Identification of Polypeptides in Neurofibrillary Tangles and Total Homogenates of Brains with Alzheimer's Disease by Tandem Mass Spectrometry

Article type: Research Article

Authors: Minjarez, Benito^a | Rustarazo, Ma. Luz Valero^b | Sanchez del Pino, Manuel M.^{b; 1} | González-Robles, Arturo^c | Sosa-Melgarejo, Jorge A.^d | Luna-Muñoz, Jose^e | Mena, Raul^e | Luna-Arias, Juan Pedro^{a; *}

Affiliations: [a] Departamento de Biología Celular, Cinvestav-IPN, Col. San Pedro Zacatenco, México, D.F., México | [b] Unidad de Proteómica, Centro de Investigación Príncipe Felipe, Valencia, España | [c] Departamento de Infectómica y Patogénesis Molecular, Cinvestav-IPN, Col. San Pedro Zacatenco, México, D.F., México | [d] Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Col. Doctores, México, D.F., México | [e] Departamento de Fisiología, Biofísica y Neurociencias, Cinvestav-IPN, Col. San Pedro Zacatenco, México, D.F., México

Correspondence: [*] Correspondence to: Dr. Juan Pedro Luna-Arias, Departamento de Biología Celular, Cinvestav-IPN, Av. Instituto Politécnico Nacional 2508, Col. San Pedro Zacatenco, 07360 México, D.F., México. Tel.: +52 55 57473800, Ext. 5512 and 5567; Fax: +52 55 57473393; E-mail: [email protected].

Note: [1] Current Address: Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Valencia, Burjassot, Valencia, España.

Abstract: Alzheimer's disease (AD) is the most common cause of dementia in the elderly. AD brains are characterized by the presence of neurofibrillary tangles (NFTs) and neuritic plaques. NFTs are constituted of paired helical filaments, which are structurally composed by assembled hyperphosphorylated and truncated tau polypeptides. To date, the integral constituents of NFTs remain unknown mainly due to the high insolubility of NFTs. The aim of this study was to identify by tandem mass spectrometry, the polypeptides contained in both isolated NFTs by laser capture microdissection and total homogenates, using tissue sections from paraformaldehyde-fixed AD brains. In the first case, we isolated 2,000 NFTs from tissue samples of hippocampus from each of the three Mexican AD brains used in our study. These were previously stained with anti-hyperphosphorylated tau AT-100 antibodies. After the removal of paraformaldehyde and delipidation with organic solvents, we tested three solubilization methods. We identified 102 polypeptides from total homogenates and 41 from isolated NFTs. We selected UCH-L1, transferrin, and GAPDH polypeptides to be studied by immunofluorescence and confocal microscopy. Only UCH-L1 and GAPDH colocalized with hyperphosphorylated tau in NFTs.

Keywords: Alzheimer's disease, neurofibrillary tangles, proteomics, tandem mass spectrometry, tau protein

DOI: 10.3233/JAD-121480

Journal: Journal of Alzheimer's Disease, vol. 34, no. 1, pp. 239-262, 2013