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

Open AccessPublished:February 28, 2021DOI:https://doi.org/10.1016/j.jbc.2021.100489
      Genetic, biochemical, and anatomical grounds led to the proposal of the amyloid cascade hypothesis centered on the accumulation of amyloid beta peptides (Aβ) to explain Alzheimer's disease (AD) etiology. In this context, a bulk of efforts have aimed at developing therapeutic strategies seeking to reduce Aβ levels, either by blocking its production (γ- and β-secretase inhibitors) or by neutralizing it once formed (Aβ-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 Aβ-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 Aβ-based clinical trials could be to envision the pathological contribution of the direct precursor of Aβ, the β-secretase-derived C-terminal fragment of APP, βCTF, 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 Aβ-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.

      Keywords

      Abbreviations:

      (amyloid β), AD (Alzheimer's disease), AICD (APP intracellular domain), APH (anterior pharynx defective), BACE (β-site APP cleaving enzyme), βAPP (amyloid precursor protein), CSF (cerebrospinal fluid), CTF (C-terminal fragment), EAL (endosomal–lysosomal–autophagy), ER (endoplasmic reticulum), FAD (familial Alzheimer's disease), GSM (γ-secretase modulators), LTP and LTD (long-term potentiation and depression), MAM (mitochondrial membrane associated), NCT (nicastrin), PEN (PS enhancer), PS (presenilin), ROS (reactive oxygen species), SAD (sporadic Alzheimer's disease), TFEB (transcriptional factor EB)
      Alzheimer's disease (AD) is the most frequent age-related neurodegenerative disease. After initial clinical characterization, histopathological analysis revealed the presence of two major anatomical lesions signing this pathology: senile plaques that are extracellular protein aggregates and neurofibrillary tangles that are intracellular neuronal lesions (
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      Aging and Alzheimer's disease pathology.
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      Alzheimer's disease: Initial report of the purification and characterization of a novel cerebrovascular amyloid protein.
      ) and neurofibrillary tangles of hyperphosphorylated and cleaved forms of the microtubule-associated protein tau (
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      ). The cloning of the Aβ precursor, the β-amyloid precursor protein (APP), in the late 80s was a key step in the understanding of the pathology. APP was found to be localized on chromosome 21 (
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      ), thus explaining the development of early-onset dementia and the presence of senile plaques in Down syndrome patients carrying an extra copy of APP due to a duplication of this chromosome (
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      Down syndrome and beta-amyloid deposition.
      ). Furthermore, the identification of APP mutations responsible for autosomal dominant form of AD (FAD), including the “Dutch” (
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      Seggregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease.
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      ), showed that the functional consequences of these mutations were to augment the load of Aβ and/or to shift Aβ production to more aggregating Aβ peptides (
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      Mutation of the b-amyloid precursor protein in familial Alzheimer's disease increases b-protein production.
      ,
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      • Golde T.E.
      • Younkin S.G.
      Release of excess amyloid b protein from a mutant amyloid b protein precursor.
      ). The role of Aβ in AD etiology was further confirmed by the discovery few years later of the first mutations in presenilins that were found to be involved in Aβ production and, similarly to APP mutations, seemed to exacerbate Aβ accumulation in cells, animal models as well as in AD-affected human brains (
      • Wolfe M.S.
      Unraveling the complexity of gamma-secretase.
      ,
      • Escamilla-Ayala A.
      • Wouters R.
      • Sannerud R.
      • Annaert W.
      Contribution of the Presenilins in the cell biology, structure and function of gamma-secretase.
      ,
      • Lleo A.
      • Berezovska O.
      • Growdon J.H.
      • Hyman B.T.
      Clinical, pathological, and biochemical spectrum of Alzheimer disease associated with PS-1 mutations.
      ). A last, but very important genetic evidence of a key role of Aβ in AD etiology was the recent findings of the Icelandic APP mutation that was shown to be protective by reducing cognitive decline and Aβ load by about 40% (
      • Jonsson T.
      • Atwal J.K.
      • Steinberg S.
      • Snaedal J.
      • Jonsson P.V.
      • Bjornsson S.
      • Stefansson H.
      • Sulem P.
      • Gudbjartsson D.
      • Maloney J.
      • Hoyte K.
      • Gustafson A.
      • Liu Y.
      • Lu Y.
      • Bhangale T.
      • et al.
      A mutation in APP protects against Alzheimer's disease and age-related cognitive decline.
      ).
      Thus, this set of histopathological, genetic, and biochemical data concurred to support the view that Aβ accumulation could be the etiological cause of the pathology, as stated in the amyloid cascade hypothesis that was proposed in 1992 by Hardy and Higgings (
      • Hardy J.A.
      • Higgins G.A.
      Alzheimer's disease: The amyloid cascade hypothesis.
      ). Indeed, mutations in three distinct proteins, namely APP, PS1, and PS2, are all responsible for FAD and have in common to modulate both APP processing and Aβ production. In this context, one can understand the huge efforts aimed at determining the mechanisms and enzymes involved in Aβ production and designing potent, specific, and bioavailable inhibitors of these enzymes (
      • Nie P.
      • Vartak A.
      • Li Y.M.
      gamma-Secretase inhibitors and modulators: Mechanistic insights into the function and regulation of gamma-Secretase.
      ,
      • Moussa-Pacha N.M.
      • Abdin S.M.
      • Omar H.A.
      • Alniss H.
      • Al-Tel T.H.
      BACE1 inhibitors: Current status and future directions in treating Alzheimer's disease.
      ) or Aβ neutralizing antibodies. However, until so far, the outcomes of these Aβ-centered strategies have been extremely disappointing in our quest for meaningful treatments (
      • Huang Y.M.
      • Shen J.
      • Zhao H.L.
      Major clinical trials failed the amyloid hypothesis of Alzheimer's disease.
      ,
      • Panza F.
      • Lozupone M.
      • Logroscino G.
      • Imbimbo B.P.
      A critical appraisal of amyloid-beta-targeting therapies for Alzheimer disease.
      ) (Table 1). This has led to question the validity of the amyloid cascade hypothesis (
      • Herrup K.
      The case for rejecting the amyloid cascade hypothesis.
      ) or to discuss in a more cautious and balanced manner the ins, outs, and limitations of the procedures of clinical trials (
      • Haass C.
      • Levin J.
      [Did Alzheimer research fail entirely? Failure of amyloid-based clinical studies].
      ). It remains that before “throwing out the baby and the bath water,” one should try to reconcile undoubted genetic evidences linking APP processing to AD and failures of Aβ-based clinical trials. In this context, a way to reconcile these observations could be to envisage the possible contribution of other APP-derived fragments distinct from Aβ itself to AD pathology. Indeed, growing evidence proposes that the direct precursor of Aβ (see below), the β-secretase-derived fragment, C99, could be an early and main contributor to AD. Thus, in this review, we address the possibility that the failure of Aβ-centric clinical trials could be explained, at least partly, by their lack of effect on C99. To go further, we describe clues and evidences suggesting that γ-secretase should be considered as a beneficial C99-inactivating enzyme and argument against therapeutic strategies targeting this enzyme. We instead propose alternative strategies seeking to circumvent C99 accumulation, which would then have the advantage to reduce both C99 and Aβ levels.
      Table 1Principal antiamyloid clinical drugs and strategies
      StrategyDrug/specific targetAβ modulation in treated AD patientsFDA statute and participantsSide effects/cognitive readoutReference
      Active immunotherapyAN-1792 (synthetic Aβ42, Janssen)≥ 60–70% Aβ load reduction in the brain (post-mortem immonustaining)Discontinued in 2002 (mild to moderate AD patients)Meningoencephalitis(
      • Nicoll J.A.R.
      • Wilkinson D.
      • Holmes C.
      • Steart P.
      • Markham H.
      • Weller R.O.
      Neuropathology of human Alzheimer disease after immunization with amyloid-b peptide: A case report.
      )

      (
      • Holmes C.
      • Boche D.
      • Wilkinson D.
      • Yadegarfar G.
      • Hopkins V.
      • Bayer A.
      • Jones R.W.
      • Bullock R.
      • Love S.
      • Neal J.W.
      • Zotova E.
      • Nicoll J.A.
      Long-term effects of Abeta42 immunisation in Alzheimer's disease: Follow-up of a randomised, placebo-controlled phase I trial.
      )

      (
      • Boche D.
      • Donald J.
      • Love S.
      • Harris S.
      • Neal J.W.
      • Holmes C.
      • Nicoll J.A.
      Reduction of aggregated Tau in neuronal processes but not in the cell bodies after Abeta42 immunisation in Alzheimer's disease.
      )
      CAD106 (multiple copies of Aβ1-6 peptide, Novartis)1.3% Aβ reduction in brain PET scan (florbetapir)

      2–3-fold increase in plasma Aβ40 at 450 mg
      Discontinued in 2019 (asymptomatic carriers of APOE-4)Worsens cognition, headache, nasopharyngitis, pyrexia, hypertension, back pain…(
      • Vandenberghe R.
      • Riviere M.E.
      • Caputo A.
      • Sovago J.
      • Maguire R.P.
      • Farlow M.
      • Marotta G.
      • Sanchez-Valle R.
      • Scheltens P.
      • Ryan J.M.
      • Graf A.
      Active Abeta immunotherapy CAD106 in Alzheimer's disease: A phase 2b study.
      )
      Passive immunotherapyCrenezumab (monomers, oligomers, and fibrils of Aβ, Roche)≥ 70% Aβ42 increase in CSF at 15 mg/kgPhase II ongoing in asymptomatic carriers of PS mutationsLack of efficacy

      in mild to moderate AD
      (
      • Cummings J.L.
      • Cohen S.
      • van Dyck C.H.
      • Brody M.
      • Curtis C.
      • Cho W.
      • Ward M.
      • Friesenhahn M.
      • Rabe C.
      • Brunstein F.
      • Quartino A.
      • Honigberg L.A.
      • Fuji R.N.
      • Clayton D.
      • Mortensen D.
      • et al.
      Abby: A phase 2 randomized trial of crenezumab in mild to moderate Alzheimer disease.
      )
      Solanezumab (monomeric and soluble forms of Aβ Eli Lilly)170- and 18-fold increase in plasma Aβ40 and Aβ42 respectively no Aβ modulation in brain PET scan (florbetapir) at 400 mgPhase III ongoing in asymptomatic people who have biomarker evidence of brain amyloid depositionLack of efficacy

      in mild to moderate AD

      and in asymptomatic carriers of APP and PS mutations
      (
      • Honig L.S.
      • Vellas B.
      • Woodward M.
      • Boada M.
      • Bullock R.
      • Borrie M.
      • Hager K.
      • Andreasen N.
      • Scarpini E.
      • Liu-Seifert H.
      • Case M.
      • Dean R.A.
      • Hake A.
      • Sundell K.
      • Poole Hoffmann V.
      • et al.
      Trial of solanezumab for mild dementia due to Alzheimer's disease.
      )
      Aducanumab (oligomers, and fibrils of Aβ, Biogen)80% Aβ reduction in brain PET scan (florbetapir) at 10 mg/kgPhase III ongoing in an open-label extension study in mild to moderate AD patientsOne of the two trials (EMERGE) was positive with a significant reduction in cognitive decline in mild to moderate AD a biologics license application was submitted to the FDA for approval on July 2020www.alzforum.org/therapeutics/aducanumab
      Gantenerumab (oligomers, and fibrils of Aβ, Roche)≥ 15%, 35% and 78% Aβ reduction in brain PET scan (florbetapir) at 60, 200 and 1200 mg respectivelyPhase III ongoing in an open-label extension study in mild to moderate AD patients (SCarlet RoAD, Marguerite RoAD, and GRADUATE) and in asymptomatic carriers of APP and PS mutations (Dian-Tu)A directional trend for slower clinical decline in mild to moderate AD(
      • Ostrowitzki S.
      • Deptula D.
      • Thurfjell L.
      • Barkhof F.
      • Bohrmann B.
      • Brooks D.J.
      • Klunk W.E.
      • Ashford E.
      • Yoo K.
      • Xu Z.X.
      • Loetscher H.
      • Santarelli L.
      Mechanism of amyloid removal in patients with Alzheimer disease treated with gantenerumab.
      )

      (
      • Klein G.
      • Delmar P.
      • Voyle N.
      • Rehal S.
      • Hofmann C.
      • Abi-Saab D.
      • Andjelkovic M.
      • Ristic S.
      • Wang G.
      • Bateman R.
      • Kerchner G.A.
      • Baudler M.
      • Fontoura P.
      • Doody R.
      Gantenerumab reduces amyloid-beta plaques in patients with prodromal to moderate Alzheimer's disease: A PET substudy interim analysis.
      )
      BAN2401 (soluble Aβ protofibrils, Biogen)≥ 120% Aβ40 increase in plasma

      300 fold Aβ42 increase in CSF

      93% Aβ reduction in brain PET scan (florbetapir) at 10 mg/kg
      Phase III ongoing in early symptomatic AD patients (Clarity AD) and in asymptomatic people who have biomarker evidence of brain amyloid deposition (AHEAD 3–45)47% and 30% reduction in cognitive decline as judged by the ADAS-Cog and the ADCOMS respetively(
      • Logovinsky V.
      • Satlin A.
      • Lai R.
      • Swanson C.
      • Kaplow J.
      • Osswald G.
      • Basun H.
      • Lannfelt L.
      Safety and tolerability of BAN2401--a clinical study in Alzheimer's disease with a protofibril selective Abeta antibody.
      )

      www.alzforum.org/therapeutics/ban2401
      β-secretase inhibitorsVerubecestat (MK-8931Merck)≥ 57–84% Aβ reduction in CSF at 12–60 mgDiscontinued in 2018 (prodromal, mild to moderate AD patients)Worsens cognition, anxiety, depression, and sleep problems(
      • Kennedy M.E.
      • Stamford A.W.
      • Chen X.
      • Cox K.
      • Cumming J.N.
      • Dockendorf M.F.
      • Egan M.
      • Ereshefsky L.
      • Hodgson R.A.
      • Hyde L.A.
      • Jhee S.
      • Kleijn H.J.
      • Kuvelkar R.
      • Li W.
      • Mattson B.A.
      • et al.
      The BACE1 inhibitor verubecestat (MK-8931) reduces CNS beta-amyloid in animal models and in Alzheimer's disease patients.
      )

      (
      • Egan M.F.
      • Kost J.
      • Voss T.
      • Mukai Y.
      • Aisen P.S.
      • Cummings J.L.
      • Tariot P.N.
      • Vellas B.
      • van Dyck C.H.
      • Boada M.
      • Zhang Y.
      • Li W.
      • Furtek C.
      • Mahoney E.
      • Harper Mozley L.
      • et al.
      Randomized trial of verubecestat for prodromal Alzheimer's disease.
      )
      Atabecestat (Janssen)≥ 67–90% Aβ reduction in CSF at 10–50 mgDiscontinued in 2018 (asymptomatic people)Worsens cognition, elevated liver enzymes, depression, anxiety, and sleep problems(
      • Timmers M.
      • Streffer J.R.
      • Russu A.
      • Tominaga Y.
      • Shimizu H.
      • Shiraishi A.
      • Tatikola K.
      • Smekens P.
      • Borjesson-Hanson A.
      • Andreasen N.
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      • Boada M.
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      • et al.
      Pharmacodynamics of atabecestat (JNJ-54861911), an oral BACE1 inhibitor in patients with early Alzheimer's disease: Randomized, double-blind, placebo-controlled study.
      )

      (
      • Henley D.
      • Raghavan N.
      • Sperling R.
      • Aisen P.
      • Raman R.
      • Romano G.
      Preliminary results of a trial of atabecestat in preclinical Alzheimer's disease.
      )
      Lanabecestat (AZD3293,Eli Lilly)≥64% to 78% Aβ reduction in plasma at 15–50 mg

      ≥51% to 76% Aβ reduction in CSF at 15–50 mg
      Discontinued in 2018 (prodromal and mild AD patients)Lack of efficacy on cognition, neuropsychiatric adverse events, weight loss, hair color changes(
      • Cebers G.
      • Alexander R.C.
      • Haeberlein S.B.
      • Han D.
      • Goldwater R.
      • Ereshefsky L.
      • Olsson T.
      • Ye N.
      • Rosen L.
      • Russell M.
      • Maltby J.
      • Eketjall S.
      • Kugler A.R.
      AZD3293: Pharmacokinetic and pharmacodynamic effects in healthy subjects and patients with Alzheimer's disease.
      )

      (
      • Wessels A.M.
      • Tariot P.N.
      • Zimmer J.A.
      • Selzler K.J.
      • Bragg S.M.
      • Andersen S.W.
      • Landry J.
      • Krull J.H.
      • Downing A.M.
      • Willis B.A.
      • Shcherbinin S.
      • Mullen J.
      • Barker P.
      • Schumi J.
      • Shering C.
      • et al.
      Efficacy and safety of Lanabecestat for treatment of early and mild Alzheimer disease: The AMARANTH and DAYBREAK-ALZ randomized clinical trials.
      )
      Umibecestat (Novartis)≥ 90% Aβ reduction in CSF at 85 mgDiscontinued in 2019 (asymptomatic carriers of APOE-4)Worsens cognition, brain atrophy and weight loss(
      • Neumann U.
      • Ufer M.
      • Jacobson L.H.
      • Rouzade-Dominguez M.L.
      • Huledal G.
      • Kolly C.
      • Luond R.M.
      • Machauer R.
      • Veenstra S.J.
      • Hurth K.
      • Rueeger H.
      • Tintelnot-Blomley M.
      • Staufenbiel M.
      • Shimshek D.R.
      • Perrot L.
      • et al.
      The BACE-1 inhibitor CNP520 for prevention trials in Alzheimer's disease.
      )

