¹Facultad de Farmacia, Universidad CEU San Pablo, Alcorcón, Madrid, Spain; ²Facultad de Medicina, Universidad de Castilla-la Mancha, Albacete, Spain; ³Facultad de Farmacia, Instituto de Estudio de las Adicciones, Universidad CEU San Pablo, Alcorcón, Madrid, Spain

Abstract

Food-related disorders are increasingly common in developed societies, and the psychological component of these disorders has been gaining increasing attention. Both overnourishment with high-fat diets and perinatal undernourishment in mice have been linked to a higher motivation toward food, resulting in an alteration in food intake. Clusterin (CLU), a multifaced protein, is overexpressed in the nucleus accumbens (NAc) of over-fed rats, as well as in those that suffered chronic undernutrition. Moreover, an increase of this protein was observed in the plasma of obese patients with food addiction, suggesting the implication of CLU in this eating disorder. To characterize CLU’s cellular mechanisms, in vitro experiments of undernutrition were performed using dopaminergic SH-SY5Y cells. To mimic in vivo dietary conditions, cells were treated with different fetal bovine serum (FBS) concentrations, resulting in control (C group) diet (10% FBS), undernourishment (U group) diet (0.5% FBS), and undernourishment diet followed by restoration of control diet (UC group) (0.5 + 10% FBS). Undernourishment compromised cell viability and proliferation, and concomitantly increased CLU secretion as well as the cytosolic pool of the protein, while decreasing the mitochondrial level. The restoration of normal conditions tended to recover cell physiology, and the normal levels and distribution of CLU. This research study is a step forward toward the characterization of clusterin as a potential marker for food addiction and nutritional status.

Keywords: clusterin; food addiction; undernutrition; mitochondria; cell survival

Obesity has become a worldwide public health problem as its prevalence continues to rise dramatically. Studies suggest that more than 30% of the world population will be overweight and 20% will become obese by 2030 (1, 2, 3). Obesity is caused by an energy imbalance between the calories consumed and the calories expended, resulting in an excess body weight. The central nervous system (CNS) plays an essential role in controlling energy homeostasis through the integration of hormonal and nutritional metabolic signals, thereby establishing the central control of food ingestion (4, 5). New theories have introduced the idea of a compulsive behavior for food ingestion, leading to the concept of food addiction (6). In fact, drug and food addictions share neurobiological and behavioral similarities. According to this hypothesis, mesolimbic dopaminergic hypofunction may underlie both drug and food addictions in humans and animal models (7, 8). Hence, food and drugs may have analog effects on the activity of the reward system. As a result, some individuals are more prone to develop eating disorders, leading to obesity. Among foods, those that are more palatable increase the release of dopamine in the nucleus accumbens (NAc) as drugs of abuse do, leading to pleasant sensation (9, 10). Moreover, high-fat and sugar diets produce neurochemical modifications in the NAc, such as decrease of D1 and D2 dopamine receptors, leading to an alteration in the reward system pathway (8, 11, 12) and lowest D2 values in individuals with the largest body mass index (BMI) (13). However, undernourishment during perinatal periods has been described to cause changes in reward-related brain structures, leading to an increase in addiction vulnerability and rewarding potential of both drugs of abuse and palatable foods (11, 14–16).

These findings highlight the idea that ‘food addiction’ may be responsible for some types of obesity and eating disorders. Indeed, it has been found that 40% of obese patients seeking bariatric surgery have food addiction, according to psychological parameters (17). Therefore, the identification of novel biomarkers associated with food addiction could be essential for the correct diagnosis and treatment of obesity.

Using a proteomic-based study, we have recently identified clusterin (CLU) as a potential candidate to measure food addiction. Clusterin levels were found to be higher in the plasma of patients with morbid obesity and uncontrollable food intake compared with those with better eating control (18). Clusterin is an extracellular chaperone that also showed a strong correlation with changes in body fat mass (19). In the hypothalamic areas that control energy metabolism and body weight, CLU mRNA is highly expressed. Among its several functions, CLU was identified as a plasma leptin-binding protein, regulating the hypothalamic leptin pathway. Leptin, a regulatory weight hormone, acts on the hypothalamus, reducing the food intake and increasing the energy expenditure (20, 21). Intracerebellar (ICV) administration of CLU in mice produced weight loss, acting as an anorexigenic molecule, whereas its inhibition stimulated food intake and weight gain (20).

