Hippocampal Astrocyte Cultures from Adult and Aged Rats Reproduce Changes in Glial Functionality Observed in the Aging Brain

Abstract

Astrocytes are dynamic cells that maintain brain homeostasis, regulate neurotransmitter systems, and process synaptic information, energy metabolism, antioxidant defenses, and inflammatory response. Aging is a biological process that is closely associated with hippocampal astrocyte dysfunction. In this sense, we demonstrated that hippocampal astrocytes from adult and aged Wistar rats reproduce the glial functionality alterations observed in aging by evaluating several senescence, glutamatergic, oxidative and inflammatory parameters commonly associated with the aging process. Here, we show that the p21 senescence-associated gene and classical astrocyte markers, such as glial fibrillary acidic protein (GFAP), vimentin, and actin, changed their expressions in adult and aged astrocytes. Age-dependent changes were also observed in glutamate transporters (glutamate aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1)) and glutamine synthetase immunolabeling and activity. Additionally, according to in vivo aging, astrocytes from adult and aged rats showed an increase in oxidative/nitrosative stress with mitochondrial dysfunction, an increase in RNA oxidation, NADPH oxidase (NOX) activity, superoxide levels, and inducible nitric oxide synthase (iNOS) expression levels. Changes in antioxidant defenses were also observed. Hippocampal astrocytes also displayed age-dependent inflammatory response with augmentation of proinflammatory cytokine levels, such as TNF-α, IL-1β, IL-6, IL-18, and messenger RNA (mRNA) levels of cyclo-oxygenase 2 (COX-2). Furthermore, these cells secrete neurotrophic factors, including glia-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), S100 calcium-binding protein B (S100B) protein, and transforming growth factor-β (TGF-β), which changed in an age-dependent manner. Classical signaling pathways associated with aging, such as nuclear factor erythroid-derived 2-like 2 (Nrf2), nuclear factor kappa B (NFκB), heme oxygenase-1 (HO-1), and p38 mitogen-activated protein kinase (MAPK), were also changed in adult and aged astrocytes and are probably related to the changes observed in senescence marker, glutamatergic metabolism, mitochondrial dysfunction, oxidative/nitrosative stress, antioxidant defenses, inflammatory response, and trophic factors release. Together, our results reinforce the role of hippocampal astrocytes as a target for understanding the mechanisms involved in aging and provide an innovative tool for studies of astrocyte roles in physiological and pathological aging brain.

Characterization of human iPSC-derived astrocytes with potential for disease modeling and drug discovery

“…Moreover, treatment with TNFa, IL-1a, and C1q led to increased levels of two genes, EAAT1 and GLUL, previously shown to be induced in reactive astrocytes (Bellaver et al, 2017;Beschorner et al, 2007) (Fig. 4C-D).…”

“…In particular, human iPSC-derived astrocytes can respond to stimulation with a pro-inflammatory combination of TNFa, IL-1a, and C1q, which are secreted by activated microglia during neuroinflammatory processes and can lead to astrocyte activation [Liddelow et al, 2017], by upregulating the levels of a number of transcripts encoding molecules previously associated with a reactive astrocyte phenotype. Interestingly, elevated levels of genes previously associated with neurotoxic reactive astrocytes, such as SERGLYCIN, SERPING1, GLUL and TNFa [Bellaver et al, 2017;Fernandes et al, 2016;Liddelow et al, 2017] is not accompanied by a concurrent increase in the levels of transcripts encoding intermediate filament proteins like GFAP and VIMENTIN. Increased levels of GFAP and VIMENTIN are considered a typical feature of pro-inflammatory reactive astrocytes (Clarke et al, 2018;Liddelow et al, 2017;Zamanian et al, 2012).…”

Anti-aging effects of guanosine in glial cells

“…Thus, to study features of adult and aged brains, our group has established a routine technique for the primary culture of astrocytes obtained from non-neonatal rats, because the metabolic, oxidative, and inflammatory properties of cells derived from newborn and adult/aged animals are distinctive [21,22,28,29]. We have previously shown that this tool can be employed to study different astrocytic roles in the CNS, as the cells present classical astroglial markers, such as glial fibrillary acidic protein (GFAP), S100B, and glutamine synthetase (GS) expression and activity.…”

In Vitro Adult Astrocytes are Derived From Mature Cells and Reproduce in Vivo Redox Profile

“…Cell cultures have significantly contributed to the understanding of brain properties. In this sense, our group recently characterized the main glial functions of cultured astrocytes from mature Wistar rats [Bellaver et al, 2016;Souza et al, 2013Souza et al, , 2016b. Here, we compared our in vitro astrocytes with the ex vivo tissue that was used to prepare the cultures, and showed that mature astrocytes were derived from mature cells (Fig.…”

“…We have previously demonstrated that adult/aged astrocytes present significant alterations in astrocytes marker proteins, metabolic and amino acid profiles, redox homeostasis, inflammatory response, and signaling pathways in adult rat astrocyte cultures [Bellaver et al, 2016;Souza et al, 2013Souza et al, , 2016b. Our previous findings also point to different responses to neuroprotective and/or neurotoxic compounds, according to the culture's maturity degree Souza et al, 2016a].…”