      (
      • Lopez Lopez C.
      • Tariot P.N.
      • Caputo A.
      • Langbaum J.B.
      • Liu F.
      • Riviere M.E.
      • Langlois C.
      • Rouzade-Dominguez M.L.
      • Zalesak M.
      • Hendrix S.
      • Thomas R.G.
      • Viglietta V.
      • Lenz R.
      • Ryan J.M.
      • Graf A.
      • et al.
      The Alzheimer's Prevention Initiative Generation Program: Study design of two randomized controlled trials for individuals at risk for clinical onset of Alzheimer's disease.
      ) www.alzforum.org/therapeutics/umibecestat
      Elenbecestat (Biogen)≥5.8% and ≥13.6% Aβ reduction in brain PET scan (florbetaben and florbetapir respectively) at 50 mgDiscontinued in 2019 (prodromal, mild to moderate AD patients)Weight loss, skin rashes and neuropsychiatric adverse events(
      • Lynch S.Y.
      • Kaplow J.
      • Zhao J.
      • Dhadda S.
      • Luthman J.
      • Albala B.
      Elenbecestat, E2609, a Bace inhibitor: Results from a phase-2 study in subjects with mild cognitive impairment and mild-to-moderate dementia due to Alzheimer's disease.
      )

      (
      • Imbimbo B.P.
      • Watling M.
      Investigational BACE inhibitors for the treatment of Alzheimer's disease.
      )
      γ-Secretase inhibitorsSemagacestat (Eli Lilly)≥58.2% to 64.6% Aβ reduction in plasma at 100–140 mg

      ≥47–84% newly Aβ reduction in CSF at 100–280 mg
      Discontinued in 2011 (AD patients)Worsens cognition, skin cancer and infections.(
      • Fleisher A.S.
      • Raman R.
      • Siemers E.R.
      • Becerra L.
      • Clark C.M.
      • Dean R.A.
      • Farlow M.R.
      • Galvin J.E.
      • Peskind E.R.
      • Quinn J.F.
      • Sherzai A.
      • Sowell B.B.
      • Aisen P.S.
      • Thal L.J.
      Phase 2 safety trial targeting amyloid beta production with a gamma-secretase inhibitor in Alzheimer disease.
      )

      (
      • Bateman R.J.
      • Siemers E.R.
      • Mawuenyega K.G.
      • Wen G.
      • Browning K.R.
      • Sigurdson W.C.
      • Yarasheski K.E.
      • Friedrich S.W.
      • Demattos R.B.
      • May P.C.
      • Paul S.M.
      • Holtzman D.M.
      A gamma-secretase inhibitor decreases amyloid-beta production in the central nervous system.
      )

      (
      • Doody R.S.
      • Raman R.
      • Farlow M.
      • Iwatsubo T.
      • Vellas B.
      • Joffe S.
      • Kieburtz K.
      • He F.
      • Sun X.
      • Thomas R.G.
      • Aisen P.S.
      • Siemers E.
      • Sethuraman G.
      • Mohs R.
      Alzheimer's Disease Cooperative Study Steering CommitteeSemagacestat Study Group
      A phase 3 trial of semagacestat for treatment of Alzheimer's disease.
      )
      Avagacestat (Bristol-Myers Squibb)≥40% Aβ reduction in CSF at 125 mgDiscontinued in 2012 (prodromal AD)Worsens cognition, skin cancers, diarrhea, nausea, vomiting, and rash(
      • Coric V.
      • van Dyck C.H.
      • Salloway S.
      • Andreasen N.
      • Brody M.
      • Richter R.W.
      • Soininen H.
      • Thein S.
      • Shiovitz T.
      • Pilcher G.
      • Colby S.
      • Rollin L.
      • Dockens R.
      • Pachai C.
      • Portelius E.
      • et al.
      Safety and tolerability of the gamma-secretase inhibitor avagacestat in a phase 2 study of mild to moderate Alzheimer disease.
      )
      γ-Secretase modulatorsRofecoxib (Merck)Not determinedDiscontinued in 2004 (mild to moderate AD patients)Lack of efficacy, cardiovascular damage(
      • Reines S.
      • Block G.
      • Morris J.
      • Liu G.
      • Nessly M.
      • Lines C.
      • Norman B.
      • Baranak C.
      Rofecoxib: No effect on Alzheimer's disease in a 1-year, randomized, blinded, controlled study.
      )
      Tarenflurbil (Myriad Genetics)No Aβ42 modulation in plasma and CSFDiscontinued in 2009 (mild AD patients)Worsens cognition, dizziness, upper respiratory tract infection and constipation.(
      • Galasko D.R.
      • Graff-Radford N.
      • May S.
      • Hendrix S.
      • Cottrell B.A.
      • Sagi S.A.
      • Mather G.
      • Laughlin M.
      • Zavitz K.H.
      • Swabb E.
      • Golde T.E.
      • Murphy M.P.
      • Koo E.H.
      Safety, tolerability, pharmacokinetics, and Abeta levels after short-term administration of R-flurbiprofen in healthy elderly individuals.
      )

      (
      • Green R.C.
      • Schneider L.S.
      • Amato D.A.
      • Beelen A.P.
      • Wilcock G.
      • Swabb E.A.
      • Zavitz K.H.
      Tarenflurbil Phase 3 Study Group
      Effect of tarenflurbil on cognitive decline and activities of daily living in patients with mild Alzheimer disease: A randomized controlled trial.
      )
      Naproxen (Procter & Gamble)No Aβ42 modulations in CSFDiscontinued in 2019 (asymptomatic carriers of APP and PS mutations)Lack of efficacy, hypertension, gastrointestinal, and vascular or cardiac problems(
      • Meyer P.F.
      • Tremblay-Mercier J.
      • Leoutsakos J.
      • Madjar C.
      • Lafaille-Maignan M.E.
      • Savard M.
      • Rosa-Neto P.
      • Poirier J.
      • Etienne P.
      • Breitner J.
      • Group P.-A.R.
      INTREPAD: A randomized trial of naproxen to slow progress of presymptomatic Alzheimer disease.
      )
      ADAS-Cog, Alzheimer's Disease Assessment Scale-Cognitive Subscale; ADCOMS, Alzheimer's Disease Composite Score; CSF, cerebrospinal fluid; FDA, US Food and Drug Administration; MCI, mild cognitive impairment.

      APP processing and APP mutations

      Aβ is derived from APP that undergoes sequential limited proteolysis catalyzed by proteases called “secretases” (Fig. 1A) (
      • Checler F.
      Processing of the b-amyloid precursor protein and its regulation in Alzheimer's disease.
      ). In the nonamyloidogenic pathway, cleavage by α-secretase and γ-secretase ends up with the production of three fragments: a large secreted N-terminal fragment (sAPPα), a small soluble peptide p3, and the APP intracellular domain (AICD) (Fig. 1A). In the amyloidogenic pathway, a first cleavage of APP by the β-secretase is followed by γ-secretase cleavage. Again, the cleavage by β-secretase generates a large soluble extracellular secreted domain (sAPPβ) and the remaining membrane stub, the β-secretase-derived fragment, C99, undergoes γ-secretase cleavage, thus liberating Aβ and its C-terminal counterpart AICD (Fig. 1B). Other noncanonical cleavages on APP have also been more recently described (
      • Andrew R.J.
      • Kellett K.A.
      • Thinakaran G.
      • Hooper N.M.
      A Greek tragedy: The growing complexity of Alzheimer amyloid precursor protein proteolysis.
      ). Among them, the ƞ-secretase activity, carried by the matrix metalloproteinases (MT1-MMP and MT5-MMP), cleaves APP in its extracellular domain, thus producing a soluble fragment (sAPPη) and a membrane-bound C-terminal fragment, ηCTF. This latter fragment can subsequently be processed by α- or β-secretase, which will generate Anα and C83 and Anβ and C99, respectively (Fig. 1C) (
      • Willem M.
      • Tahirovic S.
      • Busche M.A.
      • Ovsepian S.V.
      • Chafai M.
      • Kootar S.
      • Hornburg D.
      • Evans L.D.
      • Moore S.
      • Daria A.
      • Hampel H.
      • Muller V.
      • Giudici C.
      • Nuscher B.
      • Wenninger-Weinzierl A.
      • et al.
      eta-Secretase processing of APP inhibits neuronal activity in the hippocampus.
      ,
      • Baranger K.
      • Marchalant Y.
      • Bonnet A.E.
      • Crouzin N.
      • Carrete A.
      • Paumier J.M.
      • Py N.A.
      • Bernard A.
      • Bauer C.
      • Charrat E.
      • Moschke K.
      • Seiki M.
      • Vignes M.
      • Lichtenthaler S.F.
      • Checler F.
      • et al.
      MT5-MMP is a new pro-amyloidogenic proteinase that promotes amyloid pathology and cognitive decline in a transgenic mouse model of Alzheimer's disease.
      ).
      Figure thumbnail gr1
      Figure 1APP metabolism. A, represents the α/γ-secretase pathway in which APP is first cleaved by the α-secretase producing the soluble fragment sAPPα and the membrane-embedded C-terminal fragment, C83 that is then cleaved by γ-secretase to release the cytosolic AICD fragment and p3 peptide, respectively. B, represents the β or β′/γ-secretase pathway in which APP is first cleaved by the β-secretase at β or β′ site to liberate the soluble fragments sAPPβ or sAPPβ’ and the membrane-embedded C99 or C89 fragments, respectively. Then γ-secretase cleavage releases the cytosolic AICD fragment and Aβ peptides (Aβ1-40 or Aβ1-42) or Aβ11–40, respectively. Note that in physiological conditions, β-secretase mainly cleaves at the β’ site, but some FAD mutations shift the cleavage to the β-site to produce more C99. C, represents the ƞ/γ-secretase pathway in which ƞ-secretase cleavage releases the soluble fragment sAPPƞ and the membrane-embedded ƞCTF, which is processed by α- or β-secretase, thus generating Aƞα or Aƞβ peptides, respectively.
      Until so far, 28 pathogenic APP mutations have been identified with all, except the Swedish mutation, lying within the sequence of C99 (www.alzforum.org/mutations). The Swedish variant (APPswe, KM670/671NL), although not located within C99 but two residues upstream, strongly increases the levels of this fragment, as well as those of Aβ peptides, by boosting β-secretase cleavage (
      • Citron M.
      • Oltersdorf T.
      • Haass C.
      • McConlogue L.
      • Hung A.Y.
      • Seubert P.
      • Vigo-Pelfrey C.
      • Lieberburg I.
      • Selkoe D.J.
      Mutation of the b-amyloid precursor protein in familial Alzheimer's disease increases b-protein production.
      ,
      • Cai X.-D.
      • Golde T.E.
      • Younkin S.G.
      Release of excess amyloid b protein from a mutant amyloid b protein precursor.
      ,
      • Citron M.
      • Vigo-Pelfrey C.
      • Teplow D.B.
      • Miller C.
      • D S.
      • Johnston J.
      • Winblad B.
      • Venizelos N.
      • Lannfelt L.
      • Selkoe D.J.
      Excessive production of amyloid -protein by peripheral cells of symptomatic and presymptomatic patients carrying the Swedish familial Alzheimer disease mutation.
      ). All pathogenic APP mutations lie either close to the α-secretase cleavage site (the middle part of the Aβ domain of C99), within the γ-secretase cleavage sites, or near the β-secretase cleavage site (N-terminal part), thus modifying the cleavages of APP by either of these secretases. While the knowledge about the exact effects of the pathogenic APP mutations is still limited, it seems that mutations close to the β-secretase (such as the Swedish and Leuven (E682K) mutations) favor C99 production, those close to the α-secretase site (such as the Flemish [E693Q] or Arctic [E693G] mutations) lead to the generation of particular aggregation-prone Aβ species, while mutations lying within the γ-secretase cleavage site elevate the Aβ42/Aβ40 ratio. In all cases, the development of AD pathology linked to these mutations is believed to be caused by an excessive Aβ deposition (
      • Chavez-Gutierrez L.
      • Szaruga M.
      Mechanisms of neurodegeneration - insights from familial Alzheimer's disease.
      ). Nevertheless, the recent study by Xu et al. (
      • Xu T.H.
      • Yan Y.
      • Kang Y.
      • Jiang Y.
      • Melcher K.
      • Xu H.E.
      Alzheimer's disease-associated mutations increase amyloid precursor protein resistance to gamma-secretase cleavage and the Abeta42/Abeta40 ratio.
      ) showed that most (20 out of 28) APP mutations not only alter Aβ production, but are also less efficiently processed by γ-secretase and thereby also enhance the levels of C99.

      The β-secretase

      The nature of β-secretase was described in 1999 in four independent works and was referred to as BACE1 (β-site APP cleaving enzyme), memapsin 2 or ASP2 (
      • Vassar R.
      • Bennett B.D.
      • Babu-Khan S.
      • Khan S.
      • Mendiaz E.A.
      • Denis P.
      • Teplow D.B.
      • Ross S.
      • Amarante P.
      • Loeloff R.
      • Luo Y.
      • Fisher S.
      • Fuller J.
      • Edenson S.
      • Lile J.
      • et al.
      b-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE.
      ,
      • Sinha S.
      • Anderson J.P.
      • Barbour R.
      • Basi G.S.
      • Caccavello R.
      • Davis D.
      • Doan M.
      • Dovey H.F.
      • Frigon N.
      • Hong J.
      • Jacobson-Croak K.
      • Jewett N.
      • Keim P.
      • Knops J.
      • Lieberburg I.
      • et al.
      Purification and cloning of amyloid precursor protein b-secretase from human brain.
      ,
      • Yan R.
      • Bienkowski M.J.
      • Shuck M.E.
      • Miao H.
      • Tory M.C.
      • Pauley A.M.
      • Brashler J.R.
      • Stratman N.C.
      • Mathews W.R.
      • Buhl A.E.
      • Carter D.B.
      • Tomasselli A.G.
      • Parodi L.A.
      • Heinrikson R.L.
      • Gurney M.E.
      Membrane-anchored aspartyl protease with Alzheimer's disease b-secretase activity.
      ,
      • Hussain I.
      • Powell D.
      • Howlett D.R.
      • Tew D.G.
      • Meek T.D.
      • Chapman C.
      • Gloger I.S.
      • Murphy K.E.
      • Southan C.D.
      • Ryan D.M.
      • Smith T.S.
      • Simmons D.L.
      • Walsh F.S.
      • Dingwall C.
      • Christie G.
      Identification of a novel aspartic protease (Asp2) as b-secretase.
      ). BACE1 is an aspartic protease cleaving mainly within the Golgi and endosomes due to its optimal enzymatic activity at acidic pH (
      • Cole S.L.
      • Vassar R.
      The Alzheimer's disease Beta-secretase enzyme, BACE1.
      ). It cleaves APP at two distinct sites called the β and β′ site, respectively. While only the cleavage on the β-site (Asp1) leads to Aβ production (Fig. 1B), BACE1 is considered to cut APP primarily at the β′-site (Glu11) (Fig. 1B), thus generating the C89 fragment and a truncated nonpathological Aβ peptide (Aβ 11-x) (
      • Cole S.L.
      • Vassar R.
      The Alzheimer's disease Beta-secretase enzyme, BACE1.
      ). Until so far, no mutations have been found in β-secretase, but the Swedish and the Leuven APP mutations are known to shift the cleavage from the β′-site to the β-site, thus leading to a much higher C99 and Aβ production (
      • Citron M.
      • Oltersdorf T.
      • Haass C.
      • McConlogue L.
      • Hung A.Y.
      • Seubert P.
      • Vigo-Pelfrey C.
      • Lieberburg I.
      • Selkoe D.J.
      Mutation of the b-amyloid precursor protein in familial Alzheimer's disease increases b-protein production.
      ,
      • Cai X.-D.
      • Golde T.E.
      • Younkin S.G.
      Release of excess amyloid b protein from a mutant amyloid b protein precursor.
      ,
      • Zhou L.
      • Brouwers N.
      • Benilova I.
      • Vandersteen A.
      • Mercken M.
      • Van Laere K.
      • Van Damme P.
      • Demedts D.
      • Van Leuven F.
      • Sleegers K.
      • Broersen K.
      • Van Broeckhoven C.
      • Vandenberghe R.
      • De Strooper B.
      Amyloid precursor protein mutation E682K at the alternative beta-secretase cleavage beta'-site increases Abeta generation.
      ,
      • Seubert P.
      • Oltersdorf T.
      • Lee M.G.
      • Barbour R.
      • Blomquist C.
      • Davis D.L.
      • Bryant K.
      • Fritz L.C.
      • Galasko D.
      • Thal L.J.
      • Lieberburg I.
      • Schenk D.B.
      Secretion of b-amyloid precursor protein cleaved at the amino terminus of the b-amyloid peptide.
      ). Inversely, the Icelandic mutation (A673T) seems to be protective against AD by decreasing β-secretase cleavage and thus reducing by 40% the amyloidogenic fragments sAPPβ and Aβ (
      • Jonsson T.
      • Atwal J.K.
      • Steinberg S.
      • Snaedal J.
      • Jonsson P.V.
      • Bjornsson S.
      • Stefansson H.
      • Sulem P.
      • Gudbjartsson D.
      • Maloney J.
      • Hoyte K.
      • Gustafson A.
      • Liu Y.
      • Lu Y.
      • Bhangale T.
      • et al.
      A mutation in APP protects against Alzheimer's disease and age-related cognitive decline.
      ,
      • Maloney J.A.
      • Bainbridge T.
      • Gustafson A.
      • Zhang S.
      • Kyauk R.
      • Steiner P.
      • van der Brug M.
      • Liu Y.
      • Ernst J.A.
      • Watts R.J.
      • Atwal J.K.
      Molecular mechanisms of Alzheimer disease protection by the A673T allele of amyloid precursor protein.
      ).

