Intraneuronal aggregation of the βCTF fragment of

Authors: Inger Lauritzen Raphaëlle PardossiPiquard Alexandre Bourgeois Sophie Pagnotta MariaGrazia Biferi Martine Barkats Pascale Lacor William Klein Charlotte Bauer Frederic Checler

Publish Date: 2016/04/30

Volume: 132, Issue: 2, Pages: 257-276

PDF Link

Abstract

Endosomalautophagiclysosomal EAL dysfunction is an early and prominent neuropathological feature of Alzheimers’s disease yet the exact molecular mechanisms contributing to this pathology remain undefined By combined biochemical immunohistochemical and ultrastructural approaches we demonstrate a link between EAL pathology and the intraneuronal accumulation of the βsecretasederived βAPP fragment C99 in two in vivo models 3xTgAD mice and adenoassociated viralmediated C99infected mice We present a pathological loop in which the accumulation of C99 is both the effect and causality of impaired lysosomalautophagic function The deleterious effect of C99 was found to be linked to its aggregation within EALvesicle membranes leading to disrupted lysosomal proteolysis and autophagic impairment This effect was Aβ independent and was even exacerbated when γsecretase was pharmacologically inhibited No effect was observed in inhibitortreated wildtype animals suggesting that lysosomal dysfunction was indeed directly linked to C99 accumulation In some brain areas strong C99 expression also led to inflammatory responses and synaptic dysfunction Taken together this work demonstrates a toxic effect of C99 which could underlie some of the earlystage anatomical hallmarks of Alzheimer’s disease pathology Our work also proposes molecular mechanisms likely explaining some of the unfavorable sideeffects associated with γsecretase inhibitordirected therapiesAlzheimer’s disease AD is the most prevalent neurodegenerative disease and cause of dementia in elderly Histopathologically it is characterized by the deposition of extracellular amyloid plaques and intraneuronal neurofibrillary tangles as well as synaptic pathology and neurodegeneration 7 Growing evidence supports that these alterations are preceded and may be even caused by a defective endolysosomal/autophagic degradation 33 36 39 43 50 Indeed endolysosomal/autophagic anomalies are wellrecognized early neuropathological features of AD marked by enlarged endosomes lysosomal alterations and progressive accumulation of autophagic vacuoles AVs 33 34 35 39 43 50 It is also well established that endosomalautophagiclysosomal EAL compartments constitute the main sites of proteolytic processing of the amyloidβ precursor protein βAPP 43 and that amyloidogenic species can accumulate inside organelles of the EAL machinery 12 20 24 26 62 Moreover the accumulation of the aggregateprone Aβ42 within EAL organelles was found to disturb normal EAL function suggesting that it could be both a consequence and a cause of endolysosomalautophagic dysfunction 43EAL dysfunction has been extensively described in transgenic mice displaying ADlike anatomopathology 11 13 47 51 55 61 including in the widely used 3xTgAD mouse APPSwe TauP301L PS1M146V 10 11 37 It is also well known that this mouse model displays an early accumulating and particularly high intraneuronal amyloidlike immunostaining within the subiculum of the hippocampus which was firstly accounted for Aβ 37 Yet we recently demonstrated that this staining corresponded to the βsecretasederived fragment C99 βCTF rather than to Aβ which was only detected at low levels and at late stages in these mice 24 We found that C99 accumulated in enlarged cathepsin Bpositive structures suggesting a link between C99 and EAL pathology 24 In this work we establish that C99 accumulation is the consequence of impaired lysosomalautophagic degradation but that C99 in turn also contributes to lysosomal dysfunction Interestingly these effects of C99 were observed in the 3xTgAD mouse in which C99 is generated by proteolytically APP processing but also in a direct transgenic C99expressing mouse C99 fragments were found