reconcile undoubted genetic evidences linking processing of APP to AD and a consistent failure of Aß-based clinical trials could be to envision the pathological contribution of the direct precursor of Aß, the ß-secretase-derived C-terminal fragment of APP, ßCTF, also referred to as C99. In this review, we summarize scientific evidences pointing to C99 as an early contributor to AD and postulate that γ-secretase should be considered as not only an Aß-generating protease, but also a beneficial C99-inactivating enzyme. In that sense, we discuss the limitations of molecules targeting γ-secretase and propose alternative strategies seeking to reduce C99 levels by other means and notably by enhancing its lysosomal degradation.

Accumulation of amyloid precursor protein C-terminal fragments triggers mitochondrial structure, function, and mitophagy defects in Alzheimer's disease models and human brains.

Several lines of recent evidence indicate that the amyloid precursor protein-derived C-terminal fragments (APP-CTFs) could correspond to an etiological trigger of Alzheimer's disease (AD) pathology. Altered mitochondrial homeostasis is considered an early event in AD development. However, the specific contribution of APP-CTFs to mitochondrial structure, function, and mitophagy defects remains to be established. Here, we demonstrate in neuroblastoma SH-SY5Y cells expressing either APP Swedish mutations, or the ß-secretase-derived APP-CTF fragment (C99) combined with ß- and γ-secretase inhibition, that APP-CTFs accumulation independently of Aß triggers excessive mitochondrial morphology alteration (i.e., size alteration and cristae disorganization) associated with enhanced mitochondrial reactive oxygen species production. APP-CTFs accumulation also elicit basal mitophagy failure illustrated by enhanced conversion of LC3, accumulation of LC3-I and/or LC3-II, non-degradation of SQSTM1/p62, inconsistent Parkin and PINK1 recruitment to mitochondria, enhanced levels of membrane and matrix mitochondrial proteins, and deficient fusion of mitochondria with lysosomes. We confirm the contribution of APP-CTFs accumulation to morphological mitochondria alteration and impaired basal mitophagy in vivo in young 3xTgAD transgenic mice treated with γ-secretase inhibitor as well as in adeno-associated-virus-C99 injected mice. Comparison of aged 2xTgAD and 3xTgAD mice indicates that, besides APP-CTFs, an additional contribution of Aß to late-stage mitophagy activation occurs. Importantly, we report on mitochondrial accumulation of APP-CTFs in human post-mortem sporadic AD brains correlating with mitophagy failure molecular signature. Since defective mitochondria homeostasis plays a pivotal role in AD pathogenesis, targeting mitochondrial dysfunctions and/or mitophagy by counteracting early APP-CTFs accumulation may represent relevant therapeutic interventions in AD.

Intraneuronal aggregation of the ß-CTF fragment of APP (C99) induces Aß-independent lysosomal-autophagic pathology.

Endosomal-autophagic-lysosomal (EAL) dysfunction is an early and prominent neuropathological feature of Alzheimers's disease, yet the exact molecular mechanisms contributing to this pathology remain undefined. By combined biochemical, immunohistochemical and ultrastructural approaches, we demonstrate a link between EAL pathology and the intraneuronal accumulation of the ß-secretase-derived ßAPP fragment (C99) in two in vivo models, 3xTgAD mice and adeno-associated viral-mediated C99-infected mice. We present a pathological loop in which the accumulation of C99 is both the effect and causality of impaired lysosomal-autophagic function. The deleterious effect of C99 was found to be linked to its aggregation within EAL-vesicle membranes leading to disrupted lysosomal proteolysis and autophagic impairment. This effect was Aß independent and was even exacerbated when γ-secretase was pharmacologically inhibited. No effect was observed in inhibitor-treated wild-type animals suggesting that lysosomal dysfunction was indeed directly linked to C99 accumulation. In some brain areas, strong C99 expression also led to inflammatory responses and synaptic dysfunction. Taken together, this work demonstrates a toxic effect of C99 which could underlie some of the early-stage anatomical hallmarks of Alzheimer's disease pathology. Our work also proposes molecular mechanisms likely explaining some of the unfavorable side-effects associated with γ-secretase inhibitor-directed therapies.