      The γ-secretase and role of PS mutations

      The first hint of the identity of the protease yielding the C-terminal end of Aβ, the γ-secretase, was the discovery in 1995 of presenilin 1 (PS1) (
      • Schellenberg G.D.
      • Bird T.D.
      • Wijsman E.M.
      • Orr H.T.
      • Anderson L.
      • Nemens E.
      • White J.A.
      • Bonnycastle L.
      • Weber J.L.
      • Alonso M.E.
      • Potter H.
      • Heston L.L.
      • Martin G.M.
      Genetic linkage evidence for a familial Alzheimer's disease locus on chromosome 14.
      ,
      • St. George-Hyslop P.
      • Haines J.
      • Rogaev E.
      • Mortilla M.
      • Vaula G.
      • Pericak-Vance M.
      • Foncin J.F.
      • Montesi M.
      • Bruni A.
      • Sorbi S.
      • Rainero I.
      • Pinessi L.
      • Pollen D.
      • Polinsky R.
      • Nee L.
      • et al.
      Genetic evidence for a novel familial Alzheimer's disease locus on chromosome 14.
      ,
      • Van Broeckhoven C.
      • Backhovens H.
      • Cruts M.
      • De Winter G.
      • Bruyland M.
      • Cras P.
      • Martin J.J.
      Mapping of a gene predisposing to early-onset Alzheimer's disease to chromosome 14q24.3.
      ) and its family member presenilin 2 (PS2) (
      • Rogaev E.I.
      • Sherrington R.
      • Rogaeva E.A.
      • Levesque G.
      • Ikeda M.
      • Liang Y.
      • Chi H.
      • Lin C.
      • Holman K.
      • Tsuda T.
      • Mar L.
      • Sorbi S.
      • Nacmias B.
      • Piacentini S.
      • Amaducci L.
      • et al.
      Familial Alzheimer's disease in kindreds with missense mutations in a gene on chromosome 1 related to the Alzheimer's disease type 3 gene.
      ,
      • Sherrington R.
      • Rogaev E.I.
      • Liang Y.
      • Rogaeva E.A.
      • Levesque G.
      • Ikeda M.
      • Chi H.
      • Lin C.
      • Li G.
      • Holman K.
      • Tsuda T.
      • Mar L.
      • Foncin J.F.
      • Bruni A.C.
      • Montesi M.P.
      • et al.
      Cloning of a gene bearing missense mutations in early-onset familial Alzheimer's disease.
      ) proposed to constitute this enzyme (Fig. 2A). The definitive demonstration that presenilins could be involved in γ-secretase activity came from subsequent functional characterization showing that their depletion fully prevents Aβ production (
      • De Strooper B.
      • Saftig P.
      • Craessaerts K.
      • Vanderstichele H.
      • Guhde G.
      • Annaert W.
      • Von Figura K.
      • Van Leuven F.
      Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein.
      ,
      • Herreman A.
      • Serneels L.
      • Annaert W.
      • Collen D.
      • Schoonjans L.
      • De Strooper B.
      Total inactivation of g-secretase activity in presenilin-deficient embryonic stem cells.
      ). However, later studies revealed that γ-secretase is a multiproteic complex built of not only PS1 or PS2 but also of nicastrin, the anterior pharynx defective 1 (Aph1), and presenilin enhancer 2 (PEN2) (
      • Edbauer D.
      • Winkler E.
      • Regula J.T.
      • Pesold B.
      • Steiner H.
      • Haass C.
      Reconstitution of g-secretase activity.
      ,
      • Kimberly W.T.
      • LaVoie M.J.
      • Ostaszewski B.L.
      • Ye W.
      • Wolfe M.S.
      • Selkoe D.J.
      g-secretase is a membrane protein complex comprised of presenilin, nicastrin, aph-1 and pen-2.
      ,
      • Takasugi N.
      • Tomita T.
      • Hayashi I.
      • Tsuruoka M.
      • Niimura M.
      • Takahashi Y.
      • Thinakaran G.
      • Iwatsubo T.
      The role of presenilin cofactors in the g-secretase complex.
      ) (Fig. 2B), but the exact molecular structure of this complex was revealed by crystallography only in 2014 (
      • Lu P.
      • Bai X.C.
      • Ma D.
      • Xie T.
      • Yan C.
      • Sun L.
      • Yang G.
      • Zhao Y.
      • Zhou R.
      • Scheres S.H.W.
      • Shi Y.
      Three-dimensional structure of human gamma-secretase.
      ). Further studies also revealed a very complex cleavage of APP-CTFs by γ-secretase, since it takes place within the membrane and successively at several sites (ɛ, ζ-, γ- and γ′). The first endopeptidase cleavage (ɛ-cleavage) can occur at two distinct sites leading to the generation of Aβ48 and Aβ49, which is then shortened by sequential carboxypeptidase cleavages (trimming) at ζ, γ-and γ′-sites, occurring at each three or four residues and via two distinct product lines (Aβ49 > Aβ46 > Aβ43 > Aβ40 > Aβ37) or (Aβ48 > Aβ45 > Aβ42 > Aβ38) and the final occurrence of peptides of varying lengths but two major species of 40 or 42 amino acid residues, respectively (
      • Wolfe M.S.
      Substrate recognition and processing by gamma-secretase.
      ). Whereas Aβ40 is the most abundant peptide, Aβ42 is considered as the most pathogenic due to its high aggregation propensity.
      Figure thumbnail gr2
      Figure 2γ-secretase. A, displays a schematic representation of presenilins (PS1 or PS2) composed of nine transmembrane domains and harboring the catalytic core of the complex with two aspartyl residues in the transmembrane domains TM-6 and TM-7. During maturation, presenilin undergoes endoproteolysis, and the resulting N-terminal fragment (PS-NTF) and C-terminal fragment (PS-CTF) remain associated. B, represents the multimeric γ-secretase complex composed of four membrane proteins: presenilin (green), nicastrin (blue) composed of a single transmembrane and a large glycosylated N-terminal ectodomain, Aph-1 (purple) composed of seven transmembrane domains, and Pen-2 (pink) composed of two transmembrane domains. The substrate C99 (yellow) interacts with γ-secretase to form an enzyme substrate complex. C, represents the successive proteolysis of C99 transmembrane domain by γ-secretase. First, an endoproteolytic cleavage occurs close to the membrane–cytosol interface at two possible ε sites yielding long Aβ peptides Aβ48 or Aβ49, respectively. Then a carboxypeptidase activity of γ-secretase trims Aβ48 and Aβ49 each 3 or 4 amino acid at successive ζ-, γ, and γ′-sites leading to two Aβ product lines: Aβ49→Aβ46→Aβ43→Aβ40 and Aβ48→Aβ45→Aβ42→ Aβ38, respectively.
      So far, more than 300 mutations in presenilins are proposed to be responsible for early onset and familial forms of AD (FAD) and are now believed to comprise at least 70% of all FAD cases with the vast majority on PS1 (www.alzforum.org/mutations). Although a huge effort has been made to try to understand the pathogenic mechanisms linked to these mutations, their exact functional consequences remain enigmatic and are probably not explained by one common mechanism. Two main, but not exclusive, hypotheses have been proposed and are still hotly debated. First, Hardy and Selkoe proposed, accordingly to the amyloid cascade hypothesis, that the effect of PS mutations should be a gain of function of γ-secretase (
      • Hardy J.
      • Selkoe D.
      The amyloid hypothesis of Alzheimer's disease: Progress and problems on the road to therapeutics.
      ). However, later studies revealed that most PS mutations are actually loss-of-function mutants, and a revised version of this hypothesis became focused on the relative levels of Aβ42 to Aβ40 (Aβ42/Aβ40) rather than absolute increases in Aβ42 (
      • Selkoe D.J.
      • Hardy J.
      The amyloid hypothesis of Alzheimer's disease at 25 years.
      ). It was suggested that partial loss of PS mutations shifts the cleavage specificity of the mutant enzyme to favor Aβ42 production and that even a minor increase in Aβ42 levels could be sufficient for seed formation (
      • Selkoe D.J.
      • Wolfe M.S.
      Presenilin: Running with scissors in the membrane.
      ,
      • Steiner H.
      • Fluhrer R.
      • Haass C.
      Intramembrane proteolysis by gamma-secretase.
      ). In that way, loss of function should be considered as gain of “toxic function” because of a relative increase in more aggregation-prone Aβ (
      • Wolfe M.S.
      When loss is gain: Reduced presenilin proteolytic function leads to increased Abeta42/Abeta40. Talking point on the role of presenilin mutations in Alzheimer disease.
      ). On the other hand, Shen and Kelleher proposed an alternative hypothesis “the presenilin hypothesis,” stating that loss of general PS function should be the trigger of neurodegeneration and defects in cognitive function (
      • Shen J.
      • Kelleher 3rd., R.J.
      The presenilin hypothesis of Alzheimer's disease: Evidence for a loss-of-function pathogenic mechanism.
      ). They proposed that pathogenic PS mutations behave as dominant-negative mutation due to a mechanism in which mutant PS interferes with the activity of wild-type PS, thereby reducing their physiological functions (
      • Heilig E.A.
      • Gutti U.
      • Tai T.
      • Shen J.
      • Kelleher 3rd., R.J.
      Trans-dominant negative effects of pathogenic PSEN1 mutations on gamma-secretase activity and Abeta production.
      ,
      • Xia D.
      • Watanabe H.
      • Wu B.
      • Lee S.H.
      • Li Y.
      • Tsvetkov E.
      • Bolshakov V.Y.
      • Shen J.
      • Kelleher 3rd., R.J.
      Presenilin-1 knockin mice reveal loss-of-function mechanism for familial Alzheimer's disease.
      ). In agreement with this hypothesis, the extensive work of Sun and colleagues (
      • Sun L.
      • Zhou R.
      • Yang G.
      • Shi Y.
      Analysis of 138 pathogenic mutations in presenilin-1 on the in vitro production of Abeta42 and Abeta40 peptides by gamma-secretase.
      ), in which 138 pathogenic PS mutations were analyzed, revealed that more than 90% of them led to reduced production of both Aβ40 and Aβ42, and this is in a dominant negative manner (
      • Sun L.
      • Zhou R.
      • Yang G.
      • Shi Y.
      Analysis of 138 pathogenic mutations in presenilin-1 on the in vitro production of Abeta42 and Abeta40 peptides by gamma-secretase.
      ). This conclusion of Sun and colleagues was based on purified γ-secretases harboring PS mutations and assay of C99 cleavage in vitro or in a cell-based assay. In contrast, Szaruga and colleagues (
      • Szaruga M.
      • Veugelen S.
      • Benurwar M.
      • Lismont S.
      • Sepulveda-Falla D.
      • Lleo A.
      • Ryan N.S.
      • Lashley T.
      • Fox N.C.
      • Murayama S.
      • Gijsen H.
      • De Strooper B.
      • Chavez-Gutierrez L.
      Qualitative changes in human gamma-secretase underlie familial Alzheimer's disease.
      ) measured γ-secretase activity in brain extracts from patients carrying PS1 mutations (one healthy and one disease allele) and found a variable effect on endopeptidase cleavage but a consistent reduction in carboxypeptidase activity, seen as Aβ38/Aβ42. These data therefore seemed to fit with the postulate that a qualitative shift in Aβ profiles toward the generation of longer aggregation-prone peptides (>Aβ42) should be the common denominator of AD (
      • Szaruga M.
      • Veugelen S.
      • Benurwar M.
      • Lismont S.
      • Sepulveda-Falla D.
      • Lleo A.
      • Ryan N.S.
      • Lashley T.
      • Fox N.C.
      • Murayama S.
      • Gijsen H.
      • De Strooper B.
      • Chavez-Gutierrez L.
      Qualitative changes in human gamma-secretase underlie familial Alzheimer's disease.
      ) and were in agreement with the earlier work from Saito and Saido, demonstrating a particular high toxicity of Aβ43 (
      • Saito T.
      • Suemoto T.
      • Brouwers N.
      • Sleegers K.
      • Funamoto S.
      • Mihira N.
      • Matsuba Y.
      • Yamada K.
      • Nilsson P.
      • Takano J.
      • Nishimura M.
      • Iwata N.
      • Van Broeckhoven C.
      • Ihara Y.
      • Saido T.C.
      Potent amyloidogenicity and pathogenicity of Abeta43.
      ) and the initial statements of a role of aggregating-prone Aβ in seed formation (
      • Borchelt D.R.
      • Thinakaran G.
      • Eckman C.B.
      • Lee M.K.
      • Davenport F.
      • Ratovitsky T.
      • Prada C.-M.
      • Kim G.
      • Seekins S.
      • Yager D.
      • Slunt H.H.
      • Wang R.
      • Seeger M.
      • Levey A.I.
      • Gandy S.E.
      • et al.
      Familial Alzheimer's disease-linked presenilin 1 variants elevate Ab1-42/1-40 in vitro and in vivo.
      ,
      • Duff K.
      • Eckman C.
      • Zehr C.
      • Yu X.
      • Prada C.-M.
      • Perez-Tur J.
      • Hutton M.
      • Buee L.
      • Harigaya Y.
      • Yager D.
      • Morgan D.
      • Gordon M.N.
      • Holcomb L.
      • Refolo L.
      • Zenk B.
      • et al.
      Increased amyloid-b42(43) in brains expressing mutant presenilin 1.
      ,
      • Scheuner D.
      • Eckman C.
      • Jensen M.
      • Song X.
      • Citron M.
      • Suzuki N.
      • Bird T.D.
      • Hardy J.
      • Hutton M.
      • Kukull W.
      • Larson E.
      • Levy-lahad E.
      • Viitanen M.
      • Peskind E.
      • Poorkaj P.
      • et al.
      Secreted amyloid b-protein similar to that in the senile plaques of Alzheimer's disease is increased in vivo by the presenilin 1 and 2 and APP mutations linked to familial Alzheimer's disease.
      ). However, arguing against a direct and unique role of the Aβ42/Aβ40 ratio was the findings in the work of Sun and colleagues that observed that the age of onset of FAD did not seem to correlate well with the change in this ratio (
      • Sun L.
      • Zhou R.
      • Yang G.
      • Shi Y.
      Analysis of 138 pathogenic mutations in presenilin-1 on the in vitro production of Abeta42 and Abeta40 peptides by gamma-secretase.
      ) in contradiction to what was previous proposed (
      • Kumar-Singh S.
      • Theuns J.
      • Van Broeck B.
      • Pirici D.
      • Vennekens K.
      • Corsmit E.
      • Cruts M.
      • Dermaut B.
      • Wang R.
      • Van Broeckhoven C.
      Mean age of onset of familial Alzheimer disease caused by presenilin mutations correlates with both increased Ab42 and decreased Ab40.
      ). A recent study indicated that the pathogenicity of PS1 mutations could be related to an intracellular mislocalization of the γ-secretase complex composed of these mutants (
      • Sannerud R.
      • Esselens C.
      • Ejsmont P.
      • Mattera R.
      • Rochin L.
      • Tharkeshwar A.K.
      • De Baets G.
      • De Wever V.
      • Habets R.
      • Baert V.
      • Vermeire W.
      • Michiels C.
      • Groot A.J.
      • Wouters R.
      • Dillen K.
      • et al.
      Restricted location of PSEN2/gamma-secretase determines substrate specificity and generates an intracellular Abeta pool.
      ,
      • Meckler X.
      • Checler F.
      Presenilin 1 and presenilin 2 target gamma-secretase complexes to distinct cellular compartments.
      ). Indeed, in contrary to wild-type PS1 having a broad cellular distribution, mutated PS1 was found to be restricted to endosomal/lysosomal compartments (
      • Sannerud R.
      • Esselens C.
      • Ejsmont P.
      • Mattera R.
      • Rochin L.
      • Tharkeshwar A.K.
      • De Baets G.
      • De Wever V.
      • Habets R.
      • Baert V.
      • Vermeire W.
      • Michiels C.
      • Groot A.J.
      • Wouters R.
      • Dillen K.
      • et al.
      Restricted location of PSEN2/gamma-secretase determines substrate specificity and generates an intracellular Abeta pool.
      ,
      • Meckler X.
      • Checler F.
      Presenilin 1 and presenilin 2 target gamma-secretase complexes to distinct cellular compartments.
      ). Hence, it was postulated that mutated PS would favor intraneuronal Aβ production, which could be particularly toxic (
      • Gouras G.K.
      • Tsai J.
      • Naslund J.
      • Vincent B.
      • Edgar M.
      • Checler F.
      • Greenfield J.P.
      • Haroutunian V.
      • Buxbaum J.D.
      • Xu H.
      • Greenghard P.
      • Relkin N.R.
      Intraneuronal Ab42 accumulmation in human brain.
      ,
      • Chui D.H.
      • Dobo E.
      • Makifuchi T.
      • Akiyama H.
      • Kawakatsu S.
      • Petit A.
      • Checler F.
      • Araki W.
      • Takahashi K.
      • Tabira T.
      Apoptotic neurons in Alzheimer's disease frequently show intracellular Abeta42 labeling.
      ,
      • Chui D.H.
      • Tanahashi H.
      • Ozawa K.
      • Ikeda S.
      • Checler F.
      • Ueda O.
      • Suzuki H.
      • Araki W.
      • Inoue H.
      • Shirotani K.
      • Takahashi K.
      • Gallyas F.
      • Tabira T.
      Transgenic mice with Alzheimer presenilin 1 mutations show accelerated neurodegeneration without amyloid plaque formation.
      ). Moreover, the acidity of endosomal/lysosomal compartments could be less propitious for carboxypeptidase activity, thus leading to the generation of longer and more aggregating Aβ peptides (
      • Sannerud R.
      • Esselens C.
      • Ejsmont P.
      • Mattera R.
      • Rochin L.
      • Tharkeshwar A.K.
      • De Baets G.
      • De Wever V.
      • Habets R.
      • Baert V.
      • Vermeire W.
      • Michiels C.
      • Groot A.J.
      • Wouters R.
      • Dillen K.
      • et al.
      Restricted location of PSEN2/gamma-secretase determines substrate specificity and generates an intracellular Abeta pool.
      ) and the acidic pH and high concentrations of Aβ in these compartments could be particularly propitious for Aβ aggregation and thus toxicity.
      A more recent hypothesis suggests that the pathogenicity of PS mutations could also be linked, or at least partly, to their effect on C99 accumulation. Indeed, although few studies have investigated the effects of FAD mutations on C99 levels, recent studies seem to reveal a consistent increase in C99 in the presence of most pathogenic PS mutants (
      • Li N.
      • Liu K.
      • Qiu Y.
      • Ren Z.
      • Dai R.
      • Deng Y.
      • Qing H.
      Effect of presenilin mutations on APP cleavage; insights into the pathogenesis of FAD.
      ,
      • Kwart D.
      • Gregg A.
      • Scheckel C.
      • Murphy E.A.
      • Paquet D.
      • Duffield M.
      • Fak J.
      • Olsen O.
      • Darnell R.B.
      • Tessier-Lavigne M.
      A large panel of isogenic APP and PSEN1 mutant human iPSC neurons reveals shared endosomal abnormalities mediated by APP beta-CTFs, not Abeta.
      ,
      • Cacquevel M.
      • Aeschbach L.
      • Houacine J.
      • Fraering P.C.
      Alzheimer's disease-linked mutations in presenilin-1 result in a drastic loss of activity in purified gamma-secretase complexes.
      ). This was even the case in neurons derived from pluripotent stem cells (iPSCs) harboring a wide panel of pathogenic PS mutations in the absence of overexpression (
      • Kwart D.
      • Gregg A.
      • Scheckel C.
      • Murphy E.A.
      • Paquet D.
      • Duffield M.
      • Fak J.
      • Olsen O.
      • Darnell R.B.
      • Tessier-Lavigne M.
      A large panel of isogenic APP and PSEN1 mutant human iPSC neurons reveals shared endosomal abnormalities mediated by APP beta-CTFs, not Abeta.
      ). Whether this effect on C99 is explained by a loss of function (reduced γ-secretase activity) and/or other PS-associated side effects remains unclear. Indeed, accumulating evidence indicates that both PS1 and PS2 are involved in essential cellular processes, independently of their role in γ-secretase, and proposed to be due notably to their function as chaperons and/or control of calcium release from the endoplasmic reticulum (for reviews see (
      • Duggan S.P.
      • McCarthy J.V.
      Beyond gamma-secretase activity: The multifunctional nature of presenilins in cell signalling pathways.
      ,
      • Oikawa N.
      • Walter J.
      Presenilins and gamma-secretase in membrane proteostasis.
      )). Some of these functions have been suggested to be modified by FAD mutations, although again, many contrasting findings have been reported. Indeed, most studies on noncatalytic functions of PS have been carried out on knockout models, thus in the absence of PS, whereas fewer works have investigated the impact of FAD mutations on these functions. One of the most consistently described noncatalytic functions of PS is their involvement in the regulation of both autophagy and lysosomal function (
      • Duggan S.P.
      • McCarthy J.V.
      Beyond gamma-secretase activity: The multifunctional nature of presenilins in cell signalling pathways.
      ,
      • Oikawa N.
      • Walter J.
      Presenilins and gamma-secretase in membrane proteostasis.
      ) known also to be involved in C99 degradation (see below). Whether these γ-secretase-independent functions contribute to, or are responsible for, C99 accumulation, in cells expressing PS mutations, remains to be established.

