1 INTRODUCTION

Aging is characterized by a progressive change in the physiology of brain cells that may contribute to cognitive deficits, leading ultimately to dementia and impairment of quality of life. The unique complexity of the human brain, as well as the increasing longevity of humans, represents additional challenges to prevent and postpone aging effects.

It is estimated that by 2050 the number of people aged 60 and older is double from now, reaching nearly 2.1 billion worldwide (Nations, 2017). In this context, a substantial increase in the incidence of age-associated diseases, such as cancer, diabetes, and neurodegenerative diseases, is expected for the next few years (Hou et al., 2019). However, the mechanisms underlying the transition from a healthy fully functional brain to an elderly increasingly dysfunctional brain are not well understood.

Among the causal factors of aging, cellular senescence is an important tumor suppression mechanism, but it also leads to a harmful cellular phenotype which affects tissue homeostasis and regeneration (Campisi, 2005; Lopez-Otin et al., 2013). Several studies have reported age-related accumulation of senescent cells in humans, non-human primates, and rodents (Herbig et al., 2006; Wang et al., 2009; Yousefzadeh et al., 2020). In the aging brain, neural stem cells, neurons, and glial cells display several features of cellular senescence and have been implicated in the pathogenesis of age-related neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD; Cohen & Torres, 2019; Martínez-Cué & Rueda, 2020). However, so far little is known about the impact of glial cell senescence on normal brain aging.

Astrocytes form a large and heterogeneous glial cell population in the central nervous system (CNS), playing several vital roles in brain homeostasis and physiology (Verkhratsky & Nedergaard, 2018). In contrast, astrocyte dysfunction has been associated with many brain disorders, such as AD and PD (Diniz et al., 2019; Matias et al., 2019; Osborn et al., 2016), in which senescent astrocytes accumulate and are linked to neurodegeneration and cognitive and motor impairments in mouse models (Bhat et al., 2012; Bussian et al., 2018; Chinta et al., 2018). Nevertheless, despite the amount of evidence pointing to the involvement of astrocyte senescence in age-related pathologies, the phenotypic and functional changes of senescent astrocytes in the context of physiological brain aging remain to be elucidated. This is at least partially due to the lack of senescence-associated biomarkers for glial cells. Moreover, most of our knowledge of astrocyte biology and brain aging comes from rodent models. A growing number of studies show the unique complexity of human astrocytes (Barbar et al., 2020; Oberheim et al., 2009) and thus it is important to study whether the mechanisms underlying rodent brain aging also apply to the human brain.

The senescent state usually requires a coordinated activation of well-known molecular changes, which identify senescent cells in vitro and in vivo (Sharpless & Sherr, 2015). Moreover, senescent cells undergo important morphological changes, including altered nuclear size and shape (Mehta et al., 2007), as well as reduced levels of lamin-B1 (Freund et al., 2012).

Lamin-B1, together with the other nuclear lamins, constitute the major components of the nuclear lamina, where they form a dense filamentous meshwork essential for maintaining the structural and functional integrity of the nucleus (de Leeuw et al., 2018). Mutations in lamin genes cause several devastating human diseases, collectively known as laminopathies, among which are premature aging syndromes (Schreiber & Kennedy, 2013). Interestingly, nuclear deformations and reduced levels of B-type lamins are present in post-mortem human brain tissue of AD and frontotemporal dementia (FTD) patients (Frost et al., 2016; Paonessa et al., 2019). Besides, lamin-B1 loss was found specifically within astrocytes in PD brain tissue, suggesting the contribution of senescent astrocytes to PD pathology (Chinta et al., 2018). Still, it remains to be elucidated whether lamin-B1 levels and nuclear morphology are altered in astrocytes during normal brain aging.

Here, we investigated whether lamin-B1 loss and changes in nuclear morphology accompany astrocyte senescence in vitro and in the mouse and human aging brain. Altogether, our findings suggest that lamin-B1 loss and defective nuclear morphology are hallmarks of astrocyte senescence in vitro and human hippocampal aging.

2 RESULTS 2.1 Age-related loss of lamin-B1 in the mouse hippocampus

Loss of lamin-B1 has been described as a senescence-associated biomarker in some tissues in vivo (Saito et al., 2019; Wang et al., 2017; Yue et al., 2019), although it remains less known whether this also applies to the aged brain. To address this question, we first examined with immunofluorescence the levels of lamin-B1 in the hippocampal dentate gyrus (granular cell layer and molecular layer) of C57Bl/6 mice at 2–3 months and 18–24 months of age. We observed a steady reduction of more than 75% of lamin-B1 intensity in aged mice compared with young ones (Figure 1a–g). Interestingly, in young mice, lamin-B1 intensity was higher at the subgranular zone and in a few cells of the molecular layer (Figure 1c–c′). In contrast, there was a widespread decline of lamin-B1 throughout the molecular and granular cell layers of the dentate gyrus in aged mice (Figure 1f–f′ and Figure S1).