CLU is a multifaced protein, encoded from a single gene located in chromosome 8 in humans, which is highly conserved among species and expressed in multiple tissues (17). The role of CLU is not well understood, but it seems to be highly dependent on its localization. Secreted CLU is a heterodimeric complex of two 40−45 kDa subunits (α- and β-subunits) interlinked by disulfide bonds (22, 23). Extracellular CLU acts as a chaperone exerting a scavenging and clearance activity under physiological and pathological conditions (24–26). Apart from secreted CLU, several intracellular isoforms and post-splicing modifications that play different roles have been reported (27–31). Among these, a hypoglycosylated form of CLU is produced under endoplasmic reticulum stress and translocated to the mitochondria using the chaperone BiP (GRP78), where induces apoptosis (32). This anti-apoptotic role of intracellular CLU has been linked to its interaction with apoptosis-related proteins, Bax and Bcl-xL (33–36).

In this study, we aim to establish a relationship between CLU cellular localization and undernutrition status in a human neuronal in vitro model to clarify the potential role of CLU in an undernourished brain.

Materials and methods

Cell cultures and treatment

Human neuroblastoma cells SHSY-5Y were cultured in Roswell Park Memorial Institute medium (RPMI) supplemented with 10% (v/v) of heat-inactivated fetal bovine serum (FBS), penicillin or streptomycin (100 U/mL), and 2 mM L-glutamine at 37°C under a humidified atmosphere with 5% CO₂.

The FBS content in culture medium was modified to mimic underfeeding conditions of proteins, lipids, micronutrients, and growth factors. Two consecutive treatments were established for such modifications: first treatment of 48 h incubation and a second treatment of 72 h incubation. Briefly, control (C) cells were incubated with the medium containing 10% FBS for 48 h followed by a second treatment with fresh medium containing 10% FBS for 72 h. Undernourished (U) cells were incubated with the medium containing 0.5% FBS for 48 h followed by a second treatment with fresh medium containing 0.5% FBS for 72 h. Undernourished-control (UC) cells were incubated with the medium containing 0.5% FBS for 48 h followed by a second treatment with fresh medium containing 10% FBS for 72 h. Control-overnourished cells were incubated with the medium containing 10% FBS for 48 h followed by a second treatment with fresh medium containing 45% FBS for 72 h (CO). In addition, undernourished-overnourished cells were incubated with the medium containing 0.5% FBS for 48 h followed by a second treatment with fresh medium containing 45% FBS for 72 h (Undernourished-Overnourished [UO])

Biochemistry analysis

For Western blotting analysis of the whole-cell content, cells were lysed with Radioimmunoprecipitation assay buffer (RIPA) buffer (2% Sodium Dodecil Sulphate (SDS), 150 mM NaCl, 2 mM Ethylenediaminetetraacetic acid (EDTA), and 10 mM Hepes, pH 7.4). For Western blotting analysis of subcellular fractions, total cell lysate was fractioned by differential centrifugation as previously described (37). Protein concentration was measured using the Bradford protein assay method. Equal amounts of protein were subjected to 10% Tris-HCl SDS-PAGE gels. The gels were blotted onto 0.2 mm nitrocellulose membranes (Trans-Blot-Turbo transfer Pack, Bio-Rad) using a Transblot-Turbo Transfer System (Bio-Rad). Western blotting signals were detected using the enhanced chemiluminescence reagent (ECL Prime Western Blotting Reagent, GE Healthcare, Amersham, UK). Quantifications were performed using ImageLab software (Bio-Rad). Antibodies used were anti-clusterin (NBP1-68308 1:1000, Novusbio, Littleton, CO, USA), anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA), anti-rabbit (Santa Cruz Biotechnology, Dallas, TX, USA) and anti-mouse (Santa Cruz Biotechnology, Dallas, TX, USA).

