Women in Molecular Mechanisms of Aging Research

Editorial

05 August 2024

Editorial: Women in molecular mechanisms of aging

Aurélie Ledreux

and

Consuelo Borrás

1,051 views

0 citations

Editors

CONSUELO BORRAS

University of Valencia

Aurélie Ledreux

University of Colorado Anschutz Medical Campus

Impact

Simufilam treatment attenuated basal mTORC1 overactivation and restored its response to insulin in lymphocytes from AD subjects. Representative blot images from two AD and five healthy control subjects (A,E) show increased basal activation of mTORC1 by elevated levels of mTOR phosphorylation at S2448 (A) and by increased basal activation of mTORC1’s downstream kinase p70S6K, indicated by phosphorylation at T1389 (E), in AD subjects versus healthy controls. Blots were analyzed by densitometric quantitation (B,D,F). With heightened basal activity of the mTORC1 signaling pathway in AD subject lymphocytes, its response to in vitro insulin was blunted. The elevated basal p70S6K, still lower than insulin-stimulated control levels, also showed a weak response to insulin. Oral simufilam treatment decreased the elevated basal mTORC1 activation and restored its response to insulin across both measures, with insulin-stimulated p70S6K levels slightly higher than untreated healthy controls on Day 28. The simufilam treatment effect is also seen in the dramatic improvement in percent stimulation by insulin (C,G). mTOR association with Raptor was lower in healthy controls but unaffected by insulin or simufilam (D). Data are means ± SEM. N = 13 for AD, 18 for control. *p < 0.05, ***p < 0.001, vs. respective value in healthy control by two-sided two-sample t-test; ###p < 0.001 vs. respective AD value at Day 0 by two-sided paired t-test.

Original Research

29 June 2023

Simufilam suppresses overactive mTOR and restores its sensitivity to insulin in Alzheimer’s disease patient lymphocytes

Hoau-Yan Wang

, 6 more and

Lindsay H. Burns

Introduction: Implicated in both aging and Alzheimer’s disease (AD), mammalian target of rapamycin (mTOR) is overactive in AD brain and lymphocytes. Stimulated by growth factors such as insulin, mTOR monitors cell health and nutrient needs. A small molecule oral drug candidate for AD, simufilam targets an altered conformation of the scaffolding protein filamin A (FLNA) found in AD brain and lymphocytes that induces aberrant FLNA interactions leading to AD neuropathology. Simufilam restores FLNA’s normal shape to disrupt its AD-associated protein interactions.

Methods: We measured mTOR and its response to insulin in lymphocytes of AD patients before and after oral simufilam compared to healthy control lymphocytes.

Results: mTOR was overactive and its response to insulin reduced in lymphocytes from AD versus healthy control subjects, illustrating another aspect of insulin resistance in AD. After oral simufilam, lymphocytes showed normalized basal mTOR activity and improved insulin-evoked mTOR activation in mTOR complex 1, complex 2, and upstream and downstream signaling components (Akt, p70S6K and phosphorylated Rictor). Suggesting mechanism, we showed that FLNA interacts with the insulin receptor until dissociation by insulin, but this linkage was elevated and its dissociation impaired in AD lymphocytes. Simufilam improved the insulin-mediated dissociation. Additionally, FLNA’s interaction with Phosphatase and Tensin Homolog deleted on Chromosome 10 (PTEN), a negative regulator of mTOR, was reduced in AD lymphocytes and improved by simufilam.

Discussion: Reducing mTOR’s basal overactivity and its resistance to insulin represents another mechanism of simufilam to counteract aging and AD pathology. Simufilam is currently in Phase 3 clinical trials for AD dementia.

7,454 views

6 citations

Histology of Normal Human Skin: (A) Normal human skin from a young donor (22 yo). The dotted lines separate the epidermis (Ep), papillary (PD), and reticular (RD) dermis. (B) Histological colorations of human skin from breast young (i, iv, and vii) and old (ii, v, and viii) skin (respectively 22 and 74 yo) and cheek old (iii, vi, and ix) skin (75 yo). Different types of colorations were performed: Hematoxylin Eosin Saffron–HES (i–iii), Sirius red (iv–vi), and Orcein (vii–ix). Scale Bar = 50 µm. Arrows highlight the rete ridges.