      Physiological function of γ-secretase

      FAD cases correspond to only a very small subset of patients (about 1%) (
      • Cacace R.
      • Sleegers K.
      • Van Broeckhoven C.
      Molecular genetics of early-onset Alzheimer's disease revisited.
      ). Thus, both Aβ and C99 accumulation in FAD could reflect an exacerbated process or a dysfunction occurring only in few cases. The situation seems to be different in sporadic AD (SAD) that corresponds to most cases. In SAD, Aβ accumulation is mostly believed to be linked to age-related impairment in Aβ clearance, rather than to an increased Aβ biogenesis. Thus, Aβ should be considered firstly as a physiological product derived from normal processing of APP (
      • Shoji M.
      • Golde T.E.
      • Ghiso J.
      • Cheung T.T.
      • Estus S.
      • Shaffer M.
      • Cai X.D.
      • McKay D.M.
      • Tintner R.
      • Frangione B.
      • Younkin S.G.
      Production of the Alzheimer amyloid b protein by normal proteolytic processing.
      ,
      • Haass C.
      • Hung A.Y.
      • Schlossmacher M.G.
      • Oltersdorf T.
      • Teplow D.B.
      • Selkoe D.J.
      Normal cellular processing of the b-amyloid precursor protein results in the secretion of the amyloid b peptide and related molecules.
      ). Although little is still known about the exact physiological role of Aβ, the peptide is known to regulate synaptic activity, to have antimicrobial and tumor suppressing properties (for review see (
      • Kent S.A.
      • Spires-Jones T.L.
      • Durrant C.S.
      The physiological roles of tau and Abeta: Implications for Alzheimer's disease pathology and therapeutics.
      )), and to be neuroprotective in some pathological conditions (
      • Sevalle J.
      • Amoyel A.
      • Robert P.
      • Fournie-Zaluski M.C.
      • Roques B.
      • Checler F.
      Aminopeptidase A contributes to the N-terminal truncation of amyloid beta-peptide.
      ,
      • Castellani R.J.
      • Lee H.G.
      • Perry G.
      • Smith M.A.
      Antioxidant protection and neurodegenerative disease: The role of amyloid-beta and tau.
      ,
      • Brothers H.M.
      • Gosztyla M.L.
      • Robinson S.R.
      The physiological roles of amyloid-beta peptide hint at new ways to treat Alzheimer's disease.
      ). Thus, to some extent the failure of clinical trials aimed at either inhibiting Aβ production or neutralizing Aβ once formed could be explained, at least in part, by the abolition of such Aβ-mediated physiological functions.
      If considering Aβ as a physiological product, this implies that a main role of γ-secretase should be to generate bioactive molecules involved in important vital processes. Indeed, this point of view fits with genetic evidences indicating that the invalidation of γ-secretase in mice is lethal (
      • Shen J.
      • Bronson R.T.
      • Chen D.F.
      • Xia W.
      • Selkoe D.
      • Tonegawa S.
      Skeletal and CNS defects in presenilin-1- deficient mice.
      ) and that the conditional knockout of PS1 leads to neurodegeneration at the adulthood (
      • Saura C.A.
      • Choi S.-Y.
      • Beglopoulos V.
      • Malkani S.
      • Zhang D.
      • Rao B.S.S.
      • Chattarji S.
      • Kelleher III, R.J.
      • Kandel E.R.
      • Duff K.
      • Kirkwood A.
      • Shen J.
      Loss of presenilin function causes impairements of memory and synaptic plasticity followed by age-dependent neurodegeneration.
      ). Of course, these observations are probably explained by the numerous, more than 150, other γ-secretase proteolytic targets involved in physiological processes (
      • Guner G.
      • Lichtenthaler S.F.
      The substrate repertoire of gamma-secretase/presenilin.
      ). Indeed, similar to APP, most of γ-secretase substrates are type I transmembrane proteins, which undergo “Regulated Intramembrane proteolysis (RIP), a proteolytic process in which γ-secretase-mediated cleavage occurs inside the membrane ending up with the production of an intracellular fragment that shuttles to the nucleus and acts as a transcription factor (
      • Kuhnle N.
      • Dederer V.
      • Lemberg M.K.
      Intramembrane proteolysis at a glance: From signalling to protein degradation.
      ,
      • Lichtenthaler S.F.
      • Haass C.
      • Steiner H.
      Regulated intramembrane proteolysis--lessons from amyloid precursor protein processing.
      ). The seminal example of RIP was described for Notch that undergoes γ-secretase-mediated cleavage, thereby generating the Notch Intracellular Domain (NICD) (
      • Okochi M.
      • Steiner H.
      • Fukumori A.
      • Tanii H.
      • Tomita T.
      • Tanaka T.
      • Iwatsubo T.
      • Kudo T.
      • Takeda M.
      • Haass C.
      Presenilins mediate a dual intramembranous g-secretase cleavge of Notch-1.
      ), a key player of embryonic development (
      • Siebel C.
      • Lendahl U.
      Notch signaling in development, tissue homeostasis, and disease.
      ). For instance, this lack of Notch cleavage (
      • De Strooper B.
      • Annaert W.
      • Cupers P.
      • Saftig P.
      • Craessaerts K.
      • Mumm J.S.
      • Schroeter E.H.
      • Schrijvers V.
      • Wolfe M.S.
      • Ray W.J.
      • Goate A.
      • Kopan R.
      A presenilin-1-dependent g-secretase-like protease mediates release of Notch intracellular domain.
      ) is believed to explain the lethal embryologic defects observed in PS-invalidated mouse embryos (
      • Shen J.
      • Bronson R.T.
      • Chen D.F.
      • Xia W.
      • Selkoe D.
      • Tonegawa S.
      Skeletal and CNS defects in presenilin-1- deficient mice.
      ). In addition, numerous other substrates (the list is far from being exhaustive) have been reported to undergo RIP including cadherins (N and E Cadh-ICDs) (
      • Marambaud P.
      • Wen P.H.
      • Dutt A.
      • Takashima A.
      • Siman R.
      • Robakis N.
      A CPB binding transcriptional repressor produced by the PS1/e-cleavage of N-cadherin is inhibited by PS1 FAD mutations.
      ), alcadeins (
      • Araki Y.
      • Miyagi N.
      • Kato N.
      • Yoshida T.
      • Wada S.
      • Nishimura M.
      • Komano H.
      • Yamamoto T.
      • De Strooper B.
      • Yamamoto K.
      • Suzuki T.
      Coordinated metabolism of Alcadein and amyloid beta-protein precursor regulates FE65-dependent gene transactivation.
      ), CD44 (
      • Okamoto I.
      • Kawano Y.
      • Murakami D.
      • Sasayama T.
      • Araki N.
      • Miki T.
      • Wong A.J.
      • Saya H.
      Proteolytic release of CD44 intracellular domain and its role in the CD44 signaling pathway.
      ), or more recently TREM2 (
      • Glebov K.
      • Wunderlich P.
      • Karaca I.
      • Walter J.
      Functional involvement of gamma-secretase in signaling of the triggering receptor expressed on myeloid cells-2 (TREM2).
      ). Generally, the generation of the intracellular domain of these proteins is essential in the transcriptional regulation of physiological processes such as neurite outgrowth, cell adhesion and migration, or synaptogenesis (
      • Jurisch-Yaksi N.
      • Sannerud R.
      • Annaert W.
      A fast growing spectrum of biological functions of gamma-secretase in development and disease.
      ,
      • Haapasalo A.
      • Kovacs D.M.
      The many substrates of presenilin/gamma-secretase.
      ). This phenomenon is also described for APP for which the intracellular domain AICD has been found to form a transcriptional active complex with Fe65 and Tip60 (
      • Cao X.
      • Südhof T.C.
      A transcriptively active complex of APP with Fe65 and histone acetyltransferase tip60.
      ,
      • von Rotz R.C.
      • Kohli B.M.
      • Bosset J.
      • Meier M.
      • Suzuki T.
      • Nitsch R.M.
      • Konietzko U.
      The APP intracellular domain forms nuclear multiprotein complexes and regulates the transcription of its own precursor.
      ) that regulates key proteins involved in the control of cell death (p53) (
      • Alves da Costa C.
      • Sunyach C.
      • Pardossi-Piquard R.
      • Sevalle J.
      • Vincent B.
      • Boyer N.
      • Kawarai T.
      • Girardot N.
      • St George-Hyslop P.
      • Checler F.
      Presenilin-dependent gamma-secretase-mediated control of p53-associated cell death in Alzheimer's disease.
      ), Aβ degradation (Neprilysin) (
      • Pardossi-Piquard R.
      • Petit A.
      • Kawarai T.
      • Sunyach C.
      • Alves da Costa C.
      • Vincent B.
      • Ring S.
      • D'Adamio L.
      • Shen J.
      • Muller U.
      • St George Hyslop P.
      • Checler F.
      Presenilin-dependent transcriptional control of the Abeta-degrading enzyme neprilysin by intracellular domains of betaAPP and APLP.
      ,
      • Belyaev N.D.
      • Nalivaeva N.N.
      • Makova N.Z.
      • Turner A.J.
      Neprilysin gene expression requires binding of the amyloid precursor protein intracellular domain to its promoter: Implications for Alzheimer disease.
      ), as well as tau phosphorylation (GSK3β) (
      • Kim H.-S.
      • Kim E.-M.
      • Lee J.-P.
      • Park C.-H.
      • Kim S.J.
      • Seo J.-H.
      • Chang K.-A.
      • Yu E.
      • Jeong S.-J.
      • Chong Y.H.
      • Suh Y.-H.
      C-terminal fragments of amyloid precursor protein exert neurotoxicity by inducing glycogen synthase kinase-3b expression.
      ,
      • Ryan K.A.
      • Pimplikar S.W.
      Activation of GSK3 and phosphorylation of CRMP2 in transgenic mice expressing APP intracellular domain.
      ). Thus, γ-secretase cleavage should be considered as a necessary step in the formation of both Aβ and AICD. Besides this role, γ-secretase cleavage has also been proposed to behave as a “membranous proteasome” essential for the inactivation of the buildup of toxic membranous stubs (
      • Kopan R.
      • Ilagan M.X.
      Gamma-secretase: Proteasome of the membrane?.
      ). It remains unclear, whether this “proteasome-like activity” is nonselective and inactivates all substrates equally, or if some substrates are cleaved by specific protease complexes. Recent evidences seem to indicate at least some substrate specificity and that not only the exact protein composition of the secretase complex, but also the cellular context, subcellular localization, and the presence of nonessential cofactors (
      • Wong E.
      • Frost G.R.
      • Li Y.M.
      Gamma-secretase modulatory proteins: The guiding hand behind the running scissors.
      ) can be key determinants for this control. For instance, protease heterogeneity has been shown to be related to the exact protein composition of the protease and alternative splicing of Aph1 leads to further complexity (
      • Serneels L.
      • Van Biervliet J.
      • Craessaerts K.
      • Dejaegere T.
      • Horre K.
      • Van Houtvin T.
      • Esselmann H.
      • Paul S.
      • Schafer M.K.
      • Berezovska O.
      • Hyman B.T.
      • Sprangers B.
      • Sciot R.
      • Moons L.
      • Jucker M.
      • et al.
      gamma-Secretase heterogeneity in the Aph1 subunit: relevance for Alzheimer's disease.
      ,
      • Acx H.
      • Serneels L.
      • Radaelli E.
      • Muyldermans S.
      • Vincke C.
      • Pepermans E.
      • Muller U.
      • Chavez-Gutierrez L.
      • De Strooper B.
      Inactivation of gamma-secretases leads to accumulation of substrates and non-Alzheimer neurodegeneration.
      ). For instance, in mice the knockout of the 3 Aph1 subunits led to an important increase in some, but not all, membrane-bound fragments and APP-dependent neurodegeneration (
      • Serneels L.
      • Van Biervliet J.
      • Craessaerts K.
      • Dejaegere T.
      • Horre K.
      • Van Houtvin T.
      • Esselmann H.
      • Paul S.
      • Schafer M.K.
      • Berezovska O.
      • Hyman B.T.
      • Sprangers B.
      • Sciot R.
      • Moons L.
      • Jucker M.
      • et al.
      gamma-Secretase heterogeneity in the Aph1 subunit: relevance for Alzheimer's disease.
      ,
      • Acx H.
      • Serneels L.
      • Radaelli E.
      • Muyldermans S.
      • Vincke C.
      • Pepermans E.
      • Muller U.
      • Chavez-Gutierrez L.
      • De Strooper B.
      Inactivation of gamma-secretases leads to accumulation of substrates and non-Alzheimer neurodegeneration.
      ). Hu and colleagues demonstrated that nicastrin is required for APP processing but not Notch processing, whereas Aph1 is necessary for the processing of both APP and Notch (
      • Hu C.
      • Zeng L.
      • Li T.
      • Meyer M.A.
      • Cui M.Z.
      • Xu X.
      Nicastrin is required for amyloid precursor protein (APP) but not Notch processing, while anterior pharynx-defective 1 is dispensable for processing of both APP and Notch.
      ). Selectivity also seems to be determined by the subcellular localization of the protease, since, as described above, PS1- or PS2-containing complexes do not have the same cellular localization and do not cleave APP identically (
      • Sannerud R.
      • Esselens C.
      • Ejsmont P.
      • Mattera R.
      • Rochin L.
      • Tharkeshwar A.K.
      • De Baets G.
      • De Wever V.
      • Habets R.
      • Baert V.
      • Vermeire W.
      • Michiels C.
      • Groot A.J.
      • Wouters R.
      • Dillen K.
      • et al.
      Restricted location of PSEN2/gamma-secretase determines substrate specificity and generates an intracellular Abeta pool.
      ,
      • Meckler X.
      • Checler F.
      Presenilin 1 and presenilin 2 target gamma-secretase complexes to distinct cellular compartments.
      ). Interestingly, mutations in PS1 change the localization toward that of PS2-containing complexes (
      • Sannerud R.
      • Esselens C.
      • Ejsmont P.
      • Mattera R.
      • Rochin L.
      • Tharkeshwar A.K.
      • De Baets G.
      • De Wever V.
      • Habets R.
      • Baert V.
      • Vermeire W.
      • Michiels C.
      • Groot A.J.
      • Wouters R.
      • Dillen K.
      • et al.
      Restricted location of PSEN2/gamma-secretase determines substrate specificity and generates an intracellular Abeta pool.
      ).

