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. Author manuscript; available in PMC: 2026 Mar 31.
Published in final edited form as: Neurobiol Aging. 2024 Sep 7;144:43–55. doi: 10.1016/j.neurobiolaging.2024.09.003

Age-dependent sex differences in cofilin1 pathway (LIMK1/SSH1) and its association with AD biomarkers after chronic systemic inflammation in mice

Amsha S Alsegiani 1, Zahoor A Shah 1,*,1
PMCID: PMC13034451  NIHMSID: NIHMS2154477  PMID: 39265451

Abstract

Chronic systemic inflammation (CSI) results in neuroinflammation and neurodegeneration. Cofilin1 is a stress protein that activates microglia and induces neuroinflammation, but its role in CSI at different aging stages remains unidentified. Therefore, the study aims to identify cofilin1 and its upstream regulators LIMK1 and SSH1 after CSI in young-, middle-, and advanced-aged mice. CSI was induced by injecting the male and female mice with a sub-lethal dose of Lipopolysaccharide weekly for six weeks. The results showed that normal male mice did not show cofilin pathway dysregulation, but a significant dysregulation was observed in CSI advanced-aged mice. In females, cofilin1 dysregulation was observed in healthy and CSI advanced-aged mice, while significant cofilin1 dysregulation was observed in middle-aged mice during CSI. Furthermore, cofilin1 pathway dysregulations correlated with Alzheimer’s disease (AD) biomarkers in the brain and saliva, astrocyte activation, synaptic degeneration, neurobehavioral impairments, gut-microbiota abnormalities, and circulatory inflammation. These results provide new insights into cofilin1 sex and age-dependent mechanistic differences that might help identify targets for modulating neuroinflammation and early onset of AD.

Keywords: Cofilin1 pathway, Sex-difference, Aging, CSI, AD biomarkers

1. Introduction

A low-grade systemic inflammation in the absence of overt infection, termed inflammaging, is considered a silent killer, particularly in the older population, which is at a higher risk for morbidity and mortality (Franceschi and Campisi, 2014). Chronic systemic inflammation (CSI) can lead to cardiovascular diseases, cancer, cognitive impairments, and other age-related chronic diseases (d’Avila et al., 2018; Hou et al., 2019; Prodanovich et al., 2009; Tanaka et al., 2020). Over 50 % of the US older population has at least one chronic disease, and more than 25 % have multiple chronic diseases (Boersma et al., 2020). Persistent or CSI is associated with long-term neuroinflammation and is considered a risk factor for Alzheimer’s disease (AD) and other neurodegenerative disorders (Barnes and Yaffe, 2011). Aging brains are more susceptible to AD; however, aging alone does not amount to the onset of AD (Alsegiani and Shah, 2020; Hou et al., 2019); other factors also contribute to the pathology of the disease.

Recent studies suggest a link between neuroinflammation and AD, and it is widely believed that the dynamics of the actin cytoskeleton protein, cofilin1, is one of the causes (Bamburg and Bernstein, 2016; Rahman et al., 2014). Studies from our laboratory have found that cofilin1 plays a vital role in activating the microglia, subsequently impairing synaptic plasticity and inducing neuronal death (Alhadidi and Shah, 2018; Madineni et al., 2016). Cofilin1 has been shown to have different roles in severing and depolymerizing filamentous actin (F-actin) to generate dynamic processes of proliferation, migration, and differentiation of the actin cytoskeleton (Alsegiani and Shah, 2020). Several molecular mechanisms regulate cofilin-1 activity through phosphorylation/dephosphorylation processes. The upstream regulation by LIM kinase isoform1 (LIMK1) and slingshot phosphatase isoform1 (SSH1) show the highest substrate specificity that can affect cofilin-1 activity over the other kinases and phosphatases (Van Troys et al., 2008). Cofilin inactivation is regulated by phosphorylation of cofilin residue at (Ser3) by LIMK1 and is reactivated by dephosphorylation by SSH1 (Niwa et al., 2002; Van Troys et al., 2008). Remarkably, LIMK1 activation is regulated by phosphorylation of LIMK1 residue at (Thr508), whereas total SSH1 is inactivated by phosphorylation at residue (Ser978) (Bravo-Cordero et al., 2013). Therefore, we hypothesize that there is a gradual accumulation of cofilin1 with age, and it further increases in the presence of CSI, thus becoming a significant source of neuroinflammation and cognitive impairments. Nevertheless, the cofilin1 signaling in aging and CSI’s presence is limited, and more studies are warranted to explore the dynamics (Alsegiani and Shah, 2020).

Therefore, the present study investigates cofilin1 pathway dysregulation and its association with neuroinflammation, synaptic impairments, cognitive decline, and developing AD in normal and in the presence of CSI in young, middle, and advanced-aged mice. We also examined gut microbiota alteration, intestinal inflammation, immunosenescence, and peripheral inflammatory biomarkers that might participate in the etiology of aging and CSI.

2. Materials and methods

2.1. Experimental design

C57/B6 male and female mice, young (4–6 months old, n=15), middle-aged (12–14 months old, n=13), and advanced aged (18–20 months, n=13) were obtained from the Jackson Laboratory and National Institutes of Health (NIH). Mice were maintained in the Department of Laboratory Animal Resources, University of Toledo. All animal experiments followed the Guide for the Care and Use of Laboratory Animals. The Institutional Animal Care and Use Committee (IACUC) approved the Protocol at The University of Toledo. Mice in each group were randomly divided into controls and CSI. Low-grade CSI was induced by weekly injections of lipopolysaccharide (LPS) prepared in sterile PBS ~10 μg/mouse (Escherichia coli 0111:B4, Sigma-Aldrich, Inc.) for 6 weeks to induce CSI. Weekly injections were selected to reduce the chances of tolerance and preconditioning (d’Avila et al., 2018; López-Collazo and del Fresno, 2013). Control groups were injected with PBS. One week after the last LPS injection, all mice were subjected to behavior tests for 10 days. At the end of the study, mice were anesthetized with isoflurane; then, the blood was collected via cardiac puncture into microtainer tubes (Becton, Dickinson). After 30 min, tubes were centrifuged at 10,000 rpm for 10 min, and hemolysis-free serum was collected and stored at −80 °C. Afterward, mice brains and other tissues were dissected and stored at −80 °C.