Original Research

31 May 2023

Quantitative nanohistology of aging dermal collagen

Sophia Huang

, 10 more and

Laurent Bozec

2,876 views

6 citations

Original Research

30 June 2023

Change in circulating klotho in response to weight loss, with and without exercise, in adults with overweight or obesity

Katherine A. Collins

, 5 more and

John M. Jakicic

1,942 views

5 citations

Correction

22 August 2024

Expression of concern: Simufilam suppresses overactive mTOR and restores its sensitivity to insulin in Alzheimer’s disease patient lymphocytes

1,285 views

0 citations

Establishment of the age marker-based panel in human fibroblasts of young, mid-age and old donors. (A) Cell Model. For initial experiments, fibroblasts with different donor age was used. (B) Replicative potential of human fibroblasts. Cells were cultured under stable conditions (37°C, 5% CO2) in DMEM medium (with 15% FBS, 1% Pen-Strep). PDL was observed for max. 150 days. Fibroblasts from young (Young 1–5) and mid-age (Midage 1–3) donors display healthy growth. In contrast, fibroblasts from old donors (Old 3, Old 4, Old 5 and 6) showed slower growth except for Old 1 and Old 2. (C–K) X̅ = Mean value of all young donor fibroblasts. (C) Amount of γH2A.X foci per cell examined by IF staining to investigate DSBs in human fibroblasts. Cells of very old individuals (Old 4 and 5) displayed an increase in γH2A.X foci in comparison to the mean of all young donors. Mid-age and old (Old 1, Old 2, Old 3 and Old 6) donor fibroblast showed no DSBs. [n = 3–4 (each n tested >15 cells), mean ± SEM,*p < 0.05, **p < 0,001, ***p < 0.0001, one-way ANOVA] (D) CTCF of H3K9Me3 in human fibroblasts. Donor fibroblasts from old individuals (Old 2, Old 4, Old 5 and Old 6) displayed a decrease in CTCF of H3K9Me3 compared to the mean of young donors. There were no changes in CTCF of H3K9Me3 in mid-age cells (Midage 1–3). [n = 3–4 (each n tested >15 cells), mean ± SEM, *p < 0.05, **p < 0.001, ***p < 0.0001, one-way ANOVA] (E) Telomere length was measured by MM-qPCR in comparison to a standard of healthy mixed aged individuals. In comparison to grouped young donor cells, Old 6 displayed a shortening of telomere length. [n = 3, mean ± SEM, *p < 0.05, **p < 0.001, ***p < 0.0001, one-way ANOVA] (G) RNA expression levels measured by qRT-PCR of p21 and p16 was examined to investigate activation of cell cycle arrest. Old 4 and Old 5 displayed an increase in RNA expression level of p21. All other cells did not show altered RNA expression levels. [n = 3-4, mean ± SEM *p < 0.05, **p < 0.001, ***p < 0.0001, one-way ANOVA] (F) Quantification of SA-βGAL positive cells by X-Gal staining. In comparison to grouped young donor cells, Midage 3 and all old human fibroblasts (Old 1–6) displayed a significant increase of SA- βGAL positive cells. [n = 3–4 (each n tested >15 cells), mean ± SEM, *p < 0,05, **p < 0.001, ***p < 0.0001, one-way ANOVA] (H) CTCF of Lamin B1 expression. A significant decrease in Lamin B1 expression could be observed in mid-age (Midage 1 and Midage 3) and old (Old 1–5) cells compared to the mean of young donor fibroblasts. [n = 4 (each n tested >15 cells), mean ± SEM, *p < 0.05, **p < 0.001, ***p < 0.0001, one-way ANOVA] (I, J) Determination of cytokine secretion (IL-6, IL-8) in conditioned medium by ELISA. In comparison to grouped young donor cells, old donor fibroblasts (Old 1, Old2, Old 3, Old 4, and Old 6) showed an increase in levels of IL-6. An increase in concentration with respect to IL-8 could be observed in Midage 1 and Old 4 in comparison to grouped controls. [n = 3-4, mean ± SEM, *p < 0.05, **p < 0,001, ***p < 0.0001, one-way ANOVA] (K) Change in nucleus morphology shown as nucleus area (pixel units) in human fibroblast. Nucleus area is significantly increased in very old donor fibroblasts (Old 4 and 5) compared with grouped young controls. [n = 6–8 (each n tested >15 cells), mean ± SEM *p < 0.05, **p < 0.001, ***p < 0.0001, one-way ANOVA].