Age-related loss of lamin-B1 in the mouse hippocampus. (a–g) Densitometric analysis of lamin-B1 staining in the mouse hippocampal dentate gyrus, including the molecular layer (ML) and granular cell layer (GCL), revealed a global reduction of lamin-B1 intensity in aged mice compared with young mice (p = 0.0033). (h–n) Decreased proportion of lamin-B1 + cells in relation to the total number of GFAP + cells in the dentate gyrus of aged mice compared with young ones (p = 0.0126). Significance was determined using unpaired t test with Welch's correction. Error bars represent ±SEM. Individual data points are plotted and represent individual animals (n = 4 animals per experimental group). Scale bars, 20 µm

To further address the subcellular decrease of lamin-B1 in the aged hippocampus, we evaluated lamin-B1 staining in glial fibrillary acidic protein (GFAP)+cells in young and aged mice. While we found ~65% of lamin-B1+/GFAP+cells at hippocampal dentate gyrus of young mice, aged mice showed 25% of lamin-B1+/GFAP+cells (Figure 1h–n).

In addition, to confirm the acquisition of the senescent phenotype by aged astrocytes, we analyzed two well-established markers of DNA damage signaling and the senescence-associated secretory phenotype (SASP): p53-binding protein 1 (53BP1) and transforming growth factor beta 1 (TGF-β1), respectively (Di Micco et al., 2021; Salminen et al., 2011). We observed an increased colocalization of 53BP1 in total cell nuclei, as well as in GFAP+cells nuclei, in the hippocampal dentate gyrus of old mice (Figure S2a–d). Similarly, densitometric analysis of TGF-β1 staining revealed a five times increase in intensity in the molecular layer of the aged dentate gyrus, including a 3.1 times increase in GFAP-positive cells (Figure S2e–h).

Altogether, these results point to an age-related decline of lamin-B1 and upregulation of 53BP1 and TGF-β1 in the mouse hippocampal dentate gyrus, partially due to their regulation in astrocytes, suggesting the acquisition of the senescent phenotype by these cells.

2.2 Senescent astrocytes reveal a distinct phenotype and impaired neuritogenic and synaptogenic properties in vitro

To further investigate the molecular and functional changes of aged astrocytes, we first established a new in vitro model for astrocyte senescence by culturing mouse cortical astrocytes for 9–10 days in vitro (DIV, control group) or 30–35 DIV (senescent group) followed by evaluation of several features of cellular senescence (Figure 2a).

Characterization of a new in vitro model for astrocyte senescence. (a) Primary murine astrocyte cultures were maintained for 9–10 DIV (control group) or 30–35 DIV (senescent group) and analyzed for several senescence-associated biomarkers. (b–d) Astrocytes cultured for 30–35 DIV showed increased SA-β-galactosidase activity compared with control cultures (p = 0.0481). (e–g) Higher immunostaining for p16INK4a in senescent astrocytes compared with control cultures (p = 0.0459). (h) Senescent astrocytes showed increased expression of p16INK4a (p = 0.0471). (i) Expression levels of MMP3 and IL-6 were increased in senescent astrocyte cultures compared with control ones (p = 0.0267 and p = 0.0142, respectively). (j–l) Increased intensity of DHE labeling (level of ROS) in senescent astrocytes compared with control cultures (p = 0.0467). (m–o) Higher immunostaining for iNOS in senescent astrocytes (p = 0.0311). (p) Nitrite production was elevated in senescent astrocytes (p = 0.0100). Significance was determined using unpaired t test. Error bars represent ±SEM. Individual data points are plotted and represent individual cultures (n = 3–6 cultures per experimental group). Scale bars, 50 µm in (c) and 20 µm in (f), (k), and (n)

We first analyzed the astrocyte cultures for two classical senescence-associated biomarkers: SA-β-Gal and the expression of the tumor suppression protein, p16INK4a (Sharpless & Sherr, 2015). Accordingly, we observed increased activity of SA-β-Gal, based on the optical density of X-Gal staining per experimental condition (Figure 2b–d). Similarly, the percentage of β-Gal+cells was increased by 7.6 times in senescent astrocytes cultures (20.7% of β-Gal+cells) compared with the control group (2.7% of β-Gal+cells) (Figure S3f–h). Additionally, astrocytes cultured for 30–35 DIV showed increased intensity and expression of p16INK4a compared with control astrocytes (Figure 2e–h).