2.2. Sickness behavioral and inflammation monitoring

Sickness behavior was evaluated every week, 24 hrs after each LPS injection. As per a previous study, mice were scored as mildly, moderately, or severely affected (d’Avila et al., 2018). The scoring assessed the animal’s appearance, alertness, grip strength, and body temperature. To monitor the impact of CSI, we evaluated white blood cells (WBC) and TNF-α in the blood at several time points after LPS injections. Blood was collected from the mouse tail into EDTA-treated blood tubes (BD, Franklin Lakes, NJ, USA). The levels of monocytes and neutrophils in the peripheral blood were evaluated using the VETSCAN® HM5 apparatus (Abaxis, Union City, CA, USA).

2.3. Neurobehavioral tests

Mice were subjected to behavioral tests one week after the last LPS injection at the rate of one test per day for 3 successive days, in the following order: Open field (OF) followed by the novel objective recognition (NOR), then Y-maze test. All tests were tracked and analyzed using Any-Maze software [Stoelting Co, Wood Dale, IL, USA].

OF test is used to measure anxiety and depression-like behavior in mice. Mice were placed in an open-topped box and allowed to explore freely for 10 min. The time spent in the center of the area was tracked to measure anxiety-like behavior and the immobility time to measure depression-like behavior.

NOR test was used to assess short-term memory and performed according to the previously described method (d’Avila et al., 2018). The object recognition index was calculated as the exploration time of a novel object, the exploration time of a familiar object, and the total exploration time.

The Y-maze test evaluated spatial working memory by measuring the spontaneous alternation rate. Mice were placed in an opaque, Y-shaped maze in which the arms were symmetrically disposed at 120 angles. The mouse was placed in the center of the Y-maze and was free to explore the arena for 8 min. The spontaneous alternations rate was calculated as follows: [(number of alternations)/(number of total arm entries-2)]* 100.

2.4. Saliva collection

Saliva was extracted from mice following the previously published study (Zubeidat et al., 2022). The protein concentration of Tau proteins was measured using FastScan™ Phospho-Tau (Ser416), FastScan™ Total Tau, and PathScan® Phospho-Tau (Ser396) sandwich ELISA Kit (Cell Signaling Technology), and Aβ was measured using Aβ1–42 Elisa kit from (Novus Biologicals, Centennial CO, USA).

2.5. ELISA assay

Serum and colon inflammatory markers were measured using ELISA kits. The mouse colon was homogenized in the RIPA lysis buffer containing a 1 % phosphatase inhibitor cocktail and 1 % protease inhibitor cocktail on ice and centrifuged at 15,000 rpm at 4 C for 15 min. IL-6, IL-1b, and TNF-α cytokine levels were measured using an ELISA kit (R&D system, Minneapolis, MN-USA).

2.6. Western blotting (WB)

SDS polyacrylamide gels, 8–15 %, were used to analyze protein expression levels by WB. Proteins were transferred onto a PVDF membrane, blocked with 5 % BSA, and then incubated with antibodies overnight at 4 °C. The following primary antibodies were used in the study: rabbit anti-pcofilin1, rabbit anti-cofilin1, rabbit anti-GAPDH, rabbit anti-β-actin, rabbit anti-t-Tau, rabbit anti-p-Tau (ser416), rabbit anti-p-Tau (ser396), mouse anti-PSD-95 (1:1000–2000; Cell Signaling Technology, Danvers, MA, USA), mouse anti-LIMK1, rabbit anti-pLIMK1, rabbit anti-GFAP, rabbit anti-BDNF, rabbit anti-Syn (1:1000–3000; Abcam, Cambridge, MA, USA), rabbit anti-pSSH1 (1:1000, ECM Biosciences, Versailles, KY, USA), and anti-Aβ (1:1000, Proteintech, Rosemont, IL-USA). The proteins were detected using HRP-conjugated secondary antibodies: anti-rabbit or anti-mouse (1:5000; Cell Signaling Technology). The images were captured and analyzed using Syngene Image Lab Software.

2.7. Real-time-qPCR for total bacterial load and major phylum

The RT-qPCR amplification was performed, recorded, and analyzed using Step One Plus Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Carlsbad, CA, USA) to analyze the total bacterial load and phylum. A comparative Ct (2-ΔΔCt) mathematical model was used to calculate the data. All the primers were purchased from IDT (Newark, NJ, USA).

The fecal samples were collected freshly and directly from the anus of each mouse. Total bacterial DNA was extracted from feces (≥250 mg) using the QIAamp PowerFecal Pro DNA Kit (QIAGEN, Germantown, MD, USA) as per manufacturer instructions in a reaction mixture of bacterial DNA, SYBR green master mix, and bacterial-specific primers of predominant bacteria in mouse feces as published previously (Yang et al., 2015). The primers used were 16S rRNA, Bacteroidetes, Firmicutes, Actinobacteria, Delta and Gammaproteobacteria, Candidatus Saccharibacteria, Betaproteobacteria, and Universal. Fold change was calculated for the total bacterial load compared to the young control group in both sexes. The data were expressed as a relative value compared to the control Bacterial universal.

2.8. Limulus Amebocyte Lysate (LAL) assay

The Limulus amebocyte lysate assay (LAL) was used to evaluate serum endotoxin levels, an indirect method to determine gut permeability. First, plasma endotoxin contents were determined using the LAL kit (Genescript, Piscataway, NJ, USA) according to the manufacturer’s instructions.