The neuronal sortilin-related receptor SORL1 is genetically associated with Alzheimer disease.

The recycling of the amyloid precursor protein (APP) from the cell surface via the endocytic pathways plays a key role in the generation of amyloid beta peptide (Abeta) in Alzheimer disease. We report here that inherited variants in the SORL1 neuronal sorting receptor are associated with late-onset Alzheimer disease. These variants, which occur in at least two different clusters of intronic sequences within the SORL1 gene (also known as LR11 or SORLA) may regulate tissue-specific expression of SORL1. We also show that SORL1 directs trafficking of APP into recycling pathways and that when SORL1 is underexpressed, APP is sorted into Abeta-generating compartments. These data suggest that inherited or acquired changes in SORL1 expression or function are mechanistically involved in causing Alzheimer disease.

The amyloid precursor protein and its derived fragments concomitantly contribute to the alterations of mitochondrial transport machinery in Alzheimer's disease.

Mitochondria dysfunctions and mitophagy failure have been associated with several Alzheimer's disease (AD) related molecular actors including amyloid beta (Aß) and recently the amyloid precursor protein-C terminal fragments (APP-CTFs). The efficacy of the mitophagy process in neurons relies on regulated mitochondrial transport along axons involving a complex molecular machinery. The contribution of the amyloid precursor protein (APP) and its derived fragments to the mitochondrial transport machinery alterations in AD have not been investigated before. We report herein a change of the expression of mitochondrial transport proteins (SNPH and Miro1), motor adapters (TRANK1 and TRAK2), and components of the dynein and kinesin motors (i.e., IC1,2 and Kif5 (A, B, C) isoforms) by endogenous APP and by overexpression of APP carrying the familial Swedish mutation (APPswe). We show that APP-CTFs and Aß concomitantly regulate the expression of a set of transport proteins as demonstrated in APPswe cells treated with ß- and γ-secretase inhibitors and in cells Knock-down for presenilin 1 and 2. We further report the impact of APP-CTFs on the expression of transport proteins in AAV-injected C99 mice brains. Our data also indicate that both Aß oligomers (Aßo) and APP-CTFs impair the colocalization of mitochondria and transport proteins. This has been demonstrated in differentiated SH-SY5Y naive cells treated with Aßo and in differentiated SH-SY5Y and murine primary neurons expressing APPswe and treated with the γ-secretase inhibitor. Importantly, we uncover that the expression of a set of transport proteins is modulated in a disease-dependent manner in 3xTgAD mice and in human sporadic AD brains. This study highlights molecular mechanisms underlying mitochondrial transport defects in AD that likely contribute to mitophagy failure and disease progression.

The ß-secretase-derived C-terminal fragment of ßAPP, C99, but not Aß, is a key contributor to early intraneuronal lesions in triple-transgenic mouse hippocampus.

Triple-transgenic mice (3xTgAD) overexpressing Swedish-mutated ß-amyloid precursor protein (ßAPP(swe)), P310L-Tau (Tau(P301L)), and physiological levels of M146V-presenilin-1 (PS1(M146V)) display extracellular amyloid-ß peptides (Aß) deposits and Tau tangles. More disputed is the observation that these mice accumulate intraneuronal Aß 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 ß-secretase-derived ßAPP fragment C99 that is observed from 3 months of age and enhanced by pharmacological blockade of γ-secretase. Notably, intracellular Aß is only detectable several months later and appears, as is the case of C99, in enlarged cathepsin B-positive structures, while extracellular Aß deposits are detected ~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. Furthermore, the comparison of 3xTgAD mice with double-transgenic mice bearing the ßAPP(swe) and Tau(P301L) mutations but expressing endogenous PS1 (2xTgAD) demonstrate that C99 accumulation is not accounted for by a loss of function triggered by PS1 mutation that would have prevented C99 secondary cleavage by γ-secretase. Together, our work identifies C99 as the earliest ßAPP catabolite and main contributor to the intracellular ßAPP-related immunoreactivity in 3xTgAD mice, suggesting its implication as an initiator of the neurodegenerative process and cognitive alterations taking place in this mouse model.

The physiology of the ß-amyloid precursor protein intracellular domain AICD.