Senescent cells are known to express the SASP, linked to increased secretion of several inflammatory mediators, matrix metalloproteinases, and reactive oxygen and nitrogen species (Coppé et al., 2010). In line with this, we observed an upregulation of matrix metalloproteinase 3 (MMP3) and IL-6 mRNA in senescent astrocytes compared with the control group (Figure 2i). Moreover, senescent astrocytes also showed increased levels of reactive oxygen species (Figure 2j–l), as well as higher intensity of inducible nitric oxide synthase (iNOS, Figure 2m–o) and extracellular nitrite (NO2−), a stable metabolite of nitric oxide (NO; Figure 2p). Despite classical features of senescent cells, we did not observe evident morphological changes in astrocytes over time in vitro (Figure S3b–e).

To further investigate the functional implication of the senescent astrocyte phenotype, we have addressed two known properties of astrocytes; induction of neuritogenesis and synaptogenesis. To do that, we cultured neural progenitor cells derived from the cerebral cortex of mouse embryos (E14-15) in the presence of astrocyte conditioned medium (ACM) from control or senescent cultures for 48 h (Figure 3a). The number of primary neurites per cell in each experimental condition was quantified based on immunostaining for β-Tubulin III. As expected, ACM from control cultures (ACM-Control) induced neuritogenesis, as shown by the increased number of neurites per cell, compared with Dulbecco's minimum essential medium (DMEM; vehicle). Conversely, ACM from senescent cultures (ACM-Senescent) failed to increase neuritogenesis compared to ACM-Control (Figure 3b–e).

Senescent astrocytes present impaired neuritogenic and synaptogenic properties in vitro. (a) Neural progenitor cell cultures were maintained in DMEM (vehicle), ACM-Control, or ACM-Senescent for 2 DIV. (b–d) The number of primary neurites per cell was quantified based on β-Tubulin III immunostaining. (e) Neural progenitor cells treated with ACM-Control showed increased number of neurites compared with those treated with DMEM (p = 0.0097). In contrast, neural progenitors treated with ACM-Senescent exhibited reduced neurite number compared with ACM-Control (p = 0.0044). (a, f–h) Mature neurons (12 DIV) were treated with DMEM, ACM-Control, or ACM-Senescent for 3 h, and the number of synapses was quantified based on synaptophysin/spinophilin colocalization puncta. (i) Neurons treated with ACM-Control showed increased percentage of colocalized puncta compared with those treated with DMEM (p = 0.0083). Conversely, ACM-Senescent reduced the number of synapses compared with ACM-Control (0.0010). Significance was determined using one-way ANOVA with Tukey's multiple comparisons test. Error bars represent ±SEM. Individual data points are plotted and represent individual cultures (n = 3–9 cultures per experimental condition). Scale bars, 20 µm

Additionally, we treated mature neuronal cultures (12 DIV) with the ACM-Control or ACM-Senescent for 3 h, followed by the analysis of synaptic density, which was based on the immunostaining for pre- (synaptophysin) and post-synaptic (spinophilin) proteins. As previously shown by our group and others, the ACM-Control induces synapse formation in vitro through astrocytes’ secretion of synaptogenic factors. In contrast, the ACM-Senescent did not increase synapse formation (in comparison with DMEM), indicating that senescent astrocytes present impaired synaptogenic capacity (Figure 3f–i).

Taken together, these results indicate first, that mouse astrocytes cultured for 30–35 DIV recapitulate key features of the senescent phenotype, and validate their use as an alternative in vitro model for astrocyte senescence. In addition, these results point to a senescence-associated impairment in the neuritogenic and synaptogenic properties of astrocytes.

2.3 Nuclear deformations are associated with lamin-B1 loss in cultured senescent astrocytes

Lamin-B1 downregulation has been described as a senescence-associated biomarker in different in vitro models for cellular senescence, including in replicative, oncogene, and DNA damage-induced senescence models (Freund et al., 2012; Limbad et al., 2020). However, less is known about the regulation of lamin-B1 in aged glial cell cultures.

We thus sought to investigate lamin-B1 as an aging biomarker of astrocytes as suggested by hippocampal mouse data by analyzing its levels in control and senescent astrocyte cultures. Immunostaining, Western blotting, and quantitative RT-PCR (qPCR) assays revealed a robust reduction in lamin-B1 intensity (Figure 4a–c), protein level (Figure 4d), and expression (Figure 4e), of approximately 50%, 30%, and 40%, respectively, in senescent astrocytes compared with control cultures.