2.9. Myeloperoxidase (MPO) assay

Mouse colon was homogenized in sterile PBS buffer containing 0.5 % CTAB and centrifuged for 10 min at 14,000 g at 4 C. Myeloperoxidase (MPO) activity in the colon was assayed as previously described (Jang et al., 2018).

2.10. Serum immunoreactivity to LPS

Serum immunoreactivity to LPS was examined by ELISA as described previously (Ziegler et al., 2008).

2.11. Statistical analysis

GraphPad Prism version 9.0 was used for statistical analysis. Male and female mice were examined separately using TWO-way ANOVA followed by the Tukey test with two independent variables. Statistically significant values were represented as P < 0.05 ($), P < 0.01 ($$), and P <0.001 ($$$) in normal aging group, P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***) in CSI group and P < 0.05 (#), P < 0.01 (##), and P < 0.001 (###) in an aged-matched comparison between normal and CSI. The study results are analyzed as mean and standard errors of means (SEM).

3. Results

3.1. Cofilin1 pathway dysregulation during normal aging

Total and phosphorylated cofilin1, LIMK1, and SSH1 protein levels were analyzed in the hippocampus region of normal young, middle, and advanced-aged male and female mice. Compared with young groups, the alteration of the cofilin1 pathway was observed only in advance-aged mice during the normal aging process, and no significant effect was observed in middle-aged mice in both sexes.

No significant changes were observed in the male group in pcofilin1/cofilin1 and pLIMK1/LIMK ratios (Fig. 1a–f). However, an increase was observed in the pSSH1/SSH1 ratio (~2.6-fold, $P < 0.05) (Fig. 1g–i). In the female group, the advanced-age mice showed a significant increase in active cofilin1 (~1.4-fold, $P < 0.05) and an increase in pcofilin1 (~3.8-fold, $$$P < 0.001); however, the increase in pcofilin1 was higher than active cofilin1 level; resulting in a ~2.4-fold increase of the pcofilin1/cofilin1 ratio ($P < 0.05) (figure 1j–l). Similarly, phosphorylated LIMK1 and SSH1 were significantly increased ($$P < 0.01). Consequently, an increase (~1.6-fold, $P < 0.05) in the pLIMK1/LIMK1 and (~3.1-fold, $$$P < 0.001) in the pSSH1/SSH1 ratio were observed (Fig. 1m–r).

Fig. 1.

Fig. 1.

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Cofilin1 pathway in young, middle-aged and advanced-aged normal and CSI mice. Y: young, MA: middle-aged, and AA: advanced-aged during C: control and CSI: chronic systemic inflammation. Top panels, left-male and right-female: Western blot analyses of total protein extracts. Bottom panels: densitometric analyses of Western blot protein bands, blue-male and pink-female: Male group (a) Total cofilin1 protein levels; (b) pcofilin1 levels; (c) pcofilin1/cofilin1 ratio; (d) Total LIMK1 protein levels; (e) pLIMK1 levels; (f) pLIMK1/LIMK1 ratio; (g) Total SSH1 protein levels; (h) pSSH1 levels; (i) pSSH1/SSH1 ratio. Female group (j) Total cofilin1 protein levels; (k) pcofilin1 levels; (l) pcofilin1/cofilin1 ratio; (m) Total LIMK1 protein levels; (n) pLIMK1 levels; (o) pLIMK1/LIMK1 ratio; (p) Total SSH1 protein levels; (q) pSSH1 levels; (r) pSSH1/SSH1 ratio. Protein levels were normalized to the loading control of GAPDH. Data are expressed (n=6–7) as mean ± SEM. $The difference in control compared to the young control, *The difference in CSI compared to the young CSI, and #Difference compared to the aged-matched in control Vs. CSI. $p 0.05, $$p < 0.01, $$$p < 0.001, *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05, ##p < 0.01, ###p < 0.001.

3.2. Cofilin1 pathway dysregulation during CSI

Our data demonstrated that low doses of LPS exacerbated systemic inflammation and sickness behavior in middle-aged and advanced-aged mice (Supplementary data, Fig. 1b–e). No systemic inflammation or sickness behavior was detected in young mice in both sexes. Following LPS treatment, middle and advanced-age mice showed weight loss, muscle weakness, and changes in body appearance, including lethargy, sleepiness, bloated abdomen, piloerection, and sunken eyes, compared with the age-match group. We observed no alteration in the cofilin1 pathway in young mice suffering from CSI in either sex (Fig. 1a–r). Compared with the young CSI group, middle-aged male mice showed no changes in the pcofilin1/cofilin1 and pLIMK1/LIMK1 protein expression (Fig. 1a–f), whereas an increase in pSSH1/SSH1 ratio (~2.6-fold, *P < 0.05) (Fig. 1h and i) was observed. On the other hand, the advanced-aged male mice showed a significant increase in both cofilins (***P < 0.001), leading to an increase in the pcofilin1/cofilin1 ratio (***P < 0.001) (Fig. 1a–c). Furthermore, both pLIMK and pSSH1 were increased (***P < 0.001), and consequently, pLIMK1/LIMK1 and pSSH1/SSH1 ratios were increased (***P < 0.001) (Fig. 1c, f, h and i). In addition, the pcofilin1/cofilin1 and pLIMK1/LIMK1 ratios were significantly increased compared with the healthy aged-match group (###P < 0.001).

