Centenarian hippocampus displays high levels of astrocytic metallothioneins

Abstract

The hippocampus is a brain area linked to cognition. The mechanisms that maintain cognitive activity in humans are poorly understood. Centenarians display extreme longevity which is generally accompanied by better quality of life, lower cognitive impairment, and reduced incidence of pathologies including neurodegenerative diseases. We performed transcriptomic studies in hippocampus samples from individuals of different ages (centenarians [≥97 years], old, and young) and identified a differential gene expression pattern in centenarians compared to the other two groups. In particular, several isoforms of metallothioneins (MTs) were highly expressed in centenarians. Moreover, we identified that MTs were mainly expressed in astrocytes. Functional studies in human primary astrocytes revealed that MT1 and MT3 are necessary for their homeostasis maintenance. Overall, these results indicate that the expression of MTs specifically in astrocytes is a mechanism for protection during aging.

Keywords: astrocytes; centenarians; hippocampus; metallothioneins; transcriptomic.

FIGURE 1

Differentially expressed genes in centenarians (≥97 years) hippocampus. (a). Principal component analysis (PCA) plot based on the expression profile of all transcripts measured. (b, c) Hierarchical clustering of centenarian (n = 3) versus old (n = 8) and young individuals (n = 5). (d) The number of genes increased and decreased genes in the transcriptomic analysis (p‐value < 0.05 and FC ≥ |2|). (e) Venn diagram showing the number of overlapping differentially expressed genes between the two comparisons. (f) Representative dotplot after GO analysis associated with decreased genes in centenarians versus old and young individuals. (g) Representative dot plot after Gene Ontology (GO) analysis associated with increased genes in centenarians versus old and young individuals. (h) Statistical significance of the genes associated with the biological pathways obtained after GO in (g).

FIGURE 2

MTs are highly expressed in the hippocampus of very old individuals. (a, b) mRNA levels of indicated MT isoforms in hippocampus samples of young (n ≥ 16), elderly (n ≥ 10), and very old individuals (n ≥ 4) from cohort 1. (c) Expression of REST in same samples. (d) Representative immunohistochemistry image of MT3 in human hippocampus. (e, f) Representative immunofluorescence (IF) images of MT1 and MT3 (red) in the DG of young (n = 7), old (n = 14), and very old individuals (n = 3) from cohort 2 (scale bar = 50 μm). (g, h) Quantification of MT1 and MT3 protein levels in samples from cohort 2. The figure represents the percentage of MT1‐ or MT3‐positive cells with respect to nuclei stained with DAPI (blue). The statistical significance was assessed with the Student's t‐test (^≠ p < 0.1, *p < 0.05, **p < 0.01, ***p < 0.001).

FIGURE 3

MTs are mainly expressed by astrocytes. (a, b) Normalized expression (nTPM) of MT1 and MT3 using single‐cell RNAseq data from the whole brain from “The Human Protein Atlas” (http://www.proteinatlas.org). (c) Immunohistochemistry of MT3 in human DG. (d) Representative immunofluorescence (IF) images of GFAP (green) and MT1 (red) in the dentate gyrus (DG, scale bar = 50 μm) of hippocampal coronal sections of young (n = 7), old (n = 14) and very old individuals (n = 3) from the cohort 2. (e) Representative IF images of GFAP (green) MT3 (red) in DG samples of the same cohort (young n = 7, old n = 14, very old n = 3; scale bar = 50 μm). (f, g) Quantification (percentage) of MT1‐ and MT3‐positive cells that are also positive for GFAP (MT1+/GFAP+ or MT3+/GFAP+ cells) in the DG of the same samples. (h) Representative IF images of S100β (green) and MT1 (red) in DG samples from cohort 2 (young, n = 3; elderly, n = 11; very old, n = 3; scale bar = 50 μm). (i) Representative IF images of S100β (far red) and MT3 (red) in DG samples of the same cohort (young, n = 4; old, n = 5; very old, n = 3; scale bar = 50 μm). (j, k) Quantification (percentage) of MT1‐ or MT3‐positive cells that are also positive for S100β (MT1+/S100β+ or MT3+/S100β+ cells) in the DG of the same samples.

FIGURE 4

MTs expression decreases in cultured human astrocytes. (a, b) Representative immunofluorescence (IF) images and quantification of GFAP expression in hPSC‐derived astrocyte progenitors (day‐in vitro 60) and more mature astrocytes (day‐in vitro 110). Scale bar = 50 μm. (c) Representative IF images of MT1 and MT3 expression in astrocytes at DIV60 versus DIV110 (n = 3). Scale bar = 50 μm. (d, e) Quantification of the mean intensity of MT1 and MT3 in astrocytes at DIV60 and DIV110 (n = 3). (f) Representative images of GFAP, MT1, and MT3 in hPSC‐derived astrocytes at DIV60 (n = 3). Scale bar = 50 μm. (g) mRNA levels of MT isoforms in astrocytes at DIV53‐62 (progenitor) and DIV104‐110 (mature). (h) mRNA levels of MT isoforms in normal human astrocytes (NHA) cultured at early (1–3) and late (12–18) passages (n = 5). (i) Representative Western Blot of GFAP, MT1, MT3, and p16 ^INK4A in NHA at early and late passage (n = 3). (j) Expression of p16 ^INK4A , p21 ^CIP1 , IL6 in NHA cultured at early (2, 3) and late (15–17) passages (n = 3). The statistical significance was assessed with the Student's t‐test (^≠ p < 0.1, *p < 0.05, **p < 0.01, ***p < 0.001).

FIGURE 5

MT1 and MT3 silencing decrease astrocytic activity. MT1 and MT3 silencing was done in normal human astrocytes (NHA) using lentiviral infections. (a) Representative western blot of MT1 and MT3 in NHA with MT1 (shMT1) or MT3 silencing (shMT3) compared to controls (pLKO) (n = 3). (b) Representative immunofluorescence images of MT1 and MT3 in indicated cell types (scale bar = 50 μm). (c) mRNA levels of MT1 isoforms in shMT1 compared to pLKO cells (n = 3). (d) MT3 levels in shMT3 relative to pLKO (n = 3). (e) Quantification of Ki67⁺ cells, (f) Caspase 3⁺ cells, and (g) SA‐β‐gal activity in MT silenced compared to pLKO NHA (n = 3). (h, i) mRNA levels of p21 ^CIP1 , IL6, TNFα, IL1α, and C3 in shMT1 and shMT3 astrocytes compared to controls (n = 3). (j) Venn diagram and (k) heat map of the altered secreted common cytokines from supernatants of shMT1 and shMT3 NHA compared to pLKO astrocytes (p < 0.05, n = 2). The statistical significance was assessed with the Student's t‐test (^≠ p < 0.1, *p < 0.05, **p < 0.01, ***p < 0.001).