Nuclear deformations are associated with lamin-B1 loss in senescent astrocytes. (a–c) Reduced immunostaining for lamin-B1 in senescent astrocytes compared with the control group (p < 0.0001). (d) Lamin-B1 protein levels were diminished in senescent astrocytes in comparison with the control group (p = 0.0329). (e) Senescent astrocytes showed decreased expression of lamin-B1 (p = 0.0417). (f–i) Nuclear circularity analysis is based on the area and perimeter of the nucleus. Circularity has a maximum value of 1 and diminishes as the nuclear shape becomes increasingly convoluted, as observed in senescent cells (g, i). (j) Senescent astrocytes displayed reduced nuclear circularity compared with the control group (p < 0.0001). (k) A positive correlation was observed between lamin-B1 intensity and the nuclear circularity value (r = 0.4589; p = 0.0110). (l, m) Distinct nuclear morphological profiles were evaluated, such as regular (1), aberration (2), evagination (3), and invagination (4) in control and senescent cultured astrocytes, based on lamin-B1 staining. (n) Senescent astrocyte showed an increased incidence of invaginated nuclei (p = 0.0054) and a decreased proportion of regular nuclei (p = 0.0263). (o) A negative correlation was observed between lamin-B1 intensity and the incidence of invaginated nuclei (r = 0.5054; p = 0.0065). Significance was determined using unpaired t test with Welch's correction. Linear regression opting to show 95% confidence bands of the best-fit line. Error bars represent ±SEM. Individual data points are plotted and represent individual cultures (n = 6–11 cultures per experimental condition). Control cultures are represented by white dots and senescent cultures by gray dots in (k) and (o). Scale bars, 20 µm in (b), (b′) and (M); 10 µm in (g) and (4)

It has been shown that lamin concentration within the nucleus is essential in regulating nuclear lamina assembly and, therefore, maintaining nuclear structure and function (Guo et al., 2014; Paonessa et al., 2019). In this sense, we investigated the impact of lamin-B1 downregulation in astrocyte nuclear morphology.

We first analyzed the nuclear circularity based on lamin-B1 staining, which has a maximum value of 1 and diminishes as the nuclear shape becomes increasingly convoluted. We observed a significant reduction in nuclear circularity in senescent astrocytes compared with control ones (Figure 4f–j), which had a positive correlation with the reduced intensity of lamin-B1 in senescent cells (Figure 4k).

We next asked whether alterations of the nuclear circularity could be associated with other types of nuclear lamina abnormalities. To do so, we initially performed three-dimensional reconstruction of lamin-B1+ nuclei of astrocytes, based on z-stack fluorescence microscopy, to classify the types of nuclear deformations present in control and senescent astrocyte cultures. The nuclear morphological classification was divided into three types: invagination, when nuclei showed one clear lamin-B1 invagination (Figure S4a; Video S1); evagination, when nuclei exhibited one clear lamin-B1 protrusion from the nuclear lamina (Figure S4b; Video S2); and aberration, when nuclei presented a combination of more than one invagination, evagination or additional nuclear abnormalities (Figure S4c; Video S3). Regular nuclei were considered those without any of these deformations.

Therefore, in order to optimize the analysis, we used conventional fluorescence microscopy to image and quantify the number of nuclear deformations in control and senescent astrocyte cultures. We observed a reduced proportion of regular nuclei in senescent astrocyte cultures compared with control astrocytes (Figure 4l–n). This was mainly due to the increased incidence of nuclear invaginations in these cells (Figure 4l–n). The proportion of nuclear evaginations and aberrations was similar between control and senescent astrocytes (Figure 4l–n). Interestingly, the incidence of nuclear invaginations negatively correlated with lamin-B1 intensity in astrocytes (Figure 4o).

Together, these results corroborate previous reports on the lamin-B1 decline in cellular senescence models and provide a new hallmark for astrocyte senescence. Moreover, they strongly indicate that this process is associated with defects in nuclear morphology, represented by nuclear invaginations and reduced nuclear circularity, which correlate with reduced levels of lamin-B1 in astrocytes.

2.4 Lamin-B1 reduction in the human dentate gyrus upon aging

Whether the mechanisms underlying rodent brain aging apply to the human brain is still a matter of investigation. Thus, we further evaluated whether lamin-B1 downregulation also occurs in the human brain during normal aging. To do so, we gathered post-mortem human hippocampal tissue from two independent brain banks, both composed of non-demented controls separated into two age groups: middle-aged and elderly donors. Clinic-pathological information of all donors is present in Table S1.

Densitometric analysis of lamin-B1 immunostaining revealed an overall reduction of approximately 15% in lamin-B1 intensity at the granular cell layer of the hippocampal dentate gyrus in the elderly compared with middle-aged donors (Figure 5). It is noteworthy that this hippocampal layer is mainly composed of granule neuron cells, and at its inner boundary reside neural stem/progenitor cells and glial cells, especially astrocytes (Casse et al., 2018).