ELISA assay

The determination of extracellular CLU was carried out by enzyme-linked immunosorbent assay (ELISA) (ELISA kit ERCLU- Thermo-Fischer Scientific, Frederick, MD, USA) following manufacturer’s instructions.

Cellular viability and proliferation

MTT, NR, and LDH tests were used to evaluate the effects of undernourishment on cell proliferation and viability. Cell viability of C cells, U cells, and UC cells were measured by absorbance at 570 nm (MTT) and 540 nm (NR test) using a Versamax plate reader (BioNova Científica). An LDH test was performed following the manufacturer’s instructions (CytoTox96 Non-Radioactive Cytotoxicity assay, Promega, MD, USA)

Mitochondrial membrane potential assay

The mitochondrial membrane potential (∆Ψ_m) was detected using a MitoPTJC-1 detection kit (Immunochemistry Technologies) according to the manufacturer’s protocol, and fluorescence was quantified using a fluorescence plate reader (Varioskan Flash, Thermo Fisher Scientific). Positive control cells were induced by incubation for 75 min with [(3-chlorophenyl) hydrazono] malonitrile provided in the kit.

Morphological study

In the morphological study, cells were seeded in a 12-well plate, 50,000 cells/well. After treatment, cells were washed twice with Phosphate-buffered saline (PBS) and stained with crystal violet solution at 0.5% in 20% ethanol for 5 min. Then, the crystal violet solution was removed, and the cells were rinsed with 96% ethanol. Finally, cells were mounted on glass slides and observed under an optical inverted phase-contrast microscope (Nikon Eclipse TS1000, 40× magnification)with a digital camera (Nikon Digital Sight).

Statistical analysis

Descriptive statistics are presented as means ± SEM. One-way analyses of variance (ANOVAs) followed by Bonferroni’s test were performed for multiple comparison with GraphPad Prism 7. Results were considered to be statistically significant when P < 0.05. All the experiments presented here were repeated independently at least three times.

Results

Both cell viability and proliferation are decreased by undernourishment without mitochondrial alterations

SH-SY5Y cells were treated with FBS-restricted medium to mimic an under-protein diet, mainly based on the major protein content in FBS, albumin. After control (C), undernourishment (U) and undernourishment-control (UC) treatments, cells were stained with crystal violet dye, and no macroscopic difference was observed (Supplementary Fig. 1). After undernourishment diet, cell viability and proliferation were reduced, as shown by NR, LDH and MTT tests (Fig. 1a−c). These effects of undernourishment were partially or totally reverted after restoration of control conditions (Fig. 1a−c). A slight reduction of mitochondrial functionality was also observed in undernourished cells; however, this tendency did not reach statistical significance based on the mitochondrial membrane potential measured using the Mitotracker assay (Fig. 1d).

Fig. 1. Undernutrition decreases cell viability and proliferation. (a) Neutral Red Uptake test. n = 4 independent experiments. (b) MTT test. n = 4 independent experiments; (c) LDH assay. n =5 independent experiments; (d) Mitotracker assay. n = 5 independent experiments. C, control; U, undernourishment; UC, undernourishment control.

Undernutrition increases the release of clusterin

To elucidate whether CLU is involved in diet-related cell viability and proliferation, both the extracellular and intracellular levels of CLU were measured. After C, U, and UC treatments, cell supernatant was collected for ELISA quantification of secreted CLU, and cells were lysed for Western blot analysis of intracellular CLU. Analysis of the total CLU content did not show any differences among the treatments (Fig. 2a), while U treatment increased three times the content of CLU in the extracellular medium (Fig. 2b). Secreted CLU returned to control levels after undernourishment was discontinued (UC treatment). To check out that CLU overexpression is specifically triggered by undernourishment, we also analyzed extracellular levels of CLU after overnutrition (O) in two additional treatment groups: CO cells and UO cells. Extracellular levels of CLU in both CO and UO cells remain similar to C cells (Supplementary Fig. 2).