      Challenges and complications of targeting γ-secretase in AD

      After the discovery of γ-secretase, a huge amount of research was carried out to develop potent and bioavailable γ-secretase inhibitors (GSIs), but the clinical trials based on these inhibitors all failed (Table 1). Indeed, none of them led to improvement of AD-linked cognitive decline and even sometimes they worsened them. In most of these trials, Aβ levels in plasma or cerebrospinal fluid (CSF) were significantly decreased, thus clearly indicating that a reduction in total Aβ was not sufficient to restore cognitive function. This was the case for Semagacestat, the first GSI tested in late-stage clinical trials for AD, which was reported to reduce Aβ levels by more than 60% in the plasma (
      • Fleisher A.S.
      • Raman R.
      • Siemers E.R.
      • Becerra L.
      • Clark C.M.
      • Dean R.A.
      • Farlow M.R.
      • Galvin J.E.
      • Peskind E.R.
      • Quinn J.F.
      • Sherzai A.
      • Sowell B.B.
      • Aisen P.S.
      • Thal L.J.
      Phase 2 safety trial targeting amyloid beta production with a gamma-secretase inhibitor in Alzheimer disease.
      ), and new Aβ synthesis was decreased by more than 80% in CSF (
      • Bateman R.J.
      • Siemers E.R.
      • Mawuenyega K.G.
      • Wen G.
      • Browning K.R.
      • Sigurdson W.C.
      • Yarasheski K.E.
      • Friedrich S.W.
      • Demattos R.B.
      • May P.C.
      • Paul S.M.
      • Holtzman D.M.
      A gamma-secretase inhibitor decreases amyloid-beta production in the central nervous system.
      ). More alarming, many trials were readily stopped because of increased risk of developing skin cancer and infections, which was supposed to be tightly linked to a defective Notch signaling in the presence of the inhibitors (
      • Doody R.S.
      • Raman R.
      • Farlow M.
      • Iwatsubo T.
      • Vellas B.
      • Joffe S.
      • Kieburtz K.
      • He F.
      • Sun X.
      • Thomas R.G.
      • Aisen P.S.
      • Siemers E.
      • Sethuraman G.
      • Mohs R.
      Alzheimer's Disease Cooperative Study Steering CommitteeSemagacestat Study Group
      A phase 3 trial of semagacestat for treatment of Alzheimer's disease.
      ). Therefore, several groups started to screen for drugs having a higher affinity for APP than toward Notch, such as Avagacestat, initially reported to be 140-fold more selective for APP than Notch (
      • Albright C.F.
      • Dockens R.C.
      • Meredith Jr., J.E.
      • Olson R.E.
      • Slemmon R.
      • Lentz K.A.
      • Wang J.S.
      • Denton R.R.
      • Pilcher G.
      • Rhyne P.W.
      • Raybon J.J.
      • Barten D.M.
      • Burton C.
      • Toyn J.H.
      • Sankaranarayanan S.
      • et al.
      Pharmacodynamics of selective inhibition of gamma-secretase by avagacestat.
      ), although its Notch sparing ability remains controversial (
      • Chavez-Gutierrez L.
      • Bammens L.
      • Benilova I.
      • Vandersteen A.
      • Benurwar M.
      • Borgers M.
      • Lismont S.
      • Zhou L.
      • Van Cleynenbreugel S.
      • Esselmann H.
      • Wiltfang J.
      • Serneels L.
      • Karran E.
      • Gijsen H.
      • Schymkowitz J.
      • et al.
      The mechanism of gamma-Secretase dysfunction in familial Alzheimer disease.
      ,
      • Crump C.J.
      • Castro S.V.
      • Wang F.
      • Pozdnyakov N.
      • Ballard T.E.
      • Sisodia S.S.
      • Bales K.R.
      • Johnson D.S.
      • Li Y.M.
      BMS-708,163 targets presenilin and lacks notch-sparing activity.
      ). Avagacestat led to a 40% Aβ reduction in CSF (Table 1), but demonstrated similar side effects in clinical trials (
      • Coric V.
      • van Dyck C.H.
      • Salloway S.
      • Andreasen N.
      • Brody M.
      • Richter R.W.
      • Soininen H.
      • Thein S.
      • Shiovitz T.
      • Pilcher G.
      • Colby S.
      • Rollin L.
      • Dockens R.
      • Pachai C.
      • Portelius E.
      • et al.
      Safety and tolerability of the gamma-secretase inhibitor avagacestat in a phase 2 study of mild to moderate Alzheimer disease.
      ). Therefore, due to the huge number of different γ-secretase substrates, it has appeared difficult to target specifically and exclusively the γ-secretase-mediated APP cleavage.
      Furthermore, data from preclinical trials proposed that the lack of efficacy, or even worsening, of cognitive function in these trials was also linked to the progressive accumulation of APP-CTFs in the presence of GSIs (
      • Mitani Y.
      • Yarimizu J.
      • Saita K.
      • Uchino H.
      • Akashiba H.
      • Shitaka Y.
      • Ni K.
      • Matsuoka N.
      Differential effects between gamma-secretase inhibitors and modulators on cognitive function in amyloid precursor protein-transgenic and nontransgenic mice.
      ). To avoid such effects, therapeutic development became focused on γ-secretase modulators (GSMs) that were expected to be safer, since they interact with γ-secretase complex through the allosteric binding site, thereby modifying the enzyme activity but not blocking it (
      • Bursavich M.G.
      • Harrison B.A.
      • Blain J.F.
      Gamma secretase modulators: New Alzheimer's drugs on the Horizon?.
      ). The concept of γ-secretase modulation was discovered with some nonsteroidal anti-inflammatory drugs (NSAIDs) such as flurbiprofen, indomethacin, and ibuprofen, which are considered as GSMs, because they induce conformational changes in PS1 and shift the cleavage of C99 toward shorter Aβ species such as Aβ37 and Aβ38 (
      • Lleo A.
      • Berezovska O.
      • Growdon J.H.
      • Hyman B.T.
      Clinical, pathological, and biochemical spectrum of Alzheimer disease associated with PS-1 mutations.
      ,
      • Takeo K.
      • Tanimura S.
      • Shinoda T.
      • Osawa S.
      • Zahariev I.K.
      • Takegami N.
      • Ishizuka-Katsura Y.
      • Shinya N.
      • Takagi-Niidome S.
      • Tominaga A.
      • Ohsawa N.
      • Kimura-Someya T.
      • Shirouzu M.
      • Yokoshima S.
      • Yokoyama S.
      • et al.
      Allosteric regulation of gamma-secretase activity by a phenylimidazole-type gamma-secretase modulator.
      ). In that way, GSMs do not lead to increased APP-CTFs levels. Indeed, in AD animal models, the chronic treatment with GSMs did not lead to the worsening of cognitive function observed with GSIs (
      • Mitani Y.
      • Yarimizu J.
      • Saita K.
      • Uchino H.
      • Akashiba H.
      • Shitaka Y.
      • Ni K.
      • Matsuoka N.
      Differential effects between gamma-secretase inhibitors and modulators on cognitive function in amyloid precursor protein-transgenic and nontransgenic mice.
      ). Moreover, GSMs are also safer because they reduce the level of Aβ42 and increase shorter Aβ peptides without affecting Notch signaling (
      • Xia W.
      gamma-Secretase and its modulators: Twenty years and beyond.
      ,
      • Kukar T.L.
      • Ladd T.B.
      • Bann M.A.
      • Fraering P.C.
      • Narlawar R.
      • Maharvi G.M.
      • Healy B.
      • Chapman R.
      • Welzel A.T.
      • Price R.W.
      • Moore B.
      • Rangachari V.
      • Cusack B.
      • Eriksen J.
      • Jansen-West K.
      • et al.
      Substrate-targeting gamma-secretase modulators.
      ). Nonetheless, the first GSMs tested in clinical trials, Rofecoxib (
      • Reines S.
      • Block G.
      • Morris J.
      • Liu G.
      • Nessly M.
      • Lines C.
      • Norman B.
      • Baranak C.
      Rofecoxib: No effect on Alzheimer's disease in a 1-year, randomized, blinded, controlled study.
      ), Tarenflurbil (
      • Green R.C.
      • Schneider L.S.
      • Amato D.A.
      • Beelen A.P.
      • Wilcock G.
      • Swabb E.A.
      • Zavitz K.H.
      Tarenflurbil Phase 3 Study Group
      Effect of tarenflurbil on cognitive decline and activities of daily living in patients with mild Alzheimer disease: A randomized controlled trial.
      ) or Naproxen (
      • Meyer P.F.
      • Tremblay-Mercier J.
      • Leoutsakos J.
      • Madjar C.
      • Lafaille-Maignan M.E.
      • Savard M.
      • Rosa-Neto P.
      • Poirier J.
      • Etienne P.
      • Breitner J.
      • Group P.-A.R.
      INTREPAD: A randomized trial of naproxen to slow progress of presymptomatic Alzheimer disease.
      ), did not show efficacy, but the lack of effects seemed to be related to a very poor blood–brain barrier crossing ability of these drugs (Table 1).
      Thus, taken together, γ-secretase-based strategies have so far been unsuccessful, even if GSMs seem to be more promising (
      • Mekala S.
      • Nelson G.
      • Li Y.M.
      Recent developments of small molecule gamma-secretase modulators for Alzheimer's disease.
      ). Still, if considering γ-secretase as a C99-inactivating enzyme, we believe that one should carefully question the validity of targeting this enzyme.

      C99 is toxic and γ-secretase inhibitors potentiate pathogenic phenotypes

      Emerging data propose a pathological role of the intraneuronal accumulation of C99 (Table 2). Indeed, a very recent work showed that the accumulation of C99, rather than that of Aβ, correlates with neuronal vulnerability in AD-affected patients (
      • Pulina M.V.
      • Hopkins M.
      • Haroutunian V.
      • Greengard P.
      • Bustos V.
      C99 selectively accumulates in vulnerable neurons in Alzheimer's disease.
      ). To distinguish between C99 and Aβ in situ, which can be very challenging because the two of them have common epitopes, the group of Drs Bustos and Greengard developed a sophisticated technique adapted from the proximity ligation assay (PLA) and using both N- and C-terminal directed antibodies. This link between neuronal vulnerability and C99 in human AD was an important observation previously supported by many animal models. The first evidence of C99 accumulation in AD mouse models came from studies on the widely used 3xTg-AD model (APPswe, TauP310L, PS1M146V) developed by the group of La Ferla (
      • Oddo S.
      • Caccamo A.
      • Shepherd J.D.
      • Murphy G.
      • Golde T.E.
      • Kayed R.
      • Metherate R.
      • Mattson M.P.
      • Akbari Y.
      • LaFerla F.
      Triple-transgenic model of Alzheimer's disease with plaques and tangles: Intracellular Ab and synaptic dysfunction.
      ). In the original work, the authors used N-terminal Aβ antibodies and claimed that these mice harbor intracellular Aβ accumulation (
      • Oddo S.
      • Caccamo A.
      • Shepherd J.D.
      • Murphy G.
      • Golde T.E.
      • Kayed R.
      • Metherate R.
      • Mattson M.P.
      • Akbari Y.
      • LaFerla F.
      Triple-transgenic model of Alzheimer's disease with plaques and tangles: Intracellular Ab and synaptic dysfunction.
      ). However, years later our own work, using different and complementary approaches, indicated that this accumulated material corresponded to C99 and not to Aβ (
      • Lauritzen I.
      • Pardossi-Piquard R.
      • Bauer C.
      • Brigham E.
      • Abraham J.D.
      • Ranaldi S.
      • Fraser P.
      • St-George-Hyslop P.
      • Le Thuc O.
      • Espin V.
      • Chami L.
      • Dunys J.
      • Checler F.
      The beta-secretase-derived C-terminal fragment of betaAPP, C99, but not Abeta, is a key contributor to early intraneuronal lesions in triple-transgenic mouse hippocampus.
      ). Indeed, as stated above, it is challenging to discriminate between Aβ and C99 in situ. Hence, only a detailed characterization using N- and C-terminal antibodies, pharmacological treatments (β- and γ-secretase inhibitors), and/or genetic approaches (as PS mutations) can allow this discrimination. Most and even recent studies do not provide this detailed characterization, which may explain the lack of information concerning C99 in the literature. In the 3xTgAD model, C99 accumulates early and much before Aβ can be detected (
      • Lauritzen I.
      • Pardossi-Piquard R.
      • Bauer C.
      • Brigham E.
      • Abraham J.D.
      • Ranaldi S.
      • Fraser P.
      • St-George-Hyslop P.
      • Le Thuc O.
      • Espin V.
      • Chami L.
      • Dunys J.
      • Checler F.
      The beta-secretase-derived C-terminal fragment of betaAPP, C99, but not Abeta, is a key contributor to early intraneuronal lesions in triple-transgenic mouse hippocampus.
      ). Of importance, C99 accumulation was found to also occur in many other transgenic models including Tg-CRND8 (
      • Cavanagh C.
      • Colby-Milley J.
      • Bouvier D.
      • Farso M.
      • Chabot J.G.
      • Quirion R.
      • Krantic S.
      betaCTF-correlated burst of hippocampal TNFalpha occurs at a very early, pre-plaque stage in the TgCRND8 mouse model of Alzheimer's disease.
      ) and JA20 (
      • Mondragon-Rodriguez S.
      • Gu N.
      • Manseau F.
      • Williams S.
      Alzheimer's transgenic model is characterized by very early brain network alterations and beta-CTF fragment accumulation: Reversal by beta-secretase inhibition.
      ) mice carrying the APPswe mutation, known to increase C99 production, but also in mice displaying the APPE693Q Dutch mutation (
      • Kaur G.
      • Pawlik M.
      • Gandy S.E.
      • Ehrlich M.E.
      • Smiley J.F.
      • Levy E.
      Lysosomal dysfunction in the brain of a mouse model with intraneuronal accumulation of carboxyl terminal fragments of the amyloid precursor protein.
      ), in which a C99 overproduction is not expected to occur. Interestingly, and importantly, C99 accumulation also occurs in the knock-in models APP-NL and APP-NL-F, harboring the Swedish (KM670/671NL) mutation alone (APP-NL) or in combination with the I706F mutation (APP-NL-F), respectively (
      • Sasaguri H.
      • Nilsson P.
      • Hashimoto S.
      • Nagata K.
      • Saito T.
      • De Strooper B.
      • Hardy J.
      • Vassar R.
      • Winblad B.
      • Saido T.C.
      APP mouse models for Alzheimer's disease preclinical studies.
      ,
      • Johnson E.C.B.
      • Ho K.
      • Yu G.Q.
      • Das M.
      • Sanchez P.E.
      • Djukic B.
      • Lopez I.
      • Yu X.
      • Gill M.
      • Zhang W.
      • Paz J.T.
      • Palop J.J.
      • Mucke L.
      Behavioral and neural network abnormalities in human APP transgenic mice resemble those of App knock-in mice and are modulated by familial Alzheimer's disease mutations but not by inhibition of BACE1.
      ) (Table 2) indicating that C99 accumulation was not just an artificial phenotype linked to APP overexpression, as earlier described for other events taking place in transgenic mouse models that sometimes express very high levels of APP (
      • Sasaguri H.
      • Nilsson P.
      • Hashimoto S.
      • Nagata K.
      • Saito T.
      • De Strooper B.
      • Hardy J.
      • Vassar R.
      • Winblad B.
      • Saido T.C.
      APP mouse models for Alzheimer's disease preclinical studies.
      ). The presence of the Swedish mutation could indeed, at least to some extent, explain the C99 accumulation in these knock-in mice. However, recent studies clearly showed that humanizing APP (substituting the three amino acids G676R, F681Y, and R684H) in mice or rats is sufficient to increase C99 levels (
      • Serneels L.
      • T'Syen D.
      • Perez-Benito L.
      • Theys T.
      • Holt M.G.
      • De Strooper B.
      Modeling the beta-secretase cleavage site and humanizing amyloid-beta precursor protein in rat and mouse to study Alzheimer's disease.
      ,
      • Tambini M.D.
      • Yao W.
      • D'Adamio L.
      Facilitation of glutamate, but not GABA, release in Familial Alzheimer's APP mutant Knock-in rats with increased beta-cleavage of APP.
      ,
      • Tambini M.D.
      • Norris K.A.
      • D'Adamio L.
      Opposite changes in APP processing and human Abeta levels in rats carrying either a protective or a pathogenic APP mutation.
      ). In animals displaying rodent APP, the protein was found to be mainly processed by α-secretase, thus generating almost only C83, whereas the presence of human APP increases β-secretase cleavage and leads to enhanced C99 levels (
      • Serneels L.
      • T'Syen D.
      • Perez-Benito L.
      • Theys T.
      • Holt M.G.
      • De Strooper B.
      Modeling the beta-secretase cleavage site and humanizing amyloid-beta precursor protein in rat and mouse to study Alzheimer's disease.
      ). These data thus reveal a different APP processing of human and rodent APP, which could explain the higher levels of C99 in human as compared with rodents (
      • Vaillant-Beuchot L.
      • Mary A.
      • Pardossi-Piquard R.
      • Bourgeois A.
      • Lauritzen I.
      • Eysert F.
      • Kinoshita P.F.
      • Cazareth J.
      • Badot C.
      • Fragaki K.
      • Bussiere R.
      • Martin C.
      • Mary R.
      • Bauer C.
      • Pagnotta S.
      • et al.
      Amylois precursor protein C-terminal fragments accumulation triggers mitochondrial structure, function and mitophagy defects in Alzheimer’s disease models and human brains.
      ) and may be one of the reasons for the C99 accumulation observed even in SAD cases (
      • Pulina M.V.
      • Hopkins M.
      • Haroutunian V.
      • Greengard P.
      • Bustos V.
      C99 selectively accumulates in vulnerable neurons in Alzheimer's disease.
      ,
      • Vaillant-Beuchot L.
      • Mary A.
      • Pardossi-Piquard R.
      • Bourgeois A.
      • Lauritzen I.
      • Eysert F.
      • Kinoshita P.F.
      • Cazareth J.
      • Badot C.
      • Fragaki K.
      • Bussiere R.
      • Martin C.
      • Mary R.
      • Bauer C.
      • Pagnotta S.
      • et al.
      Amylois precursor protein C-terminal fragments accumulation triggers mitochondrial structure, function and mitophagy defects in Alzheimer’s disease models and human brains.
      ,
      • Pera M.
      • Alcolea D.
      • Sanchez-Valle R.
      • Guardia-Laguarta C.
      • Colom-Cadena M.
      • Badiola N.
      • Suarez-Calvet M.
      • Llado A.
      • Barrera-Ocampo A.A.
      • Sepulveda-Falla D.
      • Blesa R.
      • Molinuevo J.L.
      • Clarimon J.
      • Ferrer I.
      • Gelpi E.
      • et al.
      Distinct patterns of APP processing in the CNS in autosomal-dominant and sporadic Alzheimer disease.
      ). Overall, these findings ruled out the possibility that it could be “model specific” but rather suggest that it should be a common and early alteration in AD.
      Table 2C99-associated toxicity in AD mice models and human brain
      Mouse modelsMutationsC99 accumulation-associated pathologyReferences
      3xTgAD