The amyloid-ß precursor protein (ßAPP) undergoes several cleavages by enzymatic activities called secretases. Numerous studies aimed at studying the biogenesis and catabolic fate of Aß peptides, the proteinaceous component of the senile plaques that accumulate in Alzheimer's disease-affected brains. Relatively recently, another secretase-mediated ß-APP-derived catabolite called APP IntraCellular Domain (AICD) entered the game. Whether AICD corresponded to a biologically inert by-pass product of ßAPP processing or whether it could harbor its own function remained questionable. In this study, we review the mechanisms by which AICD is generated and how its production is regulated. Furthermore, we discuss the degradation mechanism underlying its rapid catabolic fate. Finally, we review putative AICD-related functions and more particularly, the numerous studies indicating that AICD could translocate to the nucleus and control at a transcriptional level, the expression of a series of proteins involved in various functions including the control of cell death and Aß degradation.

TMP21 is a presenilin complex component that modulates gamma-secretase but not epsilon-secretase activity.

The presenilin proteins (PS1 and PS2) and their interacting partners nicastrin, aph-1 (refs 4, 5) and pen-2 (ref. 5) form a series of high-molecular-mass, membrane-bound protein complexes that are necessary for gamma-secretase and epsilon-secretase cleavage of selected type 1 transmembrane proteins, including the amyloid precursor protein, Notch and cadherins. Modest cleavage activity can be generated by reconstituting these four proteins in yeast and Spodoptera frugiperda (sf9) cells. However, a critical but unanswered question about the biology of the presenilin complexes is how their activity is modulated in terms of substrate specificity and/or relative activities at the gamma and epsilon sites. A corollary to this question is whether additional proteins in the presenilin complexes might subsume these putative regulatory functions. The hypothesis that additional proteins might exist in the presenilin complexes is supported by the fact that enzymatically active complexes have a mass that is much greater than predicted for a 1:1:1:1 stoichiometric complex (at least 650 kDa observed, compared with about 220 kDa predicted). To address these questions we undertook a search for presenilin-interacting proteins that differentially affected gamma- and epsilon-site cleavage events. Here we report that TMP21, a member of the p24 cargo protein family, is a component of presenilin complexes and differentially regulates gamma-secretase cleavage without affecting epsilon-secretase activity.

p53-dependent control of transactivation of the Pen2 promoter by presenilins.

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.

TMP21 transmembrane domain regulates gamma-secretase cleavage.

TMP21 has been shown to be associated with the gamma-secretase complex and can specifically regulate gamma-cleavage without affecting epsilon-mediated proteolysis. To explore the basis of this activity, TMP21 modulation of gamma-secretase activity was investigated independent of epsilon-cleavage using an amyloid-beta precursor proteinepsilon (APPepsilon) construct which lacks the amyloid intracellular domain domain. The APPepsilon construct behaves similarly to the full-length precursor protein with respect to alpha- and beta-cleavages and is able to undergo normal gamma-processing. Co-expression of APPepsilon and TMP21 resulted in the accumulation of membrane-embedded higher molecular weight Abeta-positive fragments, consistent with an inhibition of gamma-secretase cleavage. The APPepsilon system was used to examine the functional domains of TMP21 through the investigation of a series of TMP21-p24a chimera proteins. It was found that chimeras containing the transmembrane domain bound to the gamma-secretase complex and could decrease gamma-secretase proteolytic processing. This was confirmed though investigation of a synthetic peptide corresponding to the TMP21 transmembrane helix. The isolated TMP21 TM peptide but not the homologous p24a domain was able to reduce Abeta production in a dose-dependent fashion. These observations suggest that the TMP21 transmembrane domain promotes its association with the presenilin complex that results in decreased gamma-cleavage activity.

p53 is regulated by and regulates members of the gamma-secretase complex.

