Cerebrospinal Fluid Levels of a 20-22 kDa NH2 Fragment of Human Tau Provide a Novel Neuronal Injury Biomarker in Alzheimer's Disease and Other Dementias

Giuseppina Amadoro, Veronica Corsetti, Giulia Maria Sancesario, Adele Lubrano, Gaia Melchiorri, Sergio Bernardini, Pietro Calissano, Giuseppe Sancesario

Research output: Contribution to journalArticlepeer-review

Abstract

Truncation at N-terminal domain of tau protein is early associated with neurofibrillary pathology in several human tauopathies, including Alzheimer's disease (AD). In affected subjects, the monitoring of total (t-tau) and/or phosphorylated tau (p-tau) levels in cerebrospinal fluid (CSF) provides a reliable, indirect evaluation of cellular changes occurring in vivo and the identification of additional CSF biomarkers would better assist with the clinical practice, allowing a broader profile of underlying ongoing neurodegeneration. Here we show that a 20-22 kDa NH2-truncated form of human tau (i.e., NH2htau), a neurotoxic fragment of the full length protein (htau40) that we previously found in synapses from subjects affected by different tauopathies: (i) is not a normal constituent of CSF, unlike t-tau and p-tau, being exceptionally detected in patients without cognitive impairment; (ii) discriminates, with a weak specificity of 65% but a high sensitivity of 85%, patients carrying a large spectrum of neurodegenerative diseases associated with cognitive deterioration (i.e., AD, frontotemporal lobar degeneration, Parkinson's disease with dementia, vascular dementia, mixed dementia, etc.) from subjects affected by other neurological disorders without mnesic disability; and (iii) is a neuronal injury biomarker as its levels in CSF are not related to the severity and progression of cognitive decline. The dynamic evaluation of NH2htau in CSF might add some useful hints in the ordinary clinical practice as it provides a novel, general biomarker for human tauopathies and other neurodegenerative diseases associated with dementia. Supplementary Figure 1. Specificity of CCP-NH2 4268 tau antibody was checked by RNAi-mediated knockdown of human tau protein in apoptotic NGF/dbAMP-differentiated SH-SY5Y cells. A-C) SH-SY5Y human cells were cultured in differentiation medium (DMEM/F12 medium containing N2, dbcAMP 1 mM and NGF 100 ng/ml) and after 4 h of plating were transfected with either control siRNA or tau siRNA (ThermoScientific Dharmacon, human MAPT L-012488-00), according to the manufacturer's instructions (Xfect siRNA Clontech, 63133). After 24 h of transfection, cells were transfected with either control siRNA or tau siRNA once more and cultured in differentiation medium for further 24 h. Cells treated with either control siRNA or tau siRNA were induced to apoptosis with 0.5 μM STS for 3 h, lysed in 2 × sample buffer and analyzed on SDS-PAGE probing with commercial HT7 anti-antibody (aa 159-163) (A) and with CCP-NH2 4268 tau antiserum [27] (B). α tubulin was used as loading control (C). No band of 20-22 kDa was detected by CCP-NH2 4268 tau antibody after tau RNAi-mediated knockdown in STS-treated SH-SY5Y neurons. JAD140267-Fig S1.tif Supplementary Figure 2. Centrifugal ultrafiltration of whole CSF samples successfully eliminates proteins exceeding the desired 30 kDa molecular weight cut-off. Filtrated CSF samples (up to 3 ml passed through 30 K molecular size membrane) were lyophilized, reconstituted in reducing sample buffer and then separated on SDS-PAGE 15% Bis-Tris gel. After electroblotting, the whole filter was probed with affinity-purified CCP-NH2 4268 tau in order to validate that the performed technical procedure was actually effective in the non-retention of molecular weight (MW) >30 kDa and in the recovery and enrichment of MW 2-end of full length human tau protein, also detects a band of 20-22 kDa immunoreactivity in CSF from AD and OD cohort but not from the OND one. Western blotting analysis of individual CSF from living patients affected by AD (AD) or other non-AD dementias (OD) and from cognitively healthy controls affected by other neurological disorders (OND) probed with DC39N1, a monoclonal antibody which is specific for N-terminal insert N1 (residues 45-73) of human tau protein (Sigma, T8451). A NH2htau-immunopositive band running around 20-22 kDa (arrow) was detected in CSF from AD and OD cohort but not from the OND group. IgG light chains (25-30 kDa) were also evident (asterisks).

Original languageEnglish
Pages (from-to)211-226
Number of pages16
JournalJournal of Alzheimer's Disease
Volume42
Issue number1
DOIs
Publication statusPublished - 2014

Keywords

  • Alzheimer's disease
  • biomarker
  • cerebrospinal fluid
  • tau truncation
  • tauopathies

ASJC Scopus subject areas

  • Psychiatry and Mental health
  • Geriatrics and Gerontology
  • Clinical Psychology
  • Medicine(all)

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