      2xTgAD
      APPK670N,M671L, PS1M146V, MAPTP301L

      APPK670N,M671L, MAPTP301L
      Endolysosomal and autophagic dysfunction

      Inflammation, LTP alterations

      Mitochondrial pathology
      (
      • Lauritzen I.
      • Pardossi-Piquard R.
      • Bauer C.
      • Brigham E.
      • Abraham J.D.
      • Ranaldi S.
      • Fraser P.
      • St-George-Hyslop P.
      • Le Thuc O.
      • Espin V.
      • Chami L.
      • Dunys J.
      • Checler F.
      The beta-secretase-derived C-terminal fragment of betaAPP, C99, but not Abeta, is a key contributor to early intraneuronal lesions in triple-transgenic mouse hippocampus.
      ,
      • Vaillant-Beuchot L.
      • Mary A.
      • Pardossi-Piquard R.
      • Bourgeois A.
      • Lauritzen I.
      • Eysert F.
      • Kinoshita P.F.
      • Cazareth J.
      • Badot C.
      • Fragaki K.
      • Bussiere R.
      • Martin C.
      • Mary R.
      • Bauer C.
      • Pagnotta S.
      • et al.
      Amylois precursor protein C-terminal fragments accumulation triggers mitochondrial structure, function and mitophagy defects in Alzheimer’s disease models and human brains.
      ,
      • Lauritzen I.
      • Pardossi-Piquard R.
      • Bourgeois A.
      • Pagnotta S.
      • Biferi M.G.
      • Barkats M.
      • Lacor P.
      • Klein W.
      • Bauer C.
      • Checler F.
      Intraneuronal aggregation of the beta-CTF fragment of APP (C99) induces Abeta-independent lysosomal-autophagic pathology.
      ,
      • Pardossi-Piquard R.
      • Lauritzen I.
      • Bauer C.
      • Sacco G.
      • Robert P.
      • Checler F.
      Influence of genetic background on apathy-like behavior in triple transgenic AD mice.
      ,
      • Bourgeois A.
      • Lauritzen I.
      • Lorivel T.
      • Bauer C.
      • Checler F.
      • Pardossi-Piquard R.
      Intraneuronal accumulation of C99 contributes to synaptic alterations, apathy-like behavior, and spatial learning deficits in 3xTgAD and 2xTgAD mice.
      )
      5xFADAPP KM670/671NL, APPI716V, APPV717I, PS1M146L,I286VMitochondrial pathology(
      • Lynch S.Y.
      • Kaplow J.
      • Zhao J.
      • Dhadda S.
      • Luthman J.
      • Albala B.
      Elenbecestat, E2609, a Bace inhibitor: Results from a phase-2 study in subjects with mild cognitive impairment and mild-to-moderate dementia due to Alzheimer's disease.
      )
      APP/PS1APP751 K670 N,M671L, PS1G384A

      Accumulation in MAMs(
      • Del Prete D.
      • Suski J.M.
      • Oules B.
      • Debayle D.
      • Gay A.S.
      • Lacas-Gervais S.
      • Bussiere R.
      • Bauer C.
      • Pinton P.
      • Paterlini-Brechot P.
      • Wieckowski M.R.
      • Checler F.
      • Chami M.
      Localization and processing of the amyloid-beta protein precursor in mitochondria-associated membranes.
      )
      APPE663QAPPE663QLysosomal dysfunction, inflammation(
      • Kaur G.
      • Pawlik M.
      • Gandy S.E.
      • Ehrlich M.E.
      • Smiley J.F.
      • Levy E.
      Lysosomal dysfunction in the brain of a mouse model with intraneuronal accumulation of carboxyl terminal fragments of the amyloid precursor protein.
      )
      TgCRND8APPKM670/671NL, APPV717FBrain network alterations(
      • Cavanagh C.
      • Colby-Milley J.
      • Bouvier D.
      • Farso M.
      • Chabot J.G.
      • Quirion R.
      • Krantic S.
      betaCTF-correlated burst of hippocampal TNFalpha occurs at a very early, pre-plaque stage in the TgCRND8 mouse model of Alzheimer's disease.
      ,
      • Hamm V.
      • Heraud C.
      • Bott J.B.
      • Herbeaux K.
      • Strittmatter C.
      • Mathis C.
      • Goutagny R.
      Differential contribution of APP metabolites to early cognitive deficits in a TgCRND8 mouse model of Alzheimer's disease.
      )
      J20APPKM670/671NL, APPV717FBrain network alterations(
      • Mondragon-Rodriguez S.
      • Gu N.
      • Manseau F.
      • Williams S.
      Alzheimer's transgenic model is characterized by very early brain network alterations and beta-CTF fragment accumulation: Reversal by beta-secretase inhibition.
      )
      Knock-in APP NL

      APPNL-F and APPNL-G-F

      APPHm/Hm
      APP KM670/671NL, APP KM670/671NL,I716F

      APPKM670/671NL,EI716F,E693G

      hAβ sequence (G676R,F681Y,R684H)
      Nd

      Nd
      (
      • Sasaguri H.
      • Nilsson P.
      • Hashimoto S.
      • Nagata K.
      • Saito T.
      • De Strooper B.
      • Hardy J.
      • Vassar R.
      • Winblad B.
      • Saido T.C.
      APP mouse models for Alzheimer's disease preclinical studies.
      ,
      • Johnson E.C.B.
      • Ho K.
      • Yu G.Q.
      • Das M.
      • Sanchez P.E.
      • Djukic B.
      • Lopez I.
      • Yu X.
      • Gill M.
      • Zhang W.
      • Paz J.T.
      • Palop J.J.
      • Mucke L.
      Behavioral and neural network abnormalities in human APP transgenic mice resemble those of App knock-in mice and are modulated by familial Alzheimer's disease mutations but not by inhibition of BACE1.
      ,
      • Serneels L.
      • T'Syen D.
      • Perez-Benito L.
      • Theys T.
      • Holt M.G.
      • De Strooper B.
      Modeling the beta-secretase cleavage site and humanizing amyloid-beta precursor protein in rat and mouse to study Alzheimer's disease.
      )
      Human IPSCsVarious APP and PS mutantsLysosomal dysfunction

      Endosomal pathology
      (
      • Hung C.O.Y.
      • Livesey F.J.
      Altered gamma-secretase processing of APP disrupts lysosome and autophagosome function in monogenic Alzheimer's disease.
      )
      Down syndrome fibroblastsTs65DN mice or human patientsEndosomal pathology lysosomal dysfunction

      Axonal transport defects
      (
      • Jiang Y.
      • Mullaney K.A.
      • Peterhoff C.M.
      • Che S.
      • Schmidt S.D.
      • Boyer-Boiteau A.
      • Ginsberg S.D.
      • Cataldo A.M.
      • Mathews P.M.
      • Nixon R.A.
      Alzheimer's-related endosome dysfunction in Down syndrome is Abeta-independent but requires APP and is reversed by BACE-1 inhibition.
      ,
      • Kim S.
      • Sato Y.
      • Mohan P.S.
      • Peterhoff C.
      • Pensalfini A.
      • Rigoglioso A.
      • Jiang Y.
      • Nixon R.A.
      Evidence that the rab5 effector APPL1 mediates APP-betaCTF-induced dysfunction of endosomes in Down syndrome and Alzheimer's disease.
      ,
      • Xu W.
      • Weissmiller A.M.
      • White 2nd, J.A.
      • Fang F.
      • Wang X.
      • Wu Y.
      • Pearn M.L.
      • Zhao X.
      • Sawa M.
      • Chen S.
      • Gunawardena S.
      • Ding J.
      • Mobley W.C.
      • Wu C.
      Amyloid precursor protein-mediated endocytic pathway disruption induces axonal dysfunction and neurodegeneration.
      ,
      • Jiang Y.
      • Sato Y.
      • Im E.
      • Berg M.
      • Bordi M.
      • Darji S.
      • Kumar A.
      • Mohan P.S.
      • Bandyopadhyay U.
      • Diaz A.
      • Cuervo A.M.
      • Nixon R.A.
      Lysosomal dysfunction in down syndrome is APP-dependent and mediated by APP-betaCTF (C99).
      )
      Human brainFAD