Amyloid beta-peptides is the generic term for a set of hydrophobic peptides that accumulate in Alzheimer's disease (AD)-affected brains. These amyloid-beta peptide fragments are mainly generated by an enzymatic machinery referred to as gamma-secretase complex that is built up by the association of four distinct proteins, namely presenilin 1 (PS1) or PS2, nicastrin, Aph-1 and Pen-2. AD is also characterized by exacerbated cell death that appears linked to the tumor suppressor p53. Interestingly, all members of the gamma-secretase complex control p53-dependent cell death. On the other hand, p53 appears to be able to regulate directly or indirectly the expression and transcription of PS1, PS2 and Pen-2. This review will focus on the functional cross-talk between the members of the gamma-secretase complex and p53 and will discuss the putative implication of this oncogene in AD pathology.

The Transcription Factor EB Reduces the Intraneuronal Accumulation of the Beta-Secretase-Derived APP Fragment C99 in Cellular and Mouse Alzheimer's Disease Models.

: Brains that are affected by Alzheimer's disease (AD) are characterized by the overload of extracellular amyloid ß (Aß) peptides, but recent data from cellular and animal models propose that Aß deposition is preceded by intraneuronal accumulation of the direct precursor of Aß, C99. These studies indicate that C99 accumulation firstly occurs within endosomal and lysosomal compartments and that it contributes to early-stage AD-related endosomal-lysosomal-autophagic defects. Our previous work also suggests that C99 accumulation itself could be a consequence of defective lysosomal-autophagic degradation. Thus, in the present study, we analyzed the influence of the overexpression of the transcription factor EB (TFEB), a master regulator of autophagy and lysosome biogenesis, on C99 accumulation occurring in both AD cellular models and in the triple-transgenic mouse model (3xTgAD). In the in vivo experiments, TFEB overexpression was induced via adeno-associated viruses (AAVs), which were injected either into the cerebral ventricles of newborn mice or administrated at later stages (3 months of age) by stereotaxic injection into the subiculum. In both cells and the 3xTgAD mouse model, exogenous TFEB strongly reduced C99 load and concomitantly increased the levels of many lysosomal and autophagic proteins, including cathepsins, key proteases involved in C99 degradation. Our data indicate that TFEB activation is a relevant strategy to prevent the accumulation of this early neurotoxic catabolite.

Palmitate Is Increased in the Cerebrospinal Fluid of Humans with Obesity and Induces Memory Impairment in Mice via Pro-inflammatory TNF-α.

Obesity has been associated with cognitive decline, atrophy of brain regions related to learning and memory, and higher risk of developing dementia. However, the molecular mechanisms underlying these neurological alterations are still largely unknown. Here, we investigate the effects of palmitate, a saturated fatty acid present at high amounts in fat-rich diets, in the brain. Palmitate is increased in the cerebrospinal fluid (CSF) of overweight and obese patients with amnestic mild cognitive impairment. In mice, intracerebroventricular infusion of palmitate impairs synaptic plasticity and memory. Palmitate induces astroglial and microglial activation in the mouse hippocampus, and its deleterious impact is mediated by microglia-derived tumor necrosis factor alpha (TNF-α) signaling. Our results establish that obesity is associated with increases in CSF palmitate. By defining a pro-inflammatory mechanism by which abnormal levels of palmitate in the brain impair memory, the results further suggest that anti-inflammatory strategies may attenuate memory impairment in obesity.

Presenilin-dependent transcriptional control of the Abeta-degrading enzyme neprilysin by intracellular domains of betaAPP and APLP.

Amyloid beta-peptide (Abeta), which plays a central role in Alzheimer's disease, is generated by presenilin-dependent gamma-secretase cleavage of beta-amyloid precursor protein (betaAPP). We report that the presenilins (PS1 and PS2) also regulate Abeta degradation. Presenilin-deficient cells fail to degrade Abeta and have drastic reductions in the transcription, expression, and activity of neprilysin, a key Abeta-degrading enzyme. Neprilysin activity and expression are also lowered by gamma-secretase inhibitors and by PS1/PS2 deficiency in mouse brain. Neprilysin activity is restored by transient expression of PS1 or PS2 and by expression of the amyloid intracellular domain (AICD), which is cogenerated with Abeta, during gamma-secretase cleavage of betaAPP. Neprilysin gene promoters are transactivated by AICDs from APP-like proteins (APP, APLP1, and APLP2), but not by Abeta or by the gamma-secretase cleavage products of Notch, N- or E- cadherins. The presenilin-dependent regulation of neprilysin, mediated by AICDs, provides a physiological means to modulate Abeta levels with varying levels of gamma-secretase activity.

p53-dependent control of cell death by nicastrin: lack of requirement for presenilin-dependent gamma-secretase complex.