Middle-aged CSI females showed an increase in cofilin1 expression (*P < 0.05) and pcofilin1 (***P < 0.001), leading to an increase in pcofilin1/cofilin1 ratio (*P < 0.05) compared to young CSI group (Fig. 1j–l). The upstream pathway also showed a dysregulation by an increase in pSSH1 and the ratios of pLIMK1/LIMK1 and pSSH1/SSH1 (*P < 0.05) (Fig. 1q, o and f). In addition, the cofilin1 (##P < 0.01), and pcofilin1 protein expression (#P < 0.05), and pcofilin1/cofilin1 (##P < 0.01), pLIMK1/LIMK1 (##P < 0.01), pSSH1/SSH1 (##P < 0.01) (Fig. 1j, k, l, o, q, and f) ratios were significantly increased compared to the healthy aged-match group. The advanced-aged CSI mice showed persistent cofilin1 pathway dysregulation. Compared with the aged-match group, advanced-aged CSI females showed increased protein expression of cofilin1 (#P < 0.05), pcofilin1 (##P < 0.01), and pcofilin1/cofilin1 ratio (#P < 0.05) (Fig. 1j–l). Similarly, pLIMK1 and pSSH1 (Fig. 1n and q) and their ratios, LIMK1/pLIMK1, and pSSH1/SSH1 (***P < 0.001) ratios were significantly increased in advanced-aged CSI female mice (Fig. 1o and r).

3.3. AD biomarkers

To understand if the cofilin1 pathway dysregulation correlates with AD biomarkers, WB analysis of brain tissues and non-invasive detection tests of saliva and serum were performed.

3.4. AD biomarkers in brain tissue

The tau protein has more than 25 phosphorylation sites (Buée et al., 2000). Although we tested different phosphorylation sites of tau protein, including Thr181, thr212/ser214, and ser202, only two p-tau forms at sites Ser416 and Ser396 could be detected in mice brains.

The male group with CSI showed significant upregulation of both p-tau in advanced-aged mice compared with the young CSI group and the healthy aged-match group (Fig. 2a–d). Furthermore, all the tested groups showed non-substantial changes in the t-tau expression in both sexes (Fig. 2e and k). Conversely, male groups showed no changes in p-tau expression during healthy aging (Fig. 2a–d).

Fig. 2.

Fig. 2.

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Brain and Salivary AD protein markers in young, middle-aged, and advanced-aged normal and CSI mice. Y: young, MA: middle-aged, and AA: advanced-aged during C: control and CSI: chronic systemic inflammation. Top panels, left-male and right-female: Bottom panels: densitometric analyses of Western blot protein bands, blue-male, and pink-female: Male group (a) p-Tau(Ser396) levels; (b) p-Tau(Ser396)/t-Tau ratio; (c) p-Tau(Ser416) levels; (d) p-Tau(Ser416)/t-Tau ratio; (e) t-Tau protein levels; (f) APP protein levels. Female group (g) p-Tau(Ser396) levels; (h) p-Tau(Ser396)/t-Tau ratio; (i) p-Tau(Ser396) levels; (j) p-Tau(Ser396)/t-Tau ratio; (k) t-Tau(Ser416) levels; (l) App protein levels Protein levels were normalized to the loading control β-actin. Salivary ELISA protein expression: Male group (m) p-Tau(Ser396) levels; (n) p-Tau(Ser396)/t-Tau ratio; (o) p-Tau(Ser416) levels; (p) p-Tau(Ser416)/t-Tau ratio; (q) t-Tau protein levels; (r) Aβ protein levels; Female group (s) p-Tau(Ser396) levels; (t) p-Tau(Ser396)/t-Tau ratio; (u) p-Tau(Ser416) levels; (v) p-Tau(Ser416)/t-Tau ratio; (w) t-Tau protein levels; (x) Aβ protein levels. Data are expressed (n=4–6) as mean ± SEM. $The difference in control compared to the young control, *The difference in CSI compared to the young CSI, and #Difference compared to the aged-matched in control Vs. CSI. $p < 0.05, $$p < 0.01, $$$p < 0.001, *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05, ##p < 0.01, ###p < 0.001.

Both forms of p-tau levels (ser369 and ser416) were significantly increased in advanced-aged females ($$$P < 0.001) (Fig. 2g–j), and consequently, an increase in p-tau (ser396)/t-tau ratio and p-tau (ser396)/t-t au ratio was observed ($$$P < 0.001).

Middle-aged CSI females showed a significant accumulation of p-tau (ser396) (*P < 0.05) and p-tau (ser416) (***P < 0.001) compared to the young-aged CSI. At the same time, the accumulation of p-tau (Ser396 and Ser416) levels in advanced-aged females persisted in the presence of CSI (Fig. 2g–j).

A slight increase in Aβ precursor protein (APP) was observed in the healthy advanced-aged and middle-aged CSI females but did not reach statistical significance. Both male and female advanced-aged showed an accumulation of APP after CSI compared with the young CSI group (*P < 0.05) (Fig. 2f and l), and the advanced-aged male mice showed higher APP protein expression than the healthy age-matched group (#P < 0.05).

3.5. Saliva AD biomarkers

To investigate whether non-invasive diagnostic biomarkers reflect the APP and Tau protein levels that we could detect in mice brains using Aβ, pTau (ser396 and ser416), and t-tau ELISA kits.

Salivary t-tau levels significantly increased in both sexes of advanced-aged mice during normal aging ($$P < 0.01) and with CSI in both sexes (***P < 0.001) compared with the young group (Fig. 2q and W). No significant changes were detected in p-tau proteins in the male groups during normal aging, whereas both p-tau and their ratios were increased with CSI (Fig. 2m–p). Increased salivary Aβ levels were observed in advanced-aged males with CSI compared to the young control group (***P < 0.001) and aged-match groups ($$P < 0.01) (Fig. 2r). There was an apparent increase of salivary p-tau (ser396) levels and p-tau (ser416) ($$P < 0.01) in the healthy advanced-aged females compared with a healthy young group (Fig. 2s and u), and these expressions were further increased with CSI, leading to an increase in p-tau/t-tau ratios (Fig. 2t and v). Interestingly, both p-tau levels and p-tau/t-tau ratios were significantly increased in middle-aged females with CSI compared to young mice in the same group (Fig. 2s–v). Furthermore, salivary Aβ was significantly increased in normal advanced-aged females ($P < 0.05) and persisted with CSI (**P < 0.01) (Fig. 2x).

3.6. Neuroinflammation and synaptic dysfunction

To explore astrocytic activation and synaptic dysfunction in the hippocampus region, glial fibrillary acidic protein (GFAP), brain-derived neurotrophic factor (BDNF), synaptophysin (Syn), and post-synaptic density protein 95 (PSD95) protein expression levels were evaluated using WB. Our results showed astrocytic activation only during CSI (**P < 0.01) in advanced-aged male mice, compared with the young group (Fig. 3a). In addition, the astrocytic activation was significant in healthy advanced-aged female mice ($P < 0.05) compared with a healthy young group, and this activation persisted after CSI (***P < 0.001) (Fig. 3e). The Syn, PSD95, and BDNF proteins were significantly reduced during the normal aging process in both sexes (Fig. 3b–d and f–h). Interestingly, young mice with CSI showed a significant reduction in PSD95 compared with a healthy aged-match group (###P < 0.001) in females and (#P < 0.05) in males. In both males and females, all synaptic proteins showed a significant reduction in middle-aged mice with CSI compared with the healthy aged-match group and remained decreased in the advanced-aged group after CSI.

Fig. 3.

Fig. 3.

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Synaptic plasticity and neurobehavioral outcomes in young, middle-aged and advanced-aged normal and CSI mice. Y: young, MA: middle-aged, and AA: advanced-aged during C: control and CSI: chronic systemic inflammation. Top panels, left-male and right-female: Bottom panels: densitometric analyses of Western blot protein bands: Male group (a) GFAP protein levels; (b) Syn; (c) PSD95; (d) BDNF protein levels. Female group (e) GFAP; (F) Syn; (g) PSD95; (h) BDNF. Neurobehavioral tests: Male group (i) anxiety-like behavior (time in the center); (j) depression-like behavior (immobility time); (k) short-term memory (percentage of recognition index); (l) working-like memory (spontaneous alteration rate). Female group (m) anxiety-like behavior (time in the center); (n) depression-like behavior (immobility time); (o) short-term memory (percentage of recognition index); (p) working-like memory (spontaneous alteration rate). Data are expressed (n=5–7) as mean ± SEM. $The difference in control compared to the young control, *The difference in CSI compared to the young CSI, and #Difference compared to the aged-matched in control Vs. CSI. $p < 0.05, $$p < 0.01, $$$p < 0.001, *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05, ##p < 0.01, ###p < 0.001.

3.7. Behavioral and cognitive impairment

In the open-field test, advanced-aged male and female mice exhibited significantly shorter exploration times in the central area and longer immobility time than the young group (Fig. 3i–j and m–n). During the CSI, anxiety and depressive-like behavior persisted in advanced aging compared with the young and aged-match group. In contrast, the middle-aged mice did not show significant long-term signs of anxiety and depression compared with the young normal and CSI group; however, there were significant differences in females (##P < 0.01) and males (#P < 0.05) when compared with the normal, aged-matched group.

Short-term and spatial working memory was evaluated using NOR and Y-maze tests, which demonstrated that male mice showed a slight decrease in cognition tests with aging but did not significantly differ among the healthy young group (Fig. 3k and l). In contrast to the males, the advanced-aged female mice showed a significant decrease in the percent of recognition index and spontaneous alteration rate compared with young female mice ($P < 0.05) (Fig. 3o and p). In the CSI group, middle and advanced-aged mice were more susceptible to memory impairments, as demonstrated by their poor performance in both tests. In the middle-and advanced-age male groups, there was a significant decrease in the percent of recognition index compared to the young (**P < 0.01) and the age-match group (##P < 0.01) (Fig. 3k and l). Furthermore, both middle and advanced age showed a significant decrease in spontaneous alteration rate compared to the young group (*P < 0.05) and (**P < 0.01), respectively; in addition, advanced-age mice showed a difference with the age match group (#P < 0.05). Similarly, middle-aged CSI females showed a decrease in both recognition index percentage and spontaneous alteration rate compared to the young group (*P < 0.05) and a significant reduction in recognition index compared to the aged-matched group (#P < 0.05) (Fig. 3o and p). However, Advanced-aged females with CSI led to persistent short-term and spatial working memory impairment after CSI.

3.8. Gut microbiota alteration

Next, we were interested in examining fecal bacteria load and predominant bacteria phyla using RT-qPCR. Interestingly, mice showed no sex-associated microbiome alteration (Fig. 4). In the total bacterial load analysis, microbiota diversity in the feces was slightly decreased during the normal aging process; however, the differences were statistically significant in CSI advanced-aged males and CSI middle- and advanced-aged females (Fig. 4a and e). Next, we observed a substantial alteration in fecal-predominant bacteria phyla (Fig. 4b–c and f–g, Fig. S3). Those were represented by the increased prevalence of the phyla Firmicutes, Actinobacteria, and TM7 and a reduced prevalence of Bacteroidetes in normal middle and advanced-aged, and the only difference observed in CSI was in Firmicutes. Subsequently, the ratio of Firmicutes / Bacteroidetes (F/B) was increased in healthy advanced-aged males and females ($$P < 0.01) compared with the healthy young group. In CSI, the increase in the F/B ratio was significant in middle and advanced-aged male and female mice. As expected, gut microbiota diversity and composition in middle and advanced-aged mice was significantly lower than in young groups in both sexes during the CSI (*P<0.05).

Fig. 4.

Fig. 4.