      SAD
      Nd

      Correlation with neuronal vulnerability
      (
      • Pulina M.V.
      • Hopkins M.
      • Haroutunian V.
      • Greengard P.
      • Bustos V.
      C99 selectively accumulates in vulnerable neurons in Alzheimer's disease.
      ,
      • Vaillant-Beuchot L.
      • Mary A.
      • Pardossi-Piquard R.
      • Bourgeois A.
      • Lauritzen I.
      • Eysert F.
      • Kinoshita P.F.
      • Cazareth J.
      • Badot C.
      • Fragaki K.
      • Bussiere R.
      • Martin C.
      • Mary R.
      • Bauer C.
      • Pagnotta S.
      • et al.
      Amylois precursor protein C-terminal fragments accumulation triggers mitochondrial structure, function and mitophagy defects in Alzheimer’s disease models and human brains.
      ,
      • Pera M.
      • Alcolea D.
      • Sanchez-Valle R.
      • Guardia-Laguarta C.
      • Colom-Cadena M.
      • Badiola N.
      • Suarez-Calvet M.
      • Llado A.
      • Barrera-Ocampo A.A.
      • Sepulveda-Falla D.
      • Blesa R.
      • Molinuevo J.L.
      • Clarimon J.
      • Ferrer I.
      • Gelpi E.
      • et al.
      Distinct patterns of APP processing in the CNS in autosomal-dominant and sporadic Alzheimer disease.
      ,
      • Hebert S.S.
      • Horre K.
      • Nicolai L.
      • Papadopoulou A.S.
      • Mandemakers W.
      • Silahtaroglu A.N.
      • Kauppinen S.
      • Delacourte A.
      • De Strooper B.
      Loss of microRNA cluster miR-29a/b-1 in sporadic Alzheimer's disease correlates with increased BACE1/beta-secretase expression.
      )
      APP, amyloid precursor protein; FAD, familial Alzheimer disease; IPSC, induced pluripotent stem cells; LTP, long-term potentiation; MAPT, microtubule-associated protein tau; nd, not determined; PS, presenilin; SAD, sporadic Alzheimers disease.
      In AD mouse models, such as in 3xTg-AD mice, C99 accumulation was described to be both the cause and the consequence of endosomal perturbations, ultimately leading to failure of lysosomal and autophagic processes (
      • Lauritzen I.
      • Pardossi-Piquard R.
      • Bourgeois A.
      • Pagnotta S.
      • Biferi M.G.
      • Barkats M.
      • Lacor P.
      • Klein W.
      • Bauer C.
      • Checler F.
      Intraneuronal aggregation of the beta-CTF fragment of APP (C99) induces Abeta-independent lysosomal-autophagic pathology.
      ). A similar link between C99 accumulation and lysosomal dysfunction was described in the mouse bearing the Dutch mutation APPE693Q (
      • Kaur G.
      • Pawlik M.
      • Gandy S.E.
      • Ehrlich M.E.
      • Smiley J.F.
      • Levy E.
      Lysosomal dysfunction in the brain of a mouse model with intraneuronal accumulation of carboxyl terminal fragments of the amyloid precursor protein.
      ). Moreover, in fibroblasts from Down syndrome patients or from the mouse model Ts65Dn, C99 were found to induce enlargement of Rab5 positive early endosomes, aberrant endocytosis, and impaired endosomal transport (
      • Jiang Y.
      • Mullaney K.A.
      • Peterhoff C.M.
      • Che S.
      • Schmidt S.D.
      • Boyer-Boiteau A.
      • Ginsberg S.D.
      • Cataldo A.M.
      • Mathews P.M.
      • Nixon R.A.
      Alzheimer's-related endosome dysfunction in Down syndrome is Abeta-independent but requires APP and is reversed by BACE-1 inhibition.
      ). Interestingly, these alterations were fully Aβ-independent, since they were rescued by β-secretase inhibitors or partial BACE1 genetic depletion and were enhanced by GSIs (
      • Lauritzen I.
      • Pardossi-Piquard R.
      • Bourgeois A.
      • Pagnotta S.
      • Biferi M.G.
      • Barkats M.
      • Lacor P.
      • Klein W.
      • Bauer C.
      • Checler F.
      Intraneuronal aggregation of the beta-CTF fragment of APP (C99) induces Abeta-independent lysosomal-autophagic pathology.
      ,
      • Jiang Y.
      • Mullaney K.A.
      • Peterhoff C.M.
      • Che S.
      • Schmidt S.D.
      • Boyer-Boiteau A.
      • Ginsberg S.D.
      • Cataldo A.M.
      • Mathews P.M.
      • Nixon R.A.
      Alzheimer's-related endosome dysfunction in Down syndrome is Abeta-independent but requires APP and is reversed by BACE-1 inhibition.
      ,
      • Jiang Y.
      • Rigoglioso A.
      • Peterhoff C.M.
      • Pawlik M.
      • Sato Y.
      • Bleiwas C.
      • Stavrides P.
      • Smiley J.F.
      • Ginsberg S.D.
      • Mathews P.M.
      • Levy E.
      • Nixon R.A.
      Partial BACE1 reduction in a down syndrome mouse model blocks Alzheimer-related endosomal anomalies and cholinergic neurodegeneration: Role of APP-CTF.
      ,
      • Nixon R.A.
      Amyloid precursor protein and endosomal-lysosomal dysfunction in Alzheimer's disease: Inseparable partners in a multifactorial disease.
      ). Of utmost interest, C99 accumulation has also been associated to endolysosomal dysfunction in human cellular AD models. Indeed, the earlier mentioned transcriptomic analysis of induced pluripotent stem cells (iPSCs) harboring AD-related APP or PS1 mutations indicated similar changes in endocytosis-associated genes (
      • Kwart D.
      • Gregg A.
      • Scheckel C.
      • Murphy E.A.
      • Paquet D.
      • Duffield M.
      • Fak J.
      • Olsen O.
      • Darnell R.B.
      • Tessier-Lavigne M.
      A large panel of isogenic APP and PSEN1 mutant human iPSC neurons reveals shared endosomal abnormalities mediated by APP beta-CTFs, not Abeta.
      ). Strikingly, these mutations brought divergent data concerning Aβ, but they all displayed increases in C99 levels as well as endosomal dysfunction (
      • Kwart D.
      • Gregg A.
      • Scheckel C.
      • Murphy E.A.
      • Paquet D.
      • Duffield M.
      • Fak J.
      • Olsen O.
      • Darnell R.B.
      • Tessier-Lavigne M.
      A large panel of isogenic APP and PSEN1 mutant human iPSC neurons reveals shared endosomal abnormalities mediated by APP beta-CTFs, not Abeta.
      ). The use of notably a BACE inhibitor, GSMs and GSIs, showed that endosomal pathology was linked to C99 and not to the changes in Aβ (
      • Kwart D.
      • Gregg A.
      • Scheckel C.
      • Murphy E.A.
      • Paquet D.
      • Duffield M.
      • Fak J.
      • Olsen O.
      • Darnell R.B.
      • Tessier-Lavigne M.
      A large panel of isogenic APP and PSEN1 mutant human iPSC neurons reveals shared endosomal abnormalities mediated by APP beta-CTFs, not Abeta.
      ). Resembling data were obtained in another study using iPSCs-derived neurons obtained from AD patients harboring APP or PS1 mutations, which displayed both C99 accumulation and dysfunction of the endosomal–lysosomal network (
      • Hung C.O.Y.
      • Livesey F.J.
      Altered gamma-secretase processing of APP disrupts lysosome and autophagosome function in monogenic Alzheimer's disease.
      ). As a difference to the work of Kwarts and colleagues, mutations in APP but not in PS1 led to early endosomal (Rab5 associated) pathology, but all mutations led to enlarged late endosomes and defective lysosomal degradation. These effects were APP-dependent, reversed by BACE inhibition, and exacerbated by GSIs. These results agree with the consistent data linking C99 to a dysfunction in the endolysosomal network occurring even in the presence of physiological APP levels (
      • Lauritzen I.
      • Pardossi-Piquard R.
      • Bourgeois A.
      • Becot A.
      • Checler F.
      Does intraneuronal accumulation of carboxyl-terminal fragments of the amyloid precursor protein trigger early neurotoxicity in Alzheimer's disease?.
      ). Whether C99 accumulation in these systems is linked to the partial loss of function of γ-secretase or to other PS but γ-secretase-independent mechanisms, such as impaired lysosomal-autophagic degradation, as discussed earlier in this review, remains to be established. Related to endolysosomal dysfunction, the pathogenicity of C99 could also be due to its propensity to aggregate and to spread through exosomes, small vesicles secreted from cells (
      • Lauritzen I.
      • Becot A.
      • Bourgeois A.
      • Pardossi-Piquard R.
      • Biferi M.G.
      • Barkats M.
      • Checler F.
      Targeting gamma-secretase triggers the selective enrichment of oligomeric APP-CTFs in brain extracellular vesicles from Alzheimer cell and mouse models.
      ). Indeed, exosomes originate from endosomes and C99 is detected in these vesicles (
      • Lauritzen I.
      • Becot A.
      • Bourgeois A.
      • Pardossi-Piquard R.
      • Biferi M.G.
      • Barkats M.
      • Checler F.
      Targeting gamma-secretase triggers the selective enrichment of oligomeric APP-CTFs in brain extracellular vesicles from Alzheimer cell and mouse models.
      ,
      • Vingtdeux V.
      • Hamdane M.
      • Loyens A.
      • Gele P.
      • Drobeck H.
      • Begard S.
      • Galas M.C.
      • Delacourte A.
      • Beauvillain J.C.
      • Buee L.
      • Sergeant N.
      Alkalizing drugs induce accumulation of amyloid precursor protein by-products in luminal vesicles of multivesicular bodies.
      ,
      • Miranda A.M.
      • Lasiecka Z.M.
      • Xu Y.
      • Neufeld J.
      • Shahriar S.
      • Simoes S.
      • Chan R.B.
      • Oliveira T.G.
      • Small S.A.
      • Di Paolo G.
      Neuronal lysosomal dysfunction releases exosomes harboring APP C-terminal fragments and unique lipid signatures.
      ). Our recent work showed the possible presence of C99 existing as oligomers (C99 homomers and heteromers composed of C99/C83) in exosomes, the levels of which were drastically enhanced upon γ-secretase inhibition (
      • Lauritzen I.
      • Becot A.
      • Bourgeois A.
      • Pardossi-Piquard R.
      • Biferi M.G.
      • Barkats M.
      • Checler F.
      Targeting gamma-secretase triggers the selective enrichment of oligomeric APP-CTFs in brain extracellular vesicles from Alzheimer cell and mouse models.
      ). The latter statement is of importance due to the proposed role of exosomes in prion-like transmission of neurotoxic molecules (
      • Jucker M.
      • Walker L.C.
      Self-propagation of pathogenic protein aggregates in neurodegenerative diseases.
      ).
      In addition to endosomal and lysosomal alterations, mitochondrial dysfunction is a key feature of AD with altered mitochondrial potential, increased levels of reactive oxygen species (ROS) (
      • Wang X.
      • Wang W.
      • Li L.
      • Perry G.
      • Lee H.G.
      • Zhu X.
      Oxidative stress and mitochondrial dysfunction in Alzheimer's disease.
      ,
      • Bussiere R.
      • Lacampagne A.
      • Reiken S.
      • Liu X.
      • Scheuerman V.
      • Zalk R.
      • Martin C.
      • Checler F.
      • Marks A.R.
      • Chami M.
      Amyloid beta production is regulated by beta2-adrenergic signaling-mediated post-translational modifications of the ryanodine receptor.
      ,
      • Lacampagne A.
      • Liu X.
      • Reiken S.
      • Bussiere R.
      • Meli A.C.
      • Lauritzen I.
      • Teich A.F.
      • Zalk R.
      • Saint N.
      • Arancio O.
      • Bauer C.
      • Duprat F.
      • Briggs C.A.
      • Chakroborty S.
      • Stutzmann G.E.
      • et al.
      Post-translational remodeling of ryanodine receptor induces calcium leak leading to Alzheimer's disease-like pathologies and cognitive deficits.
      ), as well as a defect in clearance of abnormal mitochondria (mitophagy) (
      • Manczak M.
      • Calkins M.J.
      • Reddy P.H.
      Impaired mitochondrial dynamics and abnormal interaction of amyloid beta with mitochondrial protein Drp1 in neurons from patients with Alzheimer's disease: Implications for neuronal damage.
      ,
      • Reddy P.H.
      • Yin X.
      • Manczak M.
      • Kumar S.
      • Pradeepkiran J.A.
      • Vijayan M.
      • Reddy A.P.
      Mutant APP and amyloid beta-induced defective autophagy, mitophagy, mitochondrial structural and functional changes and synaptic damage in hippocampal neurons from Alzheimer's disease.
      ). Our recent work proposed that in cellular models, C99 can trigger all these alterations (
      • Vaillant-Beuchot L.
      • Mary A.
      • Pardossi-Piquard R.
      • Bourgeois A.
      • Lauritzen I.
      • Eysert F.
      • Kinoshita P.F.
      • Cazareth J.
      • Badot C.
      • Fragaki K.
      • Bussiere R.
      • Martin C.
      • Mary R.
      • Bauer C.
      • Pagnotta S.
      • et al.
      Amylois precursor protein C-terminal fragments accumulation triggers mitochondrial structure, function and mitophagy defects in Alzheimer’s disease models and human brains.
      ). In agreement, mitochondrial structure alterations and mitophagy defects were observed in young preplaque 3xTg-AD mice, as well as in virus-induced C99 expressing mice (AAV10-C99 mice) and notably following GSI treatment (
      • Vaillant-Beuchot L.
      • Mary A.
      • Pardossi-Piquard R.
      • Bourgeois A.
      • Lauritzen I.
      • Eysert F.
      • Kinoshita P.F.
      • Cazareth J.
      • Badot C.
      • Fragaki K.
      • Bussiere R.
      • Martin C.
      • Mary R.
      • Bauer C.
      • Pagnotta S.
      • et al.
      Amylois precursor protein C-terminal fragments accumulation triggers mitochondrial structure, function and mitophagy defects in Alzheimer’s disease models and human brains.
      ). Other studies have demonstrated C99 accumulation in mitochondria-associated membranes (MAMs) (
      • Pera M.
      • Larrea D.
      • Guardia-Laguarta C.
      • Montesinos J.
      • Velasco K.R.
      • Agrawal R.R.
      • Xu Y.
      • Chan R.B.
      • Di Paolo G.
      • Mehler M.F.
      • Perumal G.S.
      • Macaluso F.P.
      • Freyberg Z.Z.
      • Acin-Perez R.
      • Enriquez J.A.
      • et al.
      Increased localization of APP-C99 in mitochondria-associated ER membranes causes mitochondrial dysfunction in Alzheimer disease.
      ,
      • Del Prete D.
      • Suski J.M.
      • Oules B.
      • Debayle D.
      • Gay A.S.
      • Lacas-Gervais S.
      • Bussiere R.
      • Bauer C.
      • Pinton P.
      • Paterlini-Brechot P.
      • Wieckowski M.R.
      • Checler F.
      • Chami M.
      Localization and processing of the amyloid-beta protein precursor in mitochondria-associated membranes.
      ) and have proposed its contribution to neutral lipid accumulation (
      • Del Prete D.
      • Suski J.M.
      • Oules B.
      • Debayle D.
      • Gay A.S.
      • Lacas-Gervais S.
      • Bussiere R.
      • Bauer C.
      • Pinton P.
      • Paterlini-Brechot P.
      • Wieckowski M.R.
      • Checler F.
      • Chami M.
      Localization and processing of the amyloid-beta protein precursor in mitochondria-associated membranes.
      ) and cholesterol trafficking (
      • Montesinos J.
      • Pera M.
      • Larrea D.
      • Guardia-Laguarta C.
      • Agrawal R.R.
      • Velasco K.R.
      • Yun T.D.
      • Stavrovskaya I.G.
      • Xu Y.
      • Koo S.Y.
      • Snead A.M.
      • Sproul A.A.
      • Area-Gomez E.
      The Alzheimer's disease-associated C99 fragment of APP regulates cellular cholesterol trafficking.
      ).
      Neuroinflammation is also a key feature of AD, but little is known about its molecular triggers. The contribution of C99 to neuroinflammation was proposed as early as in 1998 in mice, in which the intracerebroventricular injection of recombinant C99 (CT105) was found to induce reactive gliosis and neurodegeneration (
      • Song D.-K.
      • Won M.-H.
      • Jung J.-S.
      • Lee J.-C.
      • Kang T.-C.
      • Suh H.-W.
      • Huh S.-O.
      • Paek S.-H.
      • Kim Y.-H.
      • Kim S.-H.
      • Suh Y.-H.
      Behavioral and neuropathological changes induced by central injection of carboxyl-terminal fragment of b-amyloid precursor protein in mice.
      ). CT105 was also found to interact with inflammatory cytokines and produce a synergistic effect on working memory (
      • Matsumoto Y.
      • Watanabe S.
      • Suh Y.H.
      • Yamamoto T.
      Effects of intrahippocampal CT105, a carboxyl terminal fragment of beta-amyloid precursor protein, alone/with inflammatory cytokines on working memory in rats.
      ). Two more recent works also proposed a link between intraneuronal accumulation of C99 and astrocytic activation and astrogliosis in 3xTgAD and APPE693Q mice, respectively (
      • Lauritzen I.
      • Pardossi-Piquard R.
      • Bauer C.
      • Brigham E.
      • Abraham J.D.
      • Ranaldi S.
      • Fraser P.
      • St-George-Hyslop P.
      • Le Thuc O.
      • Espin V.
      • Chami L.
      • Dunys J.
      • Checler F.
      The beta-secretase-derived C-terminal fragment of betaAPP, C99, but not Abeta, is a key contributor to early intraneuronal lesions in triple-transgenic mouse hippocampus.
      ,
      • Kaur G.
      • Pawlik M.
      • Gandy S.E.
      • Ehrlich M.E.
      • Smiley J.F.
      • Levy E.
      Lysosomal dysfunction in the brain of a mouse model with intraneuronal accumulation of carboxyl terminal fragments of the amyloid precursor protein.
      ). Furthermore, in preplaque TgCRND8 mice, C99 was shown to correlate with a TNFα augmentation and microglial activation (
      • Cavanagh C.
      • Colby-Milley J.
      • Bouvier D.
      • Farso M.
      • Chabot J.G.
      • Quirion R.
      • Krantic S.
      betaCTF-correlated burst of hippocampal TNFalpha occurs at a very early, pre-plaque stage in the TgCRND8 mouse model of Alzheimer's disease.
      ).
      Does C99 accumulation also trigger functional and cognitive alterations? It is well established that AD is characterized by a hippocampal altered long-term potentiation (LTP) and depression (LTD) (
      • Selkoe D.J.
      Alzheimer's disease is a synaptic failure.
      ), as well as network activity dysfunctions (
      • Palop J.J.
      • Mucke L.
      Network abnormalities and interneuron dysfunction in Alzheimer disease.
      ). All of these electrophysiological signatures are linked to memory consolidation and learning ability and appear altered before senile plaques and neurodegeneration in human AD (
      • Nistico R.
      • Pignatelli M.
      • Piccinin S.
      • Mercuri N.B.
      • Collingridge G.
      Targeting synaptic dysfunction in Alzheimer's disease therapy.
      ) and many mouse models. The first evidence of a neurotoxic effect of C99 on synaptic function was demonstrated years ago in a transgenic mouse model expressing directly the C99 fragment (
      • Nalbantoglu J.
      • Tirado-Santiago G.
      • Lahsaïnl A.
      • Poirier J.
      • Goncalves O.
      • Verge G.
      • Momoll F.
      • Welner S.A.
      • Massicotte G.
      • Julien J.-P.
      • Shapiro M.L.
      Impaired learning and LTP in mice expressing the carboxy-terminus of the Alzheimer amyloid precursor protein.
      ). These mice were found to develop both LTP and memory alterations, as well as neuroinflammation and neuronal loss. Our own recent studies in AAV10-C99 mice showed that LTP was significantly reduced in preplaque mice and that GSIs did not reverse LTP alterations (
      • Lauritzen I.
      • Pardossi-Piquard R.
      • Bourgeois A.
      • Pagnotta S.
      • Biferi M.G.
      • Barkats M.
      • Lacor P.
      • Klein W.
      • Bauer C.
      • Checler F.
      Intraneuronal aggregation of the beta-CTF fragment of APP (C99) induces Abeta-independent lysosomal-autophagic pathology.
      ). Synaptic alterations also seemed to be temporarily and spatially correlated to C99 accumulation in 3xTgAD mice (
      • Pardossi-Piquard R.
      • Lauritzen I.
      • Bauer C.
      • Sacco G.
      • Robert P.
      • Checler F.
      Influence of genetic background on apathy-like behavior in triple transgenic AD mice.
      ) and in the Danish dementia mouse model, in which a deficiency in the BRI2 protein leads to increased APP levels and in which LTP alterations and memory defects were rescued by the pharmacological inhibition of β-secretase, but not γ-secretase (
      • Tamayev R.
      • Matsuda S.
      • Arancio O.
      • D'Adamio L.
      beta- but not gamma-secretase proteolysis of APP causes synaptic and memory deficits in a mouse model of dementia.
      ). Hippocampal network oscillations, as well as thêta/gamma coupling alterations, were reported at early “Aβ-free” stages in the Tg-CRND8 mouse (
      • Goutagny R.
      • Gu N.
      • Cavanagh C.
      • Jackson J.
      • Chabot J.G.
      • Quirion R.
      • Krantic S.
      • Williams S.
      Alterations in hippocampal network oscillations and theta-gamma coupling arise before Abeta overproduction in a mouse model of Alzheimer's disease.
      ). In this study, these early network defects occurred in C99-accumulating regions and were fully rescued by β-secretase inhibition (
      • Mondragon-Rodriguez S.
      • Gu N.
      • Manseau F.
      • Williams S.
      Alzheimer's transgenic model is characterized by very early brain network alterations and beta-CTF fragment accumulation: Reversal by beta-secretase inhibition.
      ). In contrast, another recent study reported an absence of rescue of network alterations by β-secretase inhibition in the J20 model, although the levels of both C99 and Aβ were clearly reduced (
      • Johnson E.C.B.
      • Ho K.
      • Yu G.Q.
      • Das M.
      • Sanchez P.E.
      • Djukic B.
      • Lopez I.
      • Yu X.
      • Gill M.
      • Zhang W.
      • Paz J.T.
      • Palop J.J.
      • Mucke L.
      Behavioral and neural network abnormalities in human APP transgenic mice resemble those of App knock-in mice and are modulated by familial Alzheimer's disease mutations but not by inhibition of BACE1.
      ). These data show that synaptic function is governed by complex mechanisms that may be difficult to rescue. First of all, in these studies β-secretase inhibition led to a decrease in C99 and Aβ, but it also led to a concomitant increase in C83 that maybe could contribute to these alterations, although this was not discussed by the authors. Second of all, and maybe more importantly, the lack of rescue by BACE1 inhibitors could be due to the suppression of important cell signaling pathways involved in synaptic function, due to the inactivation of other BACE1 substrates, such as Neuregulin-1 and Sez6 (
      • Filser S.
      • Ovsepian S.V.
      • Masana M.
      • Blazquez-Llorca L.
      • Brandt Elvang A.
      • Volbracht C.
      • Muller M.B.
      • Jung C.K.
      • Herms J.
      Pharmacological inhibition of BACE1 impairs synaptic plasticity and cognitive functions.
      ,
      • Yan R.
      • Vassar R.
      Targeting the beta secretase BACE1 for Alzheimer's disease therapy.
      ). In the same way, studies investigating the role of C99 accumulation in AD-related memory impairment in mouse AD models have reported conflicting data. In 3xTgAD mice, C99 accumulation seems to trigger both electrophysiological hippocampal defects and mild cognitive dysfunction (
      • Pardossi-Piquard R.
      • Lauritzen I.
      • Bauer C.
      • Sacco G.
      • Robert P.
      • Checler F.
      Influence of genetic background on apathy-like behavior in triple transgenic AD mice.
      ,
      • Bourgeois A.
      • Lauritzen I.
      • Lorivel T.
      • Bauer C.
      • Checler F.
      • Pardossi-Piquard R.
      Intraneuronal accumulation of C99 contributes to synaptic alterations, apathy-like behavior, and spatial learning deficits in 3xTgAD and 2xTgAD mice.
      ). Similar defects were observed in CT105-expressing mice (
      • Nalbantoglu J.
      • Tirado-Santiago G.
      • Lahsaïnl A.
      • Poirier J.
      • Goncalves O.
      • Verge G.
      • Momoll F.
      • Welner S.A.
      • Massicotte G.
      • Julien J.-P.
      • Shapiro M.L.
      Impaired learning and LTP in mice expressing the carboxy-terminus of the Alzheimer amyloid precursor protein.
      ). However, the studies from knock-in models seem to argue for an Aβ-rather than a C99-dependent contribution to synaptic alterations. In fact, the two knock-in models APP-NL and APP-NL-F, harboring the Swedish (KM670/671NL) mutation alone (APP-NL), or in combination with the I706F mutation (APP-NL-F), respectively, both accumulate C99 (
      • Sasaguri H.
      • Nilsson P.
      • Hashimoto S.
      • Nagata K.
      • Saito T.
      • De Strooper B.
      • Hardy J.
      • Vassar R.
      • Winblad B.
      • Saido T.C.
      APP mouse models for Alzheimer's disease preclinical studies.
      ), but only the APP-NL-F, displaying higher Aβ levels, develops late-stage cognitive dysfunction (
      • Sasaguri H.
      • Nilsson P.
      • Hashimoto S.
      • Nagata K.
      • Saito T.
      • De Strooper B.
      • Hardy J.
      • Vassar R.
      • Winblad B.
      • Saido T.C.
      APP mouse models for Alzheimer's disease preclinical studies.
      ,
      • Masuda A.
      • Kobayashi Y.
      • Kogo N.
      • Saito T.
      • Saido T.C.
      • Itohara S.
      Cognitive deficits in single App knock-in mouse models.
      ). Moreover, the knock-in mouse APP-NL-G-F harboring a third mutation, the E693G Arctic mutation, which generates a particular high level of oligomerized Aβ, displays cognitive dysfunction at a much younger age. Thus, taken together, these observations could suggest either that only Aβ is toxic or that Aβ and C99 contribute differently to the pathology. Whereas Aβ, as a soluble peptide, could be expected to act mainly outside cells, the toxic effect of membrane-embedded C99 should be intracellular. It is possible that intraneuronal C99 is a trigger of a more slowly developing pathological process that might be more prominent in transgenic mice that display higher levels of C99 than knock-in mice.
      In 3xTgAD mice, these electrophysiological defects are not only accompanied by alterations in spatial learning and memory, but are also associated to a decrease in spontaneous activity that is reminiscent to apathy (
      • Bourgeois A.
      • Lauritzen I.
      • Lorivel T.
      • Bauer C.
      • Checler F.
      • Pardossi-Piquard R.
      Intraneuronal accumulation of C99 contributes to synaptic alterations, apathy-like behavior, and spatial learning deficits in 3xTgAD and 2xTgAD mice.
      ), a phenotype considered as an early neuropsychiatric symptom in AD patients (
      • Robert P.
      Understanding and managing behavioural symptoms in Alzheimer's disease and related dementias: Focus on rivastigmine.
      ,
      • Robert P.H.
      • Berr C.
      • Volteau M.
      • Bertogliati-Fileau C.
      • Benoit M.
      • Guerin O.
      • Sarazin M.
      • Legrain S.
      • Dubois B.
      • Pre A.L.S.G.
      Importance of lack of interest in patients with mild cognitive impairment.
      ). Several observations suggested that this apathy-like behavior could be linked to C99 accumulation. This phenotype not only appears at a stage much before Aβ could be detected, but it similarly occurs in a 2xTg-AD model (harboring APPswe and Tau P30L), which was found to accumulate identical amounts C99 to the 3xTgAD mouse, but which display very low Aβ levels even at late stages (
      • Bourgeois A.
      • Lauritzen I.
      • Lorivel T.
      • Bauer C.
      • Checler F.
      • Pardossi-Piquard R.
      Intraneuronal accumulation of C99 contributes to synaptic alterations, apathy-like behavior, and spatial learning deficits in 3xTgAD and 2xTgAD mice.
      ).

