Disease-Specific Changes in Reelin Protein and mRNA in Neurodegenerative Diseases

Abstract

Reelin is an extracellular glycoprotein that modulates neuronal function and synaptic plasticity in the adult brain. Decreased levels of Reelin activity have been postulated as a key factor during neurodegeneration in Alzheimer´s disease (AD) and in aging. Thus, changes in levels of full-length Reelin and Reelin fragments have been revealed in cerebrospinal fluid (CSF) and in post-mortem brains samples of AD patients with respect to non-AD patients. However, conflicting studies have reported decreased or unchanged levels of full-length Reelin in AD patients compared to control (nND) cases in post-mortem brains and CSF samples. In addition, a compelling analysis of Reelin levels in neurodegenerative diseases other than AD is missing. In this study, we analyzed brain levels of RELN mRNA and Reelin protein in post-mortem frontal cortex samples from different sporadic AD stages, Parkinson's disease with dementia (PDD), and Creutzfeldt-Jakob disease (sCJD), obtained from five different Biobanks. In addition, we measured Reelin protein levels in CSF samples of patients with mild cognitive impairment (MCI), dementia, or sCJD diagnosis and a group of neurologically healthy cases. The results indicate an increase in RELN mRNA in the frontal cortex of advanced stages of AD and in sCJD(I) compared to controls. This was not observed in PDD and early AD stages. However, Reelin protein levels in frontal cortex samples were unchanged between nND and advanced AD stages and PDD. Nevertheless, they decreased in the CSF of patients with dementia in comparison to those not suffering with dementia and patients with MCI. With respect to sCJD, there was a tendency to increase in brain samples in comparison to nND and to decrease in the CSF with respect to nND. In conclusion, Reelin levels in CSF cannot be considered as a diagnostic biomarker for AD or PDD. However, we feel that the CSF Reelin changes observed between MCI, patients with dementia, and sCJD might be helpful in generating a biomarker signature in prodromal studies of unidentified dementia and sCJD.

Keywords: Alzheimer’s disease; Creutzfeldt-Jakob disease; Parkinson´s disease dementia; Reelin; a-synucleopathies; cerebrospinal fluid.

Figure 1

RELN mRNA quantification in postmortem brain samples by RT-qPCR in AD (A), PDD (B), and sCJD (type I and II) (C) compared with non-degenerative (nND) samples using GAPDH for standardization. Each dot corresponds to one postmortem sample. The values were calculated from the RELN mRNA value and normalized to GAPDH value from the same cDNA preparation; the mean ± S.D. for each group of samples is displayed. Only p values indicating statistical differences between groups are displayed in the graphs (except in (C)). p values were determined using ANOVA (Bonferroni post hoc test).

Figure 2

Examples of Reelin-immunostained cells in postmortem human samples. (A) Pyramidal neurons of layer VI of temporal cortex of an nND case. (B) Non-pyramidal neurons immunoreactive for Reelin in the stratum oriens (SO) of an nND case. (C,D) Examples of groups of Reelin-positive dystrophic neurites in the hippocampus proper (stratum oriens) in two different AD cases. (E,F) Photomicrograph illustrating Reelin-positive pyramidal-shaped neurons (arrows) in isocortical layer V in nND and PDD cases. (G) Example of Reelin positive cells in layers I-II (arrows) of the frontal cortex of an AD case. Notice the presence of a horizontally oriented Cajal-Retzius cell (arrowhead) in layer I. Abbreviations: PCL: Pyramidal cell layer. Scale bars: A–B = 75 μm; C–D = 75 μm; E–F = 100 μm; G = 100 μm.

Figure 3

Examples of Western Blotting determination of Reelin in post-mortem brain samples of representative neurodegenerative diseases AD (A), sCJD (B), and PDD (C) with respect to nND. As observed, each quantified gel contains both nND and patient samples. Each gel well was loaded with 15 μg of protein. The four-band labelling using 142 (A) or G10 (B,C) antibodies can be seen. Reelin-probed membranes were immunoblotted using antibodies against Tubulin for standardization.

Figure 4

Plots illustrating the densitometric quantification of Reelin-revealed films of different neurodegenerative diseases. PDD and AD(V-VI) (A) and sCJD (I-II) (B) with respect to nND. Each dot corresponds to one sample, and the mean ± S.D. for each group is also displayed.

Figure 5

(A) Examples of the Western Blotting determination of Reelin in the CSF of different patients (MCI, DEM, sCJD) and their controls (nND). By way of example, the upper bands of ≈420 and ≈310 kD of the revealed films of nND, MCI, DEM, and sCJD are shown. For each gel the Ponceau staining is also shown. (B) Graph illustrating the results of the densitometric measurement of the ≈420 kD band in the different CSF samples. Each dot corresponds to one sample, and the mean ± S.D. for each disease and nND is also displayed. Only p values indicating statistical differences between groups are displayed in the graphs. p values were determined using ANOVA (Bonferroni post hoc test).