Lamin-B1 reduction in the human dentate gyrus upon aging. (a–g) Densitometric analysis of lamin-B1 staining at the hippocampal granular cell layer from post-mortem human tissue revealed an overall reduction of lamin-B1 intensity in elderly cases compared with middle-aged ones (p = 0.0308). n = 16 and 13 individuals for middle-aged and elderly groups, respectively. Scale bars, 20 µm. Significance was determined using unpaired t test with Welch's correction. Error bars represent ±SEM. Individual data points are plotted and represent individual donors

Recent evidence revealed regional heterogeneity upon aging in the mouse and human brain (Boisvert et al., 2018; Soreq et al., 2017). To further investigate lamin-B1 content within the human hippocampus, we also analyzed the polymorphic layer, the innermost layer of the dentate gyrus. Curiously, we did not observe any significant difference in lamin-B1 intensity between middle-aged and elderly donors in this region (Figure S5).

Taken together, these results indicate that human aging is accompanied by a downregulation of lamin-B1 in the granular cell layer of the dentate gyrus. Moreover, they also suggest intra-regional heterogeneity in lamin-B1 regulation within the human hippocampus during aging.

2.5 Human hippocampal neural cells, including astrocytes, undergo nuclear abnormalities upon aging

Recent studies reported that lamin misregulation is associated with defects in neuronal nuclear morphology in age-related neurodegenerative diseases, including FTD and AD (Frost et al., 2016; Paonessa et al., 2019). However, it is still unknown whether these nuclear alterations also occur in neural cells during human brain aging.

Thus, we first analyzed the percentage of regular, evaginated, invaginated, and aberrant lamin-B1+ nuclei in the human hippocampal dentate gyrus from middle-aged and elderly donors. We observed a reduced proportion of regular nuclei at the hippocampal granular cell layer in elderly donors in comparison with middle-aged cases (Figure 6a–c), which was due to increased incidence of nuclear invaginations and aberrations (Figure 6a–c), accounting for a higher proportion of the total nuclear deformations in the elderly hippocampus compared with middle-aged cases (Figure 6d). We did not observe differences in the percentage of nuclear evaginations between middle-aged and elderly cases at the granular cell layer (Figure 6a–c). Therefore, these results indicate that neural cells, especially granule cells of the dentate gyrus, undergo nuclear invaginations, and aberrations upon human aging.

Neural cells, including astrocytes, from the granular cell layer of the human hippocampus undergo nuclear abnormalities upon aging. (a, b) Distinct nuclear morphological profiles were evaluated, such as regular (1), evagination (2), invagination (3), and aberration (4) at the hippocampal granular cell layer in post-mortem human tissue from middle-aged and elderly donors based on lamin-B1 staining. Scale bars, 20 µm in (b) and (b′); 10 µm in (4). (c) Elderly donors exhibited a higher incidence of invaginated (p = 0.0494) and aberrant nuclei (p = 0.0243), resulting in a decreased proportion of regular nuclei (p = 0.0102), compared with the middle-aged group. n = 16 and 14 individuals for middle-aged and elderly groups, respectively. (d) Elderly donors showed an increased proportion of total nuclear deformations (ie, evagination + invagination + aberration) (p = 0.0119). n = 16 and 14 individuals for middle-aged and elderly groups, respectively. (e–g) Nuclear circularity was quantified based on Hoechst or DAPI staining in post-mortem human tissue from middle-aged (e–e′) and elderly donors (f–f′). Elderly donors presented a reduced nuclear circularity based on the number of GFAP+cells (g; p = 0.0243; n = 12 individuals for both age groups) and on the total number of cells (h; p < 0.0001; n = 15 and 13 individuals for middle-aged and elderly groups, respectively). Scale bars, 20 µm in (f) and 10 µm in (f′). Significance was determined using unpaired t test with Welch's correction. Error bars represent ± SEM. Individual data points are plotted and represent individual donors

Human astrocytes have several distinct features from their rodent counterparts including morphological, molecular, and functional properties (Barbar et al., 2020; Oberheim et al., 2009). We then asked whether nuclear abnormalities were also evident in the aged human astrocytes.

As we previously observed, cultured senescent astrocytes presented reduced levels of lamin-B1, which correlated with increased nuclear deformation and decreased nuclear circularity in these cells. Thus, to validate this data, we investigated the nuclear circularity of astrocytes of the granular cell layer from middle-aged and elderly cases. To do so, we used the Hoechst or DAPI nuclear staining, which provided a brighter signal of the nuclei boundaries of GFAP+ astrocytes in post-mortem human tissue. We observed a significant reduction of nuclear circularity in GFAP+ astrocytes from elderly donors compared with middle-aged ones (Figure 6e–g). Additionally, considering the total number of nuclei analyzed at the granular cell layer, we also reported a global reduction of nuclear circularity in elderly donors in comparison with middle-aged donors (Figure 6h).