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Microbiota changes and peripheral inflammation in young, middle-aged, and advanced-aged normal and CSI mice. Y: young, MA: middle-aged, and AA: advanced-aged during C: control and CSI: chronic systemic inflammation. Top panels, left-male and right-female: Gut microbiota alteration: Male group (a) total bacteria load; (b) Firmicutes;(c) Bacteroidetes (d) Firmicutes/Bacteroidetes ratio. Female group (e) total bacteria load; (f) Firmicutes;(g) Bacteroidetes (h) Firmicutes/Bacteroidetes ratio. Intestinal inflammatory biomarker; (i) male MPO activity, (j) female MPO activity. Circulation system inflammation; Male group (k) LPS-endotoxin level; (l) anti-LPS immunoreactivity; (m) Lymphocyte cells number; (n) Neutrophil cells number. Female group (o) LPS-endotoxin level; (p) anti-LPS immunoreactivity; (q) Lymphocyte cells number; (r) Neutrophil cells number. Data are expressed (n=4–6) as mean ± SEM. $The difference in control compared to the young control, *The difference in CSI compared to the young CSI, and #Difference compared to the aged-matched in control Vs. CSI. $p < 0.05, $$p < 0.01, $$$p < 0.001, *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05, ##p < 0.01, ###p < 0.001.

3.9. Peripheral endotoxin and inflammation

To ascertain whether altered gut microbiota correlates with the LPS byproduct and inflammatory markers in the circulation system, we investigated inflammation in the colon by measuring the myeloperoxidase (MPO) enzyme level and serum levels of LPS, neutrophils and proinflammatory cytokines (IL-6, TNF-α, and IL-1β). We observed an increase in MPO activity in the colon during normal aging in both sexes ($P < 0.05) (Fig. 4i–j) and remained the same after CSI. The middle-aged female mice showed increased MPO activity compared with the healthy aged-match group (#P < 0.05). We found that plasma LPS concentrations were higher in healthy advanced-aged female and male mice compared to the healthy young group ($P < 0.05) (Fig. 4k and o). Furthermore, plasma endotoxemia levels remained the same in advanced aging, even after long-term inflammation.

Anti-LPS IgG immunoreactivity was measured to determine if mice had mounted anti-LPS IgG response in serum. Our results showed no sex- and-age-dependent changes in anti-LPS IgG levels during aging (Fig. 4l and p). However, after long-term CSI, the young mice showed immunoreactivity against the LPS with increased anti-LPS IgG levels in both males (#P < 0.05) and females (###P < 0.001) compared with the healthy aged-match group. In addition, we observed a reduction in lymphocyte cells in advanced-aged with and without CSI in both sexes (Fig. 4m and q). The number of neutrophils increased in healthy advanced-aging male mice but was significant only in advanced-aged CSI male mice (*P < 0.05) (Fig. 4n and r). Moreover, there was a significant elevation in the number of neutrophils in healthy advanced-aging females ($P < 0.05), which remained the same even after CSI (**P < 0.01)

4. Discussion

In the present study, we observed that healthy and advanced-aged female mice were more susceptible to cofilin1 pathway dysregulation in aged-match males. Age-dependent differences in the cofilin1 pathway dysregulation to CSI were evident in both sexes as early as middle-aged. The middle-aged mice with CSI exhibited the general characteristics of advanced-aged mice, such as cognitive decline, synaptic dysfunction, and neuroinflammation. In addition, we observed the presence of Aβ peptide and tau proteins in the saliva of CSI mice.

Our findings demonstrate that dysregulation of cofilin1 and its upstream regulators are age- and sex-dependent and correlated with cognitive decline and AD biomarkers, which were more pronounced than previously predicted (Barone et al., 2014). However, mechanisms of the cofilin1 pathway impacting aging and AD remain disputed. Some argue that excessive dephosphorylation of cofilin1 results in the formation of cofilin1–actin rods (Cichon et al., 2012; Minamide et al., 2000; Zhao et al., 2006); others argue that phosphorylation results in the formation of actin bundles and cofilin1–actin aggregates (Bellenchi et al., 2007; Heredia et al., 2006; Wang et al., 2013). Our findings are in agreement with those previously published on the inactivation of cofilin1 in aged male mice that match the mice age in this study (18–20 months) (Barone et al., 2014). We found that the dysregulation of the cofilin1 pathway occurred due to the inactivation of SSH1 and by an increased pSSH1/SSH1 ratio, and its association with age-dependent conditions signals an early onset of cofilin1 inactivation during aging in males.

For the first time, we found advanced-aged female mice have dysregulation in the cofilin1 pathway during normal aging compared with younger females. Although pcofilin1 protein was much higher than active cofilin1, the active cofilin1 protein levels were still higher overall. That means the active cofilin1 could be involved in many signaling cascades other than actin-dependent mechanisms. Our results are consistent with a study that reported the upregulation of both cofilin1 and pcofilin1 in mice brains with increased age (Tsai et al., 2021). Similarly, we have previously reported a significant increase in cofilin1 and pcofilin1 in the perihematomal region of mice subjected to intracerebral hemorrhage (ICH), leading to neuroinflammation (Alhadidi et al., 2018). Cofilin1 protein and its phosphorylated form turned out to be a marker for female brain aging and could be the possible reason for observing earlier changes in female brains than males.

Furthermore, our data demonstrates that the inactivation of cofilin1 in female brains is mediated by two mechanisms involving (i) LIMK1 overactivation and (ii) SSH1 inactivation, reflected by the increase in both pLIMK1/LIMK1 and pSSH1/SSH1 ratio. The increase in pLIMK1 levels might have resulted from the diminishing female sex hormones during aging, which negatively affects their role in the modulation of actin dynamics (Hansberg-Pastor et al., 2015). However, our results are in disagreement with earlier studies that report that estrogen hormones can increase pLIMK1 levels in the hippocampus of young female mice, and this estrogen stimulation is reduced with aging (Spencer et al., 2008; Yildirim et al., 2008; Yuen et al., 2011). These differences might be attributed to the variability in the experimental conditions of IHC staining and Western blotting of mice brains. Conversely, other studies reported that pLIMK1 and pcofilin1 levels are associated with aging and AD development (Heredia et al., 2006; Mendoza-Naranjo et al., 2012). Furthermore, aged APP/PS1 transgenic mice had the same cofilin1 pathway dysregulation we found in advanced-aged females (Barone, Mosser and Fraering, 2014), further reinforcing our findings that pLIMK1 and pcofilin1 increase with aging.