to aggregate within membranes of EAL vesicles proposing that C99 induces autophagiclysosomal dysfunction by interfering with EAL membrane integrity The pharmacological inhibition of γsecretase increased the levels of EALassociated C99 and exacerbated pathology clearly demonstrating a C99 dosedependent but Aβindependent effect Overall our work demonstrates a detrimental loop in which aggregated C99 contributes to anatomical hallmarks reminiscent of those occurring early in Alzheimer’s diseaseVirus production was performed following a protocol previously described 4 Briefly HEK293 cells were transfected with the adenovirus helper plasmid pXX6 the AAV packaging plasmid rAAV2rh10 and the AAV10 plasmid empty vector or encoding either human C99 or GFP under control of the synapsin1 promoter AAVempty AAVsynC99 and AAVsynGFP Viruses were produced purified and vector titers were determined by realtime PCR and expressed as viral genomes per ml vg/ml 43xTgAD harboring PS1M146V βAPPswe and TauP301L transgenes and nontransgenic nonTg mice 37 were generated from breeding pairs provided by Dr LaFerla Irvine USA 2xTgAD PS1wt βAPPswe and TauP301L were produced by crossing 3xTgAD with nonTg mice as described 38 For AAVmediated in vivo delivery 1dayold C57BL6 mice Janvier Labs France were injected with 4 µl of AAV virus 55 × 1012 vg/ml viral genomes per ml into the left lateral ventricle as described 21 and mice were analyzed at 2 months postAAV delivery 5monthold nonTg and 3xTgAD males or 2monthold AAVinfected mice males and females were treated daily for 12 or 30 days with the γsecretase inhibitor ELND006 referred to as D6 hereafter 30 mg/kg Elan Pharmaceuticals San Francisco USA 9 45 or with vehicle alone methylcellulose/polysorbate 80 Sigma via oral gavage as described 24 All experimental procedures were in accordance with the European Communities Council Directive of 24 November 1986 86/609/EEC and local French legislationAnimals were deeply anesthetized with a lethal dose of pentobarbital and perfused transcardially with cold phosphatebuffered saline PBS Brains were fixed in 4  Paraformaldehyde/PBS then embedded in paraffin and sliced 8 μm or cut on a vibratome 50 μm For FCA18 82E1 NU1 4G8 and 6E10 staining sections were treated with formic acid 90  or 50 /5 min for paraffin and vibratome sections respectively Sections were then incubated at 4 °C overnight with primary antibodies see supplementary table followed by Alexa Fluorconjugated antibodies Molecular Probes 11000 Cathepsin B labeling was amplified using the Vectorstain ABC kit Vector and streptavidinAlexa594 Molecular Probes 11000 Nuclei were stained with DAPI Roche 120000 and fluorescence was visualized using a confocal microscope Fluoview10 Olympus For DAB development sections were incubated with HRPconjugated secondary antibodies Jackson ImmunoResearch 1/1000 followed by DAB substrate DAB impact Vector Neuronal nuclei were then stained with cresyl violet For quantitative analysis of lamp1 staining in AAVempty and AAVC99 brain slices images were thresholded converted to mask and analyzed using Particle Analysis ImageJ pluginThe human neuroblastoma cell line SHSY5Y naive or stably expressing APPswe or pcDNA3 mock previously described 40 was exposed to the following drugs for 16–20 h D6 50 nM to 5 μM Elan Pharmaceuticals San Francisco in vehicle methylcellulose/polysorbate 80 Sigma DAPT 5 μM in DMSO Sigma leupeptin 10 μM in H2O Sigma PADK 5 μM in DMSO Bachem NH4Cl 10 mM in H2O Sigma pepstatin A 10 μM in EtOH Enzo Life sciences smer28 50 μM in DMSO Sigma and bafilomycin A1 50 nM in H20 Sigma Some cells were transfected with GFPLC3 Addgene using Lipofectamine 2000 according to standard protocols Other cells were infected with lentiviruses LV encoding mouse cathepsin B LVmCatBFUGW2 or control LVFUGW2 32 and polyclonal cell lines stably expressing mCatB naive SHSY5Y or APPsweSHSY5Y cells were obtained For immunocytochemical experiments of C99 expression COS7 cells were transfected with C99 using Lipofectamine 2000

Keywords: