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. 2017 Mar 1;4(4):226-235.
doi: 10.1002/acn3.382. eCollection 2017 Apr.

Tau Oligomers in Cerebrospinal Fluid in Alzheimer's Disease

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Free PMC article

Tau Oligomers in Cerebrospinal Fluid in Alzheimer's Disease

Urmi Sengupta et al. Ann Clin Transl Neurol. .
Free PMC article

Abstract

Objective: With an increasing incidence of Alzheimer's disease (AD) and neurodegenerative tauopathies, there is an urgent need to develop reliable biomarkers for the diagnosis and monitoring of the disease, such as the recently discovered toxic tau oligomers. Here, we aimed to demonstrate the presence of tau oligomers in the cerebrospinal fluid (CSF) of patients with cognitive deficits, and to determine whether tau oligomers could serve as a potential biomarker for AD.

Methods: A multicentric collaborative study involving a double-blinded analysis with a total of 98 subjects with moderate to severe AD (N = 41), mild AD (N = 31), and nondemented control subjects (N = 26), and two pilot studies of 33 total patients with AD (N = 19) and control (N = 14) subjects were performed. We carried out biochemical assays to measure oligomeric tau from CSF of these patients with various degrees of cognitive impairment as well as cognitively normal controls.

Results: Using a highly reproducible indirect ELISA method, we found elevated levels of tau oligomers in AD patients compared to age-matched controls. Western blot analysis confirmed the presence of oligomeric forms of tau in CSF. In addition, the ratio of oligomeric to total tau increased in the order: moderate to severe AD, mild AD, and controls.

Conclusion: These assays are suitable for the analysis of human CSF samples. These results here suggest that CSF tau oligomer measurements could be optimized and added to the panel of CSF biomarkers for the accurate and early detection of AD.

Figures

Figure 1
Figure 1
Detection and biochemical characterization of tau oligomers in ADCSF. (A, D) Representative Western blots of CSF samples from 3 AD patients and 2 controls with anti‐tau oligomer antibodies, T22 and TOMA. Immunoblotting with T22 and TOMA antibodies clearly showed tau aggregates in AD compared to controls. Tau aggregates of differing molecular weights were noted, ranging from 100 kDa and above. (B, E) Densitometric quantifications of higher molecular weight of tau from the Western blots probed with T22 and TOMA antibodies (indicated in boxes) showed elevated tau oligomers in the AD CSF samples (P = 0.01). (C, F) Densitometric quantifications of dimer/trimer revealed significantly increased tau oligomers in AD (P = 0.005 and P = 0.008, respectively) (indicated in boxes). Bands were normalized with α‐albumin, used as loading control. Bars represent median with interquartile ranges. A comparatively weaker band of tau oligomers was also noticed in the ADCSF samples at 250 kDa molecular weight with both T22 and TOMA antibodies (indicated with star). (G) Probing with Tau5 antibody showed co‐localization of signal from dimer/trimer as well as from highest band observed in the blots. (H, I) Anti‐rabbit IgG and anti‐mouse IgG secondary antibodies were used to show the specificity of the antibodies used. AU, arbitrary unit; CSF, cerebrospinal fluid.
Figure 2
Figure 2
Confirming tau oligomers in CSF from multiple independent studies by biochemical analyses. (A–E) Representative Western blot showing differing orders of tau oligomers versus monomer in CSF, using Tau 5, tau 13, Tau 12, tau, 46 and a rabbit polyclonal antibody TTC‐35. Immunolabeling with rabbit polyclonal TTC‐35 demonstrated the presence of both higher tau aggregates as well as its dimer/trimer (indicated in boxes). Tau 13 antibody detected both tau dimer/trimer and monomer (indicated in boxes). Tau 46 demonstrated high‐molecular weight tau as well as its monomer. Whereas, Tau 12 antibody explicitly demonstrated monomeric tau. A band was noted in ADCSF samples at 250 kDa molecular weight (indicated with star) that was also noticed with T22 and TOMA antibodies.CSF, cerebrospinal fluid.
Figure 3
Figure 3
The ratio of tau oligomers to total tau is decreased in AD. (A, B) CSF samples from two independent pilot studies were analyzed by ELISA, using T22 antibody. T22‐reactive tau oligomers from these ELISA assays were then quantified using a standard curve generated against recombinant tau oligomers. ADCSF samples clearly showed significant level of tau oligomers as compared to controls (P = 0.002 and P < 0.0001, Mann‐Whitney test). Data points were represented as median with interquartile ranges. (C) A double blinded analysis of large cohort of CSF samples from moderate‐severe AD (N = 41), mild AD (N = 31) and nondemented controls (N = 26) was performed using T22 antibody in ELISA. Tau oligomers were quantified from this analysis using a standard curve generated against recombinant tau oligomers. Tau oligomers were significantly increased in groups of moderate to severe AD and mild AD patients when compared with controls (P < 0.05, Mann–Whitney test). (D) The ratio of oligomeric tau to total was plotted for all the 3 groups of CSF samples. The oligomeric tau to total tau ratio was significantly increased in control groups in comparison with mild AD and then moderate to severe AD groups (P < 0.0001 between both the groups). Data were represented as median with interquartile ranges. One‐way ANOVA with Dunnets's multiple comparisons test was used for statistical analyses.CSF, cerebrospinal fluid. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

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