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.

Targeting γ-secretase triggers the selective enrichment of oligomeric APP-CTFs in brain extracellular vesicles from Alzheimer cell and mouse models.

BACKGROUND: We recently demonstrated an endolysosomal accumulation of the ß-secretase-derived APP C-terminal fragment (CTF) C99 in brains of Alzheimer disease (AD) mouse models. Moreover, we showed that the treatment with the γ-secretase inhibitor (D6) led to further increased endolysosomal APP-CTF levels, but also revealed extracellular APP-CTF-associated immunostaining. We here hypothesized that this latter staining could reflect extracellular vesicle (EV)-associated APP-CTFs and aimed to characterize these γ-secretase inhibitor-induced APP-CTFs. METHODS: EVs were purified from cell media or mouse brains from vehicle- or D6-treated C99 or APPswedish expressing cells/mice and analyzed for APP-CTFs by immunoblot. Combined pharmacological, immunological and genetic approaches (presenilin invalidation and C99 dimerization mutants (GXXXG)) were used to characterize vesicle-containing APP-CTFs. Subcellular APP-CTF localization was determined by immunocytochemistry. RESULTS: Purified EVs from both AD cell or mouse models were enriched in APP-CTFs as compared to EVs from control cells/brains. Surprisingly, EVs from D6-treated cells not only displayed increased C99 and C99-derived C83 levels but also higher molecular weight (HMW) APP-CTF-immunoreactivities that were hardly detectable in whole cell extracts. Accordingly, the intracellular levels of HMW APP-CTFs were amplified by the exosomal inhibitor GW4869. By combined pharmacological, immunological and genetic approaches, we established that these HMW APP-CTFs correspond to oligomeric APP-CTFs composed of C99 and/or C83. Immunocytochemical analysis showed that monomers were localized mainly to the trans-Golgi network, whereas oligomers were confined to endosomes and lysosomes, thus providing an anatomical support for the selective recovery of HMW APP-CTFs in EVs. The D6-induced APP-CTF oligomerization and subcellular mislocalization was indeed due to γ-secretase blockade, since it similarly occurred in presenilin-deficient fibroblasts. Further, our data proposed that besides favoring APP-CTF oligomerization by preventing C99 proteolysis, γ-secretase inhibiton also led to a defective SorLA-mediated retrograde transport of HMW APP-CTFs from endosomal compartments to the TGN. CONCLUSIONS: This is the first study to demonstrate the presence of oligomeric APP-CTFs in AD mouse models, the levels of which are selectively enriched in endolysosomal compartments including exosomes and amplified by γ-secretase inhibition. Future studies should evaluate the putative contribution of these exosome-associated APP-CTFs in AD onset, progression and spreading.

Intraneuronal accumulation of C99 contributes to synaptic alterations, apathy-like behavior, and spatial learning deficits in 3×TgAD and 2×TgAD mice.

The triple transgenic mouse model (3×TgAD: APPswe, TauP301L, PS1M146V) recapitulates both amyloid ß (Aß)- and tau-related lesions as well as synaptic and memory deficits. In these mice, we reported an early apathy-like behavior and alterations in synaptic plasticity appearing concomitantly with intraneuronal accumulation of C99 in the subiculum. To delineate the genuine contribution of C99 on the above phenotypes, we generated double transgenic mice (2×TgAD: APPswe, TauP301L) that accumulate C99 without Aß deposition or hyperphosphorylation of tau and compared them to 3×TgAD mice. Here, we show that both TgAD mice display similar decreases in long-term potentiation and in spontaneous locomotor activity measured by actimetry suggesting that the synaptic alterations and the apathy-like behavior were likely linked to C99 rather than Aß. However, spatial learning alterations, assessed by the Morris water maze task, are more pronounced in 3×TgAD than in 2×TgAD, suggesting that both Aß and C99 contribute to defects in the acquisition of spatial information. Finally, even if similar results are observed in males, cognitive and non-cognitive deficits are more severe in females.