APP mutations in the A! coding region are associated with abundant cerebral deposition of A!38

Transcription

1 APP mutations in the A coding region are associated with abundant cerebral deposition of A38 Acta Neuropathologica Maria Luisa Moro 1, Giorgio Giaccone 1 *, Raffaella Lombardi 1, Antonio Indaco 1, Andrea Uggetti 1, Michela Morbin 1, Stefania Saccucci 1,Giuseppe Di Fede 1, Marcella Catania 1, Dominic M. Walsh 2, Andrea Demarchi 3, Annemieke Rozemuller 4, Nenad Bogdanovic 5, Orso Bugiani 1, Bernardino Ghetti 6, Fabrizio Tagliavini 1. 1 IRCCS Foundation Carlo Besta Neurological Institute, Milano, Italy. 2 Brigham & Women's Hospital, Laboratory for Neurodegenerative Research, Harvard Institute of Medicine, Boston, MA, USA 3 ALS TO2, Ospedale Giovanni Bosco, Torino, Italy. 4 Department of Pathology, VU University Medical Center, Amsterdam,The Netherlands. 5 Department for Neurobiology, Caring Sciences and Society, Division of Clinical Geriatrics, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden. 6 Department of Pathology and Laboratory Medicine, Indiana Alzheimer Disease Center; Indiana University School of Medicine, Indianapolis, IN, USA. *Corresponding author: Giorgio Giaccone, MD Fondazione IRCCS Istituto Neurologico Carlo Besta, Milano, Via Celoria 11, Milano, Italy Tel.: ; fax: giaccone@istituto-besta.it

2 Online Resource SPECIFICTY OF THE ANTI-A38 ANTIBODIES MATERIAL AND METHODS Dot Blots, absorption test and Western blot separation of synthetic A38, A40, A42. In order to verify the specificity of BA1-13 and for A38, solutions of synthetic A38, A40 and A42 were prepared. Each peptide was initially dissolved to 1 mm in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and separated into aliquots, in sterile microcentrifuge tubes. HFIP was then removed by evaporation and the desiccated peptide stored at -80 C, until the day of the experiment. For dot blot, the absorption experiment and the Western Blot (WB) analysis, the synthetic peptides were dissolved in DMSO at the concentration of 5 mm and immediately diluted in sterile PBS and employed for the experiment. For dot blot, 5, 2.5, 1.25, 0.625, 0.312, µg of each peptide (without SDS sample buffer) were spotted on a PVDF membrane and dried in an incubator at 37 C for 5 min. The membrane was incubated with 100% methanol and then water for 30 sec and treated similarly to a WB: after 1 h of blocking step, the membrane was incubated over night with BA1-13 (1:1000). Following a wash step with PBS with 0.1 % Tween 20, IRDye 800 conjugated anti-rabbit was added to the membrane as the secondary antibody, for 45 min, in accordance with the manufacturer's instructions (LI-COR Biosciences, Lincoln, Nebraska, USA). The positive dots were detected at 800 nm by means of a Li-COR Odyssey near-infrared imaging system at resolutions of 84 and169 µm. As further specificity control, BA1-13 (at the same dilution used in IHC) was pre-absorbed over night at 4ºC with synthetic A38, A40 and A42. The antigen to antibody molar ratio in each tube was 20:1. The pre-absorbed antibody was then used in the IHC protocol in place of the primary antibody.

3 In order to prove that the is an effective antibody in WB and specific for A38, synthetic A38, A40 and A42 were aggregated at the concentration of 30 µm, at 4ºC. After 48h, the preparations were sonicated for 20 min and small volumes, corresponding to 250, 500 and 1000 ng of peptide were mixed with 2 x LDS sample buffer, boiled for 8 min and electrophoresed on two 4-12% Bis-Tris gel (Life Technologies, Grand Island, NY, USA). After the transferring and blocking steps, the membrane was incubated over night with clone (1:1000). Subsequently IRDye 800 conjugated anti-rabbit (LI-COR Biosciences) was used as the secondary antibody and the bands detected at 800 nm, by means of a Li-COR Odyssey near-infrared imaging system, at resolution of 169 µm. RESULTS Dot blot analysis demonstrated that BA1-13 recognizes synthetic A38 peptide and not A40 and A42. Moreover BA1-13 has no cross reactivity for the synthetic A40-Gln22, namely the Dutch E693Q APP mutation, one of the mutation investigated in the present study (Supplemental Figure a). Analogue dot blots were used to check the specificity of the other antibodies reactive to the C- terminus of A, specific for the A isoforms that end at the 40 th and 42 th amino acid. We confirmed the absence of any cross-reactivity of these antibodies for A peptides different from the antigen used to generate them. The pre-absorption of BA1-13 with synthetic A38 strongly diminished the immunostaining elicited by this immunoreagent in the amyloid of AD tissue (Supplemental Figure c, d), while BA1-13 pre-absorption with synthetic A40 and A42 had no effects (Supplemental Figure e,f). As we verified that BA1-13 is not effective in WB in denaturing conditions, we used another commercial, monoclonal antibody, identified as clone , to detect A38 monomers and LMW aggregates by WB. We confirmed by WB that recognizes monomers and aggregated forms of the synthetic A38 but not of A40 and A42 peptides (Supplemental Figure b).

4 LEGEND Supplemental Figure BA1-13 and are A38 specific antibodies. The dot blot shows that the anti-a38 antibody, clone BA1-13, recognizes synthetic peptide A38 (a, row 1, dots from left to right: 5, 2.5, 1.25, 0.625, 0.312, µg of A38 and PBS only), while it has no reactivity for the same quantity of synthetic A40 (a, row 2), A42 (a, row 3) and A40- Gln22 (a, row 4). The WB shows that anti-a38 antibody (1:1000) has no immnoreactivity for monomers and aggregates of synthetic A40 (1000, 500, 250 ng, respectively in lane 1, 2, 3 of panel b) and of A42 (1000, 500, 250 ng, respectively in lane 4, 5, 6 of panel b), while is able to recognize monomer, dimer and trimer of synthetic A38 (1000, 500, 250 ng, respectively in lane 7, 8, 9 of panel b). Frontal cortex sections of A713T APP present clear A38 immunoreactivity with BA1-13 antibody at 1:200 dilution (c). The pre-absorption of BA1-13 with synthetic A38 (d) abolishes the immunoreactivity, while no modification of the immunostaining is observed when BA1-13 is preabsorbed with synthetic A40 and A42 (e, f).

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