Fig. 2. Undernutrition increases the levels of extracellular clusterin. (a) Western blot analysis of the whole cell content of clusterin. n = 4 independent experiments. Loading control: β-actin. (b) Extracellular clusterin quantification by ELISA assay; n = 3–4 independent experiments. Quantification of clusterin was corrected by total protein content in the extracellular media. C, control; U, undernourishment; UC, undernourishment-control. Graphs: means ± SEM, one-way ANOVA was used.

Undernourishment shifts intracellular localization of clusterin from mitochondria to cytosol

As different localizations of CLU determine its cellular role, we performed a cellular fractionation of mitochondria or cytosol and posterior analysis of CLU content. Cytosolic CLU was increased six times by undernutrition (Fig. 3a), while mitochondrial CLU decreased 10 times (Fig. 3b) over the control diet. Both cytosolic and mitochondrial up-regulated levels of CLU were restored when cells returned to control conditions (Fig. 3a and b).

Fig. 3. Nutrition-dependent intracellular localization of clusterin. Western blot analysis of clusterin after cytosolic or mitochondrial fractionation. (a) Cytosolic fraction and (b) mitochondrial fraction. C, control; U, undernourishment; UC, undernourishment-control; Loading control protein: β-actin. n = 3−4 independent experiments. Graphs: means ± SEM, one-way ANOVA was used.

Discussion and conclusion

As far as we know, this is the first attempt to establish an in vitro model of undernutrition to study changes of CLU in undernourished neuronal cells. The idea is based on our previous in vivo results showing evidence that CLU may play a role in food addiction and can potentially be considered as a biomarker.

In neuronal dopaminergic cells, as SH-SY5Y, CLU is highly secreted in response to protein undernutrition and its levels return to baseline upon restoration of normal conditions. The essential role of extracellular CLU is as a chaperone with protective effects, and therefore, this increase could be interpreted as part of a homeostatic reaction to counteract the negative effects of undernutrition on cell viability and proliferation. In agreement with this idea, previous in vivo studies showed an increase of mRNA CLU in the brain in response to the oxidative damage produced by calory restrictions (38, 39). We did not detect any global change in the intracellular levels of CLU, but a specific increase in the cytosolic content and a specific decrease in the mitochondrial level. These changes could be due to a translocation of CLU from mitochondria to cytosol or due to an increase in the synthesis of the canonical cytosolic isoform associated with a decrease in the synthesis of the other intracellular isoforms. Addressing this point is not easy as there is no consensus yet about different mRNA isoforms or post-splicing forms of CLU (29).

Along with previous human and rodent studies, these new data support the relationship between nutrition and CLU, and highlight the potential importance of extracellular CLU levels and intracellular trafficking of the protein in maintaining cell homeostasis under adverse conditions.

Authors’ contributions

Carmen Rodríguez Rivera, María Dolores Pérez Carrión, and Lucía Casariego Olavarría performed the experiments and analyzed the data. Luis Fernando Alguacil Merino, María José Polanco Mora and Carmen González-Martín designed the experiments, analyzed the data, and wrote the article.