Since we found age-dependent intra-regional variations in lamin-B1 content between the granular cell layer and the polymorphic layer of the human hippocampus, we asked whether these differences may impact the nuclear morphology of the cells from these two regions during aging. To do so, we analyzed the nuclear morphology at the polymorphic layer based on lamin-B1 staining. Interestingly, we did not observe any significant differences in the proportion of invaginated, evaginated, or aberrant nuclei between middle-aged and elderly cases (Figure S6a–d). Moreover, in contrast to astrocytes from the granular cell layer, we did not observe a significant reduction of nuclear circularity in GFAP+ astrocytes, nor in the total cells analyzed in the polymorphic layer in elderly donors compared with middle-aged ones (Figure S6e–h).

Taken together, we conclude that human aging is associated with defects in the nuclear morphology of neural cells, including astrocytes, and reduction of the levels of lamin-B1. Those features are heterogeneously distributed in the aging hippocampus. Our data point to a new cellular and molecular marker to the human physiological brain aging and support the emerging concept of regional heterogeneity upon aging.

3 DISCUSSION

In the current study, we have identified that lamin-B1 loss and changes in nuclear morphology are hallmarks of astrocyte senescence in vitro and of neural cells, including astrocytes, in the aging mouse and human hippocampus. By using an in vitro model for astrocyte senescence, we observed that these cells undergo nuclear invaginations and changes in nuclear circularity, which were associated with the loss of lamin-B1. In agreement, these features were also observed in hippocampal astrocytes and granule cells in aged mice and post-mortem human hippocampal tissue from non-demented elderly. In addition, we described that mouse senescent astrocytes lose their neuritogenic and synaptogenic potential in vitro. Together, our data indicate that astrocytes undergo morphological, molecular, and functional changes during aging and suggest that they may be important targets in understanding the basic mechanisms underlying human hippocampal aging as well as in identifying new age-associated biomarkers.

The hippocampus is critical for cognitive functions and is one of the most vulnerable brain regions to aging and implicated in age-related cognitive decline (Small et al., 2011). Moreover, emerging evidence has pointed to a differential vulnerability of hippocampal subfields in aging and neurodegenerative diseases (Fu et al., 2018; Morrison & Baxter, 2012; Roussarie et al., 2020), being the dentate gyrus particularly affected during normal aging (Morrison & Baxter, 2012; Nadal et al., 2020). Although most of these studies have focused on neuronal populations, recent findings have suggested that major changes in the gene expression profile of glial cells, especially astrocytes, might predict and/or impact the selective vulnerability of brain regions to aging-associated cognitive decline (Boisvert et al., 2018; Clarke et al., 2018; Soreq et al., 2017). Nevertheless, the contribution of astrocyte senescence to aging is far less studied.

The characterization of senescence-associated biomarkers is an important and first step toward understanding the mechanisms underlying senescence itself and its role in the context of aging and age-related diseases. Emerging evidence shows that the loss of lamin-B1 is an important senescence-associated biomarker in several murine and human cell cultures/lines (Freund et al., 2012) and aging tissues, including in photoaged skin (Wang et al., 2017), thymus (Yue et al., 2019), lung (Saito et al., 2019), kidney, and liver (Yousefzadeh et al., 2020). However, relatively less is known about the potential role of lamin-B1 in brain senescence. Here, we demonstrated that lamin-B1 loss is also a hallmark of astrocyte senescence in vitro and described for the first time that its reduction also occurs in hippocampal astrocytes from aged mice, as well as in the hippocampal dentate gyrus from elderly humans. In addition, we reported an overall increase of 53BP1 and TGF-β1 levels in the mouse dentate gyrus, prominently in astrocytes. 53BP1 is one of the mediators of DNA damage checkpoint in response to DNA double-strand breaks (Di Micco et al., 2021). Therefore, it is usually upregulated in aging and cellular senescence models, including in senescent astrocytes induced by ionizing radiation (Turnquist et al., 2019), by the herbicide paraquat (Chinta et al., 2018) and H2O2 treatment (Shang et al., 2020). On the other hand, TGF-β1 is increased with aging and is considered a SASP inducer molecule that can trigger the senescence phenotype in neighboring cells through paracrine signaling (Munoz-Espin & Serrano, 2014; Tominaga & Suzuki, 2019). Thus, our data indicate that the loss of lamin-B1 is accompanied by the acquisition of the senescent phenotype by mouse hippocampal astrocytes upon aging.