      Is β-secretase a good drug target?

      BACE1 cleavage of APP represents the rate-limiting step of Aβ production, and targeting this enzyme would provide advantage to prevent production of both Aβ and C99. To date, there is no evidence of AD-linked mutations on BACE1, but a clue of an indirect importance of BACE1 in AD came from genetic studies showing that APP mutations lying close to the β-secretase cleaving site strongly affect the cleavage by this enzyme and can either cause early-onset AD (the Swedish and Leuven mutations) or be protective (the Icelandic mutation). BACE1 has also been shown to have a role in SAD as its expression was reported to be increased in AD patients. Thus, BACE1 protein was found to be increased 3-fold in cortical areas of AD patients as compared with age-matched controls (
      • Holsinger R.M.D.
      • McLean C.A.
      • Beyreuther K.
      • Masters C.L.
      • Evin G.
      Increased expression of the amyloid precursor b-secretase in Alzheimer's disease.
      ), and these data were confirmed at the mRNA level in the frontal cortex of SAD patients (
      • Holsinger R.M.D.
      • McLean C.A.
      • Beyreuther K.
      • Masters C.L.
      • Evin G.
      Increased expression of the amyloid precursor b-secretase in Alzheimer's disease.
      ,
      • Li R.
      • Lindholm K.
      • Yang L.B.
      • Yue X.
      • Citron M.
      • Yan R.
      • Beach T.
      • Sue L.
      • Sabbagh M.
      • Cai H.
      • Wong P.
      • Price D.
      • Shen Y.
      Amyloid beta peptide load is correlated with increased beta-secretase activity in sporadic Alzheimer's disease patients.
      ). Yang and colleagues also showed a significant elevation of BACE1 in temporal cortex and hippocampal samples from a cohort of clinically diagnosed and neuropathologically confirmed AD patients (
      • Yang L.-B.
      • Lindholm K.
      • Xia R.
      • Citron M.
      • Xia W.
      • Yang X.-L.
      • Beach T.
      • Sue L.
      • Wong P.C.
      • Price D.L.
      • Li R.
      • Shen Y.
      Elevated b-secretase expression and enzymatic activity detected in sporadic Alzheimer disease.
      ). This study used fluorimetric analysis assays and in vitro proteolysis to confirm that this increase in BACE1 expression led to enhanced functional enzyme. At that stage, most of the studies attempted to correlate BACE1 expression to Aβ load. However, some later studies analyzed C99 levels and reported also an increase in both FAD and SAD brains (
      • Vaillant-Beuchot L.
      • Mary A.
      • Pardossi-Piquard R.
      • Bourgeois A.
      • Lauritzen I.
      • Eysert F.
      • Kinoshita P.F.
      • Cazareth J.
      • Badot C.
      • Fragaki K.
      • Bussiere R.
      • Martin C.
      • Mary R.
      • Bauer C.
      • Pagnotta S.
      • et al.
      Amylois precursor protein C-terminal fragments accumulation triggers mitochondrial structure, function and mitophagy defects in Alzheimer’s disease models and human brains.
      ,
      • Pera M.
      • Alcolea D.
      • Sanchez-Valle R.
      • Guardia-Laguarta C.
      • Colom-Cadena M.
      • Badiola N.
      • Suarez-Calvet M.
      • Llado A.
      • Barrera-Ocampo A.A.
      • Sepulveda-Falla D.
      • Blesa R.
      • Molinuevo J.L.
      • Clarimon J.
      • Ferrer I.
      • Gelpi E.
      • et al.
      Distinct patterns of APP processing in the CNS in autosomal-dominant and sporadic Alzheimer disease.
      ,
      • Hebert S.S.
      • Horre K.
      • Nicolai L.
      • Papadopoulou A.S.
      • Mandemakers W.
      • Silahtaroglu A.N.
      • Kauppinen S.
      • Delacourte A.
      • De Strooper B.
      Loss of microRNA cluster miR-29a/b-1 in sporadic Alzheimer's disease correlates with increased BACE1/beta-secretase expression.
      ). Of most interest, the recent work from Pulina and colleagues quantifying both C99 and Aβ in either controls, mildly or severely-affected AD brains, showed that C99 accumulates specifically in vulnerable neurons and correlated with the degree of cognitive alterations, whereas Aβ load was increased in both vulnerable and resistant brain regions in AD (
      • Pulina M.V.
      • Hopkins M.
      • Haroutunian V.
      • Greengard P.
      • Bustos V.
      C99 selectively accumulates in vulnerable neurons in Alzheimer's disease.
      ). The latter data are to be put back into context with the consistent former observations that Aβ expression and distribution within brain areas do not fully match neuronal loss, cognitive defects severity, and dementia AD progression (
      • Terry R.D.
      • Masliah E.
      • Salmon D.P.
      • Butters N.
      • DeTeresa R.
      • Hill R.
      • Hansen L.A.
      • Katzman R.
      Physical basis of cognitive alterations in Alzheimer's disease: Synapse loss is the major correlate of cognitive impairment.
      ,
      • Arriagada P.V.
      • Growdon J.H.
      • Hedley-Whyte E.T.
      • Hyman B.T.
      Neurofibrillary tangles but not senile plaques parallel duration and severity of Alzheimer's disease.
      ,
      • Nelson P.T.
      • Alafuzoff I.
      • Bigio E.H.
      • Bouras C.
      • Braak H.
      • Cairns N.J.
      • Castellani R.J.
      • Crain B.J.
      • Davies P.
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      • Duyckaerts C.
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      • Hof P.R.
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      • et al.
      Correlation of Alzheimer disease neuropathologic changes with cognitive status: A review of the literature.
      ). It should be emphasized that two recent studies documented that BACE may inactivate an intravesicular domain of APP, thereby exacerbating glutamate release (
      • Yao W.
      • Tambini M.D.
      • Liu X.
      • D'Adamio L.
      Tuning of glutamate, but not GABA, release by an intrasynaptic vesicle APP domain whose function can be modulated by beta- or alpha-secretase cleavage.
      ), a process enhanced in a rat knock-in model bearing the Swedish mutation (
      • Tambini M.D.
      • Yao W.
      • D'Adamio L.
      Facilitation of glutamate, but not GABA, release in Familial Alzheimer's APP mutant Knock-in rats with increased beta-cleavage of APP.
      ).
      As stated above, BACE1 mainly cleaves APP at the β′ site to generate C89 and therefore can be considered to be protective. In addition, BACE1 is also able to cleave Aβ40 and Aβ42 into the nonamyloidogenic (
      • Shi X.P.
      • Tugusheva K.
      • Bruce J.E.
      • Lucka A.
      • Wu G.X.
      • Chen-Dodson E.
      • Price E.
      • Li Y.
      • Xu M.
      • Huang Q.
      • Sardana M.K.
      • Hazuda D.J.
      Beta-secretase cleavage at amino acid residue 34 in the amyloid beta peptide is dependent upon gamma-secretase activity.
      ) and neuroprotective (
      • Caillava C.
      • Ranaldi S.
      • Lauritzen I.
      • Bauer C.
      • Fareh J.
      • Abraham J.D.
      • Checler F.
      Study on Abeta34 biology and detection in transgenic mice brains.
      ) Aβ34. A recent study also proposed another neuroprotective role of BACE1, since the inhibition of this enzyme was found to enhance the levels of both ƞ-secretase and Aƞα, the latter was proposed to be neurotoxic (
      • Willem M.
      • Tahirovic S.
      • Busche M.A.
      • Ovsepian S.V.
      • Chafai M.
      • Kootar S.
      • Hornburg D.
      • Evans L.D.
      • Moore S.
      • Daria A.
      • Hampel H.
      • Muller V.
      • Giudici C.
      • Nuscher B.
      • Wenninger-Weinzierl A.
      • et al.
      eta-Secretase processing of APP inhibits neuronal activity in the hippocampus.
      ). Thus, in AD, the shift from a beneficial to a deleterious BACE1 cleavage could be due to the age-dependent augmentation of BACE1 expression occurring in sporadic AD or to the expression of a subset of pathogenic mutations in FAD, thereby enhancing C99 levels above physiological threshold (
      • Vassar R.
      • Bennett B.D.
      • Babu-Khan S.
      • Khan S.
      • Mendiaz E.A.
      • Denis P.
      • Teplow D.B.
      • Ross S.
      • Amarante P.
      • Loeloff R.
      • Luo Y.
      • Fisher S.
      • Fuller J.
      • Edenson S.
      • Lile J.
      • et al.
      b-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE.
      ).
      All clinical trials targeting BACE-1 that have entered in phase III have been reported to decrease significantly and dose-dependently Aβ levels in the brain, plasma, or CSF (Table 1). However, all of them have been terminated mostly due to reported toxicity. Indeed, many studies have demonstrated serious adverse effects such as an abnormal elevation in liver enzymes and clinical worsening (Table 1). The reasons for these side effects observed in human trials are not well established, but could be related to the high number of BACE1 substrates being involved in vital functions. BACE1 is known to cleave nearly 40 different neuronal membrane proteins (
      • Kuhn P.H.
      • Koroniak K.
      • Hogl S.
      • Colombo A.
      • Zeitschel U.
      • Willem M.
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      • Schepers U.
      • Imhof A.
      • Hoffmeister A.
      • Haass C.
      • Rossner S.
      • Brase S.
      • Lichtenthaler S.F.
      Secretome protein enrichment identifies physiological BACE1 protease substrates in neurons.
      ) and some of these substrates, such as Sez6 or neuron myelination like neuregulin-1, are known to be essential for synaptic function (
      • Cai H.
      • Wang Y.
      • McCarthy D.
      • Wen H.
      • Borchelt D.R.
      • Price D.L.
      • Wong P.C.
      BACE1 is the major b-secretase for generation of Ab peptides by neurons.
      ). Indeed, several studies reported that BACE1 inhibition triggers synaptic and cognitive dysfunctions (
      • Filser S.
      • Ovsepian S.V.
      • Masana M.
      • Blazquez-Llorca L.
      • Brandt Elvang A.
      • Volbracht C.
      • Muller M.B.
      • Jung C.K.
      • Herms J.
      Pharmacological inhibition of BACE1 impairs synaptic plasticity and cognitive functions.
      ,
      • Yan R.
      • Vassar R.
      Targeting the beta secretase BACE1 for Alzheimer's disease therapy.
      ). BACE1 inhibitors could also cross over with not only its homologue BACE2, but also with cathepsin D, another aspartyl protease (
      • Chevallier N.
      • Vizzavona J.
      • Marambaud P.
      • Baur C.P.
      • Spillantini M.
      • Fulcrand P.
      • Martinez J.
      • Goedert M.
      • Vincent J.P.
      • Checler F.
      Cathepsin D displays in vitro b-secretase-like specificity.