References

1. Segula D. Complications of obesity in adults: a short review of the literature. Malawi Med J 2014; 26(1): 20–4.
2. Hruby A, Hu FB. The epidemiology of obesity: a big picture. Pharmacoeconomics 2015; 33(7): 673–89. doi: 10.1007/s40273-014-0243-x
3. WHO. Obesity and overweight 2020. Available from: https://www.who.int/en/news-room/fact-sheets/detail/obesity-and-overweight.
4. Fortuna JL. The obesity epidemic and food addiction: clinical similarities to drug dependence. J Psychoactive Drugs 2012; 44(1): 56–63. doi: 10.1080/02791072.2012.662092
5. Sahu A. Minireview: a hypothalamic role in energy balance with special emphasis on leptin. Endocrinology 2004; 145(6): 2613–20. doi: 10.1210/en.2004-0032
6. Zheng H, Berthoud HR. Eating for pleasure or calories. Curr Opin Pharmacol 2007; 7(6): 607–12. doi: 10.1016/j.coph.2007.10.011
7. Volkow ND, O’Brien CP. Issues for DSM-V: should obesity be included as a brain disorder? Am J Psychiatry 2007; 164(5): 708–10. doi: 10.1176/ajp.2007.164.5.708
8. Alsio J, Olszewski PK, Levine AS, Schioth HB. Feed-forward mechanisms: addiction-like behavioral and molecular adaptations in overeating. Front Neuroendocrinol 2012; 33(2): 127–39. doi: 10.1016/j.yfrne.2012.01.002
9. Small DM, Jones-Gotman M, Dagher A. Feeding-induced dopamine release in dorsal striatum correlates with meal pleasantness ratings in healthy human volunteers. Neuroimage 2003; 19(4): 1709–15. doi: 10.1016/S1053-8119(03)00253-2
10. Beaulieu JM, Gainetdinov RR. The physiology, signaling, and pharmacology of dopamine receptors. Pharmacol Rev 2011; 63(1): 182–217. doi: 10.1124/pr.110.002642
11. de Melo Martimiano PH, da Silva GR, Coimbra VF, Matos RJ, de Souza BF, da Silva AA, et al. Perinatal malnutrition stimulates motivation through reward and enhances drd(1a) receptor expression in the ventral striatum of adult mice. Pharmacol Biochem Behav 2015; 134: 106–14. doi: 10.1016/j.pbb.2015.04.008
12. Volkow ND, Wang GJ, Telang F, Fowler JS, Thanos PK, Logan J, et al. Low dopamine striatal D2 receptors are associated with prefrontal metabolism in obese subjects: possible contributing factors. Neuroimage 2008; 42(4): 1537–43. doi: 10.1016/j.neuroimage.2008.06.002
13. Wang GJ, Volkow ND, Logan J, Pappas NR, Wong CT, Zhu W, et al. Brain dopamine and obesity. Lancet 2001; 357(9253): 354–7. doi: 10.1016/S0140-6736(00)03643-6
14. Vucetic Z, Totoki K, Schoch H, Whitaker KW, Hill-Smith T, Lucki I, et al. Early life protein restriction alters dopamine circuitry. Neuroscience 2010; 168(2): 359–70. doi: 10.1016/j.neuroscience.2010.04.010
15. da Silva AA, Borba TK, de Almeida Lira L, Cavalcante TC, de Freitas MF, Leandro CG, et al. Perinatal undernutrition stimulates seeking food reward. Int J Dev Neurosci 2013; 31(5): 334–41. doi: 10.1016/j.ijdevneu.2013.05.001
16. da Silva AA, Oliveira MM, Cavalcante TC, do Amaral Almeida LC, de Souza JA, da Silva MC, et al. Low protein diet during gestation and lactation increases food reward seeking but does not modify sucrose taste reactivity in adult female rats. Int J Dev Neurosci 2016; 49: 50–9. doi: 10.1016/j.ijdevneu.2016.01.004
17. Clark SM, Saules KK. Validation of the Yale Food Addiction Scale among a weight-loss surgery population. Eat Behav 2013; 14(2): 216–9. doi: 10.1016/j.eatbeh.2013.01.002
18. Rodriguez-Rivera C, Perez-Garcia C, Munoz-Rodriguez JR, Vicente-Rodriguez M, Polo F, Ford RM, et al. Proteomic identification of biomarkers associated with eating control and bariatric surgery outcomes in patients with morbid obesity. World J Surg 2019; 43(3): 744–50. doi: 10.1007/s00268-018-4851-z