We first reported a global reduction of lamin-B1 in the hippocampal dentate gyrus from aged mice, which was partially due to a decreased proportion of hippocampal astrocytes expressing lamin-B1 in this age group. In agreement with our data, a recent study showed age-dependent downregulation of lamin-B1 in adult neural stem/progenitors cells in the mouse hippocampus, which underlies stem cell aging and negatively impacted adult neurogenesis (Bedrosian et al., 2020). Conversely, restoration of lamin-B1 expression was sufficient to enhance progenitors cells proliferation and neurogenesis in the aged hippocampus (bin Imtiaz et al., 2021).

To further investigate lamin-B1 level in astrocytes, we took advantage of an alternative long-term cell culture model for astrocyte senescence. Astrocytes cultured for 30–35 DIV recapitulated key features of the senescent phenotype, including increased SA-β-Gal activity and p16INK4a expression, high expression of MMP3, IL-6, and reactive oxygen and nitrogen species, characteristics of SASP. These results corroborate other models previously described to study glial senescence (Bellaver et al., 2017; Bigagli et al., 2016; Pertusa et al., 2007; Souza et al., 2015) and reproduce several glial changes observed in the aging brain, including increased expression of p21, oxidative/nitrosative stress as well as an inflammatory phenotype (Bellaver et al., 2017). Accordingly, astrocytes acutely isolated from the cerebral cortex of aged mice showed a distinct gene expression signature, including an increased inflammatory phenotype (Orre et al., 2014).

It is well-known that astrocytes play several homeostatic functions in the CNS, such as modulating neuronal morphogenesis, synapse formation, and function (Verkhratsky & Nedergaard, 2018). These actions can be triggered by the secretion of neurotrophic and synaptogenic factors, as described by our group and others (Baldwin & Eroglu, 2017; Matias et al., 2019). However, so far, little is known about the functional profile of astrocytes during normal brain aging. Interestingly, hippocampal neurons co-cultured with aged astrocytes showed a decreased excitatory synaptic transmission, due to a reduced pool of readily releasable synaptic vesicles, although the number of synapses was unchanged (Kawano et al., 2012). In our study, we provide the first evidence that senescent astrocytes have impaired neuritogenic and synaptogenic capacity. Neural progenitor cells or mature neurons treated with ACM from senescent astrocytes displayed fewer neurites and synapses, respectively. These results indicate that the content of astrocyte secreted molecules changes upon senescence, suggesting the involvement of these cells in age-related synaptic decline.

Together, these results strongly support the use of long-term cell culture as an in vitro model to investigate the phenotype and function of glial cell senescence. In addition, as shown in the murine hippocampus, we reported reduced levels and expression of lamin-B1 in senescent cultured astrocytes associated with nuclear membrane malformations.

Human astrocytes are distinct from their murine counterparts, morphologically as well as molecularly, pharmacologically, and functionally (Barbar et al., 2020; Oberheim et al., 2009). Limited availability to human post-mortem samples has deeply hampered our understanding of brain aging at the cellular and molecular levels. Whether the mechanisms underlying rodent brain aging apply to the human brain is still a matter of investigation. Here, we took the advantage of analyzing post-mortem human tissue from non-demented elderly and middle-aged donors. We observed reduced intensity of lamin-B1 in hippocampal cells at the granular cell layer from non-demented elderly donors. This was followed by increased nuclear deformations and a global reduction of nuclear circularity in the elderly hippocampus compared with middle-aged cases. Similarly to senescent mouse astrocytes in vitro, we observed a significant reduction of nuclear circularity in GFAP+ astrocytes from elderly donors, suggesting that alteration in nuclear properties might be a conserved characteristic of mice and human astrocyte aging. In agreement with our results, a recent in vitro study using X-irradiation induced senescent human astrocytes showed a downregulation of the lamin-B1 gene in these cells, together with higher expression of other classical senescent-associated biomarkers (Limbad et al., 2020).

Curiously, when analyzing lamin-B1 intensity at the polymorphic layer, we did not see a clear reduction of its content, suggesting a region-dependent aging regulation of lamin-B1 levels within the human hippocampus. Recently, it has been shown organ-specific (Schaum et al., 2020) and regional-specific (Boisvert et al., 2018; Pestana et al., 2020; Soreq et al., 2017) molecular signatures during aging. However, whether this intra-regional difference is a predictor of selective vulnerability of the granular cell layer during aging is still unknown.