Sex and age-related differences in response to LPS have been reported in many studies and are associated with neuroinflammation and cognitive decline (d’Avila et al., 2018; Murtaj et al., 2019). It has been observed that aged mice are more sensitive to LPS than young mice because of sustained neuroinflammation and resistance to regulation, which could be a possible reason for inflammatory priming (Norden and Godbout, 2013). In parallel, we observed CSI-induced cofilin1 pathway dysregulation in middle-aged male and female mice showed disruptions similar to those reported in advance-aged. Thus, this supports the theory that CSI accelerates brain aging (Franceschi and Campisi, 2014). Hence, CSI in the middle-aged is potentially a sensitive and critical cause for brain aging and the early onset of neurodegenerative disease. CSI led to sustained cofilin1 dysregulation in advanced-aged female mice, and in contrast, the advanced-aged males showed cofilin1 dysregulation similar to healthy advanced-aged females. These results support our hypothesis that sex-and-age differences exist in cofilin1 pathway dysregulation in the brain, and CSI is sexually dimorphic.

Since women have a higher prevalence of developing AD (Plassman et al., 2011), the correlation between cofilin1 and its upstream mediators raised questions about its role in normal aging and during CSI and its association with the onset of AD and its progression. It has been found that active cofilin1 exacerbates p-tau expression by inhibiting tau-induced microtubule assembly (Woo et al., 2019), forming rod-shaped aggregation, which signifies events initiated early in AD development (Rahman et al., 2014). In addition, studies have reported that systemic LPS can cause Aβ deposition and tau hyperphosphorylation in AD (Lee et al., 2010; Thygesen et al., 2018; Wang et al., 2018). To our knowledge, no study has examined CSI effects on AD biomarker incidence. Our findings suggest essential age-and-sex-related differences in the early onset of AD during CSI. Based on our data, the significant increase in both p-tau ser396 and ser416 is concurrent with the substantial increase in active cofilin1 in both sexes, which are regulated by mechanisms extending beyond active cofilin1 upregulation. In addition, it has been observed that pLIMK and pSSH1 mediate Aβ-induced neurodegeneration in AD (Barone et al., 2014; Heredia et al., 2006). Our results also showed a concurrent increase in pLIMK, pSSH1, and APP accumulation in both sexes, even though the results are insignificant in CSI middle and advanced-aged females. Consistent with our results, increased endogenous levels of both APP and Aβ during normal aging are reported in the senescence-accelerated prone mice (SAMP) model (Okuma and Nomura, 1998). Based on these findings, females are at higher risk of developing AD during normal aging, which could start early in middle age in the presence of CSI.

It is now recognized that many aging-related diseases are not restricted to brain biomarkers but can be identified by peripheral biomarkers. Using a diagnostic biomarker to detect the pre-symptomatic phase of AD has attracted significant attention in recent years. Saliva is one of the simplest and most easily accessible, non-invasive body fluids among all body specimens. Tau and Aβ proteins are secreted by acinar epithelial cells in the salivary glands, and their expression is reportedly increased in AD patients (Bermejo-Pareja et al., 2010; Shi et al., 2011). We observed age-and-sex differences in detecting Aβ and tau proteins in mice saliva during normal aging and CSI. Interestingly, both sexes showed increased t-tau levels in healthy and CSI advanced-aged mice, suggesting that t-tau could possibly become a biomarker of the early stage of AD. These results are consistent with a previous study that reported total and phosphorylated tau was increased in CSF samples of AD patients (Sjogren et al., 2001). We also observed a simultaneous increase in salivary p-tau (ser416/ser396) with their levels in the brain, but no changes in tau proteins in the serum were observed. In contrast, the Aβ showed similar results in saliva. Therefore, salivary Aβ could be an alternative to the serum method. Overall, the presence of CSI in middle-aged females and advanced-aged males and females are considered high-risk factors for developing AD.

Neuroinflammation, a common cause in all CNS disorders, is critical in glial activation, cognitive decline, and accelerating brain aging (Alsegiani and Shah, 2020). Glial cell activation may be an essential factor underlying cofilin1 pathway dysregulation. We observed that astrocyte biomarkers (GFAP) upregulation correlated with cofilin1 pathway dysregulation in the brain. These findings are consistent with other studies that reported increased GFAP expression levels in aged females, suggesting a correlation with estradiol-dependent glial cell hyperactivity (Kohama et al., 1995; Murtaj et al., 2019). On the other hand, no age-related differences in astrocyte activation were found in the advanced-aged males. Since the gonadal hormones regulate glial cell activation, the effects of sex hormones and reproductive aging may be the reason for the observed sex differences (Mouton et al., 2002). Furthermore, we observed that the upregulations of GFAP in advanced-aged females remain the same after CSI, which means the glial cells respond less effectively to episodic CSI with aging. However, studies have reported that sex hormones are essential in modulating neuroinflammation through other mechanisms, such as anti-inflammatory, anti-apoptotic, and neuroprotective effects (Mallah et al., 2020; Villa et al., 2016).

Our results also show that healthy, advanced-aged females had higher proinflammatory cytokine levels than the healthy-young group and aged-match males. Similarly, earlier preclinical studies revealed that female brains showed more immune activation and upregulation of inflammatory markers than males with aging (Berchtold et al., 2008). During CSI, both sexes showed upregulation in M1 and M2 mediators with aging. The simultaneous elevation of M1 and M2 mediators produced by activated microglia could be a feedback or compensatory mechanism in the brains of aged mice during CSI (d’Avila et al., 2018; Henry et al., 2009).