Original Research

15 February 2023

Systematic estimation of biological age of in vitro cell culture systems by an age-associated marker panel

Christiane Hartmann

, 5 more and

Andreas Hermann

Aging is a process that affects almost all multicellular organisms and since our population ages with increasing prevalence of age-related diseases, it is important to study basic processes involved in aging. Many studies have been published so far using different and often single age markers to estimate the biological age of organisms or different cell culture systems. However, comparability of studies is often hampered by the lack of a uniform panel of age markers. Consequently, we here suggest an easy-to-use biomarker-based panel of classical age markers to estimate the biological age of cell culture systems that can be used in standard cell culture laboratories. This panel is shown to be sensitive in a variety of aging conditions. We used primary human skin fibroblasts of different donor ages and additionally induced either replicative senescence or artificial aging by progerin overexpression. Using this panel, highest biological age was found for artificial aging by progerin overexpression. Our data display that aging varies depending on cell line and aging model and even from individual to individual showing the need for comprehensive analyses.

6,913 views

13 citations

Methods

29 August 2022

The C. elegans Observatory: High-throughput exploration of behavioral aging

Rex A. Kerr

, 2 more and

Cynthia Kenyon

Organisms undergo a variety of characteristic changes as they age, suggesting a substantial commonality in the mechanistic basis of aging. Experiments in model organisms have revealed a variety of cellular systems that impact lifespan, but technical challenges have prevented a comprehensive evaluation of how these components impact the trajectory of aging, and many components likely remain undiscovered. To facilitate the deeper exploration of aging trajectories at a sufficient scale to enable primary screening, we have created the Caenorhabditis elegans Observatory, an automated system for monitoring the behavior of group-housed C. elegans throughout their lifespans. One Observatory consists of a set of computers running custom software to control an incubator containing custom imaging and motion-control hardware. In its standard configuration, the Observatory cycles through trays of standard 6 cm plates, running four assays per day on up to 576 plates per incubator. High-speed image processing captures a range of behavioral metrics, including movement speed and stimulus-induced turning, and a data processing pipeline continuously computes summary statistics. The Observatory software includes a web interface that allows the user to input metadata and view graphs of the trajectory of behavioral aging as the experiment unfolds. Compared to the manual use of a plate-based C. elegans tracker, the Observatory reduces the effort required by close to two orders of magnitude. Within the Observatory, reducing the function of known lifespan genes with RNA interference (RNAi) gives the expected phenotypic changes, including extended motility in daf-2(RNAi) and progeria in hsf-1(RNAi). Lifespans scored manually from worms raised in conventional conditions match those scored from images captured by the Observatory. We have used the Observatory for a small candidate-gene screen and identified an extended youthful vigor phenotype for tank-1(RNAi) and a progeric phenotype for cdc-42(RNAi). By utilizing the Observatory, it is now feasible to conduct whole-genome screens for an aging-trajectory phenotype, thus greatly increasing our ability to discover and analyze new components of the aging program.

6,295 views

14 citations

(A) Representative images of transduced ECs during the 4-h wound healing window after exposure to flow at low (2 dynes/cm2) or normal (15 dynes/cm2) magnitude. Scale bar: 200 µm. (B,C) Quantifications of wounded area covered by cells after wound formation for wild-type lamin A-expressing ECs and progerin-expressing ECs at horizontal and vertical wounds with shear stress at low or normal magnitude. Blue lines represent the recovery area of cells that were sheared at low magnitude after wound formation, and red lines represent that of cells that were sheared at normal magnitude after wound formation. *p < 0.05.

Original Research

14 March 2022

Progerin-Induced Impairment in Wound Healing and Proliferation in Vascular Endothelial Cells

Yizhi Jiang

and

Julie Y. Ji

Progerin as a mutated isoform of lamin A protein was first known to induce premature atherosclerosis progression in patients with Hutchinson-Gilford progeria syndrome (HGPS), and its role in provoking an inflammatory response in vascular cells and accelerating cell senescence has been investigated recently. However, how progerin triggers endothelial dysfunction that often occurs at the early stage of atherosclerosis in a mechanical environment has not been studied intensively. Here, we generated a stable endothelial cell line that expressed progerin and examined its effects on endothelial wound repair under laminar flow. We found decreased wound healing rate in progerin-expressing ECs under higher shear stress compared with those under low shear. Furthermore, the decreased wound recovery could be due to reduced number of cells at late mitosis, suggesting potential interference by progerin with endothelial proliferation. These findings provided insights into how progerin affects endothelial mechanotransduction and may contribute to the disruption of endothelial integrity in HGPS vasculature, as we continue to examine the mechanistic effect of progerin in shear-induced endothelial functions.

2,685 views

7 citations