Taken together, these findings strengthen the hypothesis that lamin-B1 loss, in specific brain areas, is a hallmark associated with cellular senescence, including in astrocytes, in mouse and human hippocampal aging. Although a direct link between lamin-B1 loss with changes in astrocyte phenotype and function has yet to be investigated, lamin-B1 downregulation has been recognized as a key step for the progression of full cellular senescence in other cell types and tissues (Freund et al., 2012; Saito et al., 2019; Shimi et al., 2011), which can, in turn, contribute to tissue degeneration in aging and induce pathological conditions (Saito et al., 2019; Yue et al., 2019). However, whether the lamin-B1 loss is a consequence of the senescent phenotype or a trigger of astrocyte senescence remains to be investigated.

Lamin-B1 is a component of the nuclear lamina and is required for proper organogenesis, cell proliferation, self-maintenance, chromatin structure, nuclear integrity, and gene expression (Ho & Lammerding, 2012). Misregulation of nuclear lamins and abnormalities of the nuclear shape have been widely reported in laminopathies, including premature aging syndromes (Schreiber & Kennedy, 2013), and more recently in in vivo models for age-related neurodegenerative diseases. Reduction in lamin-B protein levels and nuclear invaginations have been shown in Tau-transgenic Drosophila brains, as well as in AD human brains, events associated with an aberrant cytoskeleton-nucleoskeleton coupling and neuronal death (Frost et al., 2016). Here, we report increased nuclear invagination and abnormal nuclear circularity in senescent astrocytes in vitro and in human aged brain. Likewise, a higher incidence of nuclear lamina invaginations is present in FTD human brain tissue and FTD iPSC-derived neurons, resulting in disrupted neuronal nucleocytoplasmic transport in vitro (Paonessa et al., 2019).

Astrocyte dysfunctions have been strongly implicated in the pathogenesis of several age-related diseases, although astrocyte senescence has only recently been fully addressed (Bussian et al., 2018; Chinta et al., 2018; Cohen & Torres, 2019). Recently, it has been shown that senescent astrocytes of PD patients were lamin-B1 deficient, while non-astrocytic neighboring cells retained basal levels of lamin-B1, suggesting that astrocytes may preferentially undergo senescence in PD brain tissue (Chinta et al., 2018). In this sense, this and other studies have shown that depletion of senescent cells, including senescent glial cells, mitigates PD pathology (Chinta et al., 2018), prevents tau-dependent pathology (Bussian et al., 2018), and cognitive deficits in AD mouse models (Zhang et al., 2019).

The fact that loss of lamin-B1 has been reported in age-related neurodegenerative diseases (Chinta et al., 2018; Frost et al., 2016; Paonessa et al., 2019) suggests that this feature may represent an early sign of age-associated brain pathology. Our study demonstrated that lamin-B1 downregulation and nuclear deformations are present in astrocytes (and other neural cells) from post-mortem human tissues from elderly donors, suggesting that astrocytes may play an important role in the mechanisms underlying brain aging.

4 EXPERIMENTAL PROCEDURES 4.1 Animals

Newborn (P0) Swiss mice were used for astrocytes cultures. For in vivo experiments, we used male C57Bl/6 mice divided into two age groups: 2–3 months old (young group) and 18–24 months old (aged group). All animals were housed at standard conditions with ad libitum access to food and water. Animal handling and experimental procedures were previously approved by the Animal Use Ethics Committee of the Federal University of Rio de Janeiro (CEUA-UFRJ, approval protocol 006/18) and the Central Authority for Scientific Experiments on Animals of the Netherlands (CCD, approval protocol AVD115002016659). Experiments were performed according to Brazilian Guidelines on Care and Use of Animals for Scientific and Teaching Purposes (DBCA) and the Directive of the European Parliament and of the Council of the European Union of 22 September 2010 (2010/63/EU).

4.2 Human post-mortem brain material

Human post-mortem brain material was obtained from two brain banks to increase representation and statistical power: the Netherlands Brain Bank, Amsterdam (www.brainbank.nl) (NBB) and the Brain Bank of the Brazilian Aging Brain Study Group, University of São Paulo Medical School (http://en.gerolab.com.br/) (BBBABSG). A written informed consent for a brain autopsy and the use of the material and clinical information for research purposes had been obtained by the NBB (project number 1073) and BBBABSG (CAAE number: 30038520.0.0000.5257, approval number 3.986.070). We obtained paraffin-embedded tissue of the hippocampal area from both brain banks. A total of 30 donors were classified into two age groups: middle-aged (ranged from 50 to 60 years, n = 16) and elderly (ranged from 76 to 93 years, n = 14). Only non-demented controls were included in the analysis, based on the medical history and pathological scoring. Clinico-pathological information of all donors is present in Table S1.

4.3 Control and senescent astr