Synaptic plasticity is impaired with aging, which might explain memory deficits and disease initiation in the elderly (Petralia et al., 2014). In addition, cofilin1 activity is involved in various signaling pathways for synaptic dysfunction (Ben Zablah et al., 2020). However, the expression of the synaptic plasticity proteins regarding sex and age is also controversial; both upregulated and downregulated have been reported in different brain regions (Head et al., 2009; Proctor et al., 2010). No sex differences regarding synaptic plasticity protein expression were observed in our mice. However, a severe reduction in synaptic plasticity was observed in healthy advanced-aged mice and was associated with cofilin1 pathway dysregulation. Furthermore, synaptic dysfunction was observed during the CSI in all tested groups, even the young mice, suggesting long-term CSI negatively affects hippocampus synaptic plasticity. CSI contributes to the impairment of synaptic function and plasticity in young mice through mechanisms other than cofilin1 pathways, such as by downregulation of the Wnt/β-catenin pathway, disruption of mGluR signaling, or activation of the NMDAR signaling pathway (Han et al., 2023; Tang, 2014).

Aging involves several pathological complications that impair various neurobehavioral and cognitive faculties (d’Avila et al., 2018). Sex differences in neurobehavioral studies have been documented widely in rodents (Bettis and Jacobs, 2009). Our behavior studies showed that anxiety and depressive-like behavior were observed in advanced-aged mice in both sexes during normal aging. In contrast, cognitive behavior, such as memory and learning, were particularly impaired in the advanced-aged female mice. As expected, cognitive and behavioral impairment started in middle age and worsened with CSI, indicating that CSI affected the brain region involving cognition, including the hippocampus. Mice in the study showed behavioral and cognition impairments that correlated to synaptic dysfunction, cofilin1 dysregulation, Tau, Aβ accumulation, astrocyte activation, and increased inflammatory cytokines.

At the peripheral level, advanced-aged mice exhibited general gut inflammation, immunosenescence, and high levels of endotoxin and inflammatory markers in the circulation system, as reported in different studies (Alsegiani and Shah, 2022; Franceschi et al., 2000; Thevaranjan et al., 2017), and these signs remained the same after the CSI. Mice have shown sex-and-age differences in diversity and composition of gut microbial alteration (Thevaranjan et al., 2017). Conversely, we found no sex-associated differences in the diversity and community of fecal microbiomes in our mice with or without CSI. This may be attributed to the differences in the types of model and mice strains. Gut microbiota alteration increases susceptibility to intestinal inflammation (Jang et al., 2018). In parallel to these results, we found that advanced-aged mice had colon inflammation, which was observed by increased MPO activity in both sexes.

We also observed a significant correlation between gut inflammation and elevated LPS levels in the circulation system. The correlation between systemic LPS and the development of neurodegeneration highlights the role of chronic neuroinflammation in the pathophysiology of several neurodegenerative diseases (Batista et al., 2019). Besides the cofilin1 pathway dysregulation, several molecular mechanisms have reportedly linked systemic LPS to the progression of neurodegenerative diseases, such as oxidative stress, endoplasmic reticulum (ER) stress, mitochondrial dysfunction, and blood-brain barrier (BBB) disruption (Esteves et al., 2023; Jaeger et al., 2009; Lei et al., 2019; Xu et al., 2017).

In addition, we observed a deficiency of anti-LPS IgG immunoreactivity in middle-aged and advanced-aged mice in both sexes, suggesting an age-dependent impairment of the immune system. Consistent with our results, prior research shows that episodic stimulation of the immune system is thought to be an essential cause of immunosenescence and subsequently accelerates age-associated pathologies (Franceschi and Campisi, 2014). Furthermore, evidence from preclinical studies has shown that neutrophils significantly increase while lymphocytes decrease with age (Valiathan et al., 2016). We also observed reduced lymphocytes and increased neutrophil numbers in healthy advanced-aged females and both sexes of advanced-aged mice with CSI compared with middle-aged and young groups (Fig. 5).

Fig. 5.

Fig. 5.

Open in a new tab

Cofilin1 pathway at the centerstage of CSI. Cofilin1 pathway is playing a central role in glial activation in CSI. Cofilin1 is associated with reduced levels of PSD95, SYN, and BDNF leading to cognitive impairments and increased markers of AD.

5. Conclusion and future directions

The study suggests that cofilin1 is a potential target for treating neuroinflammation, particularly in middle-aged populations with CSI, to prevent early brain aging and AD. Furthermore, active cofilin1 seems to play a fundamental role in neuroinflammation by mechanisms other than actin-dependent mechanisms. These findings suggest that active cofilin1 and pLIMK1 could partially have a sex-specific role in AD and other neurodegenerative disorders. The data also supports the hypothesis that abnormalities in the gut and circulation system may contribute to the disruption in the central nervous system during aging and CSI. These results set the basis for further studies aimed at characterizing sex- and age-related therapies to aid in identifying novel therapeutic targets and agents. However, the study has some limitations, such as tracking the expression of active cofilin1 and exploring other possible signaling cascades in the brain to identify the mechanisms involved in this dysregulation. While our study focused primarily on age- and sex differences in cofilin1 pathway dysregulation during CSI, the correlation between CSI and the development of AD biomarkers warrants further investigation. Finally, it would be desirable if the age- and sex-specific neuroinflammation patterns were identified in humans.

Supplementary Material

Supplementary Material

Funding

This research was funded by the National Institute of Neurological Disorders and Stroke of the National Institutes of Health #R01NS112642 to ZAS. ASA was supported by the College of Pharmacy, Department of Pharmaceutical Chemistry, King Saud University, Riyadh, Saudi Arabia.

Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.neurobiolaging.2024.09.003.

Footnotes

CRediT authorship contribution statement

Amsha S. Alsegiani: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Zahoor A. Shah: Writing – review & editing, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Funding acquisition, Formal analysis, Conceptualization.

Declaration of Competing Interest

The authors declare no conflict of interest.

Informed Consent Statement

NA

Verification

This is an original article and does not contain any data already published. Authors agree to the contents of the research work.

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