19. Oberbach A, Bluher M, Wirth H, Till H, Kovacs P, Kullnick Y, et al. Combined proteomic and metabolomic profiling of serum reveals association of the complement system with obesity and identifies novel markers of body fat mass changes. J Proteome Res 2011; 10(10): 4769–88. doi: 10.1021/pr2005555
20. Gil SY, Youn BS, Byun K, Huang H, Namkoong C, Jang PG, et al. Clusterin and LRP2 are critical components of the hypothalamic feeding regulatory pathway. Nat Commun 2013; 4: 1862. doi: 10.1038/ncomms2896
21. Byun K, Gil SY, Namkoong C, Youn BS, Huang H, Shin MS, et al. Clusterin/ApoJ enhances central leptin signaling through Lrp2-mediated endocytosis. EMBO Rep 2014; 15(7): 801–8. doi: 10.15252/embr.201338317
22. Urban J, Parczyk K, Leutz A, Kayne M, Kondor-Koch C. Constitutive apical secretion of an 80-kD sulfated glycoprotein complex in the polarized epithelial Madin-Darby canine kidney cell line. J Cell Biol 1987; 105(6 Pt 1): 2735–43. doi: 10.1083/jcb.105.6.2735
23. Kapron JT, Hilliard GM, Lakins JN, Tenniswood MP, West KA, Carr SA, et al. Identification and characterization of glycosylation sites in human serum clusterin. Protein Sci 1997; 6(10): 2120–33. doi: 10.1002/pro.5560061007
24. Humphreys DT, Carver JA, Easterbrook-Smith SB, Wilson MR. Clusterin has chaperone-like activity similar to that of small heat shock proteins. J Biol Chem 1999; 274(11): 6875–81. doi: 10.1074/jbc.274.11.6875
25. Poon S, Rybchyn MS, Easterbrook-Smith SB, Carver JA, Pankhurst GJ, Wilson MR. Mildly acidic pH activates the extracellular molecular chaperone clusterin. J Biol Chem 2002; 277(42): 39532–40. doi: 10.1074/jbc.M204855200
26. Wyatt AR, Yerbury JJ, Berghofer P, Greguric I, Katsifis A, Dobson CM, et al. Clusterin facilitates in vivo clearance of extracellular misfolded proteins. Cell Mol Life Sci 2011; 68(23): 3919–31. doi: 10.1007/s00018-011-0684-8
27. Rohne P, Prochnow H, Wolf S, Renner B, Koch-Brandt C. The chaperone activity of clusterin is dependent on glycosylation and redox environment. Cell Physiol Biochem 2014; 34(5): 1626–39. doi: 10.1159/000366365
28. Carver JA, Rekas A, Thorn DC, Wilson MR. Small heat-shock proteins and clusterIn: intra- and extracellular molecular chaperones with a common mechanism of action and function? IUBMB Life 2003; 55(12): 661–8. doi: 10.1080/15216540310001640498
29. Rohne P, Prochnow H, Koch-Brandt C. The CLU-files: disentanglement of a mystery. Biomol Concepts 2016; 7(1): 1–15. doi: 10.1515/bmc-2015-0026
30. Gregory JM, Whiten DR, Brown RA, Barros TP, Kumita JR, Yerbury JJ, et al. Clusterin protects neurons against intracellular proteotoxicity. Acta Neuropathol Commun 2017; 5(1): 81. doi: 10.1186/s40478-017-0481-1
31. Nizard P, Tetley S, Le Drean Y, Watrin T, Le Goff P, Wilson MR, et al. Stress-induced retrotranslocation of clusterin/ApoJ into the cytosol. Traffic 2007; 8(5): 554–65. doi: 10.1111/j.1600-0854.2007.00549.x
32. Li N, Zoubeidi A, Beraldi E, Gleave ME. GRP78 regulates clusterin stability, retrotranslocation and mitochondrial localization under ER stress in prostate cancer. Oncogene 2013; 32(15): 1933–42. doi: 10.1038/onc.2012.212
33. Debure L, Vayssiere JL, Rincheval V, Loison F, Le Drean Y, Michel D. Intracellular clusterin causes juxtanuclear aggregate formation and mitochondrial alteration. J Cell Sci 2003; 116(Pt 15): 3109–21. doi: 10.1242/jcs.00619
34. Kim YS, Choi MY, Ryu JH, Lee DH, Jeon BT, Roh GS, et al. Clusterin interaction with Bcl-xL is associated with seizure-induced neuronal death. Epilepsy Res 2012; 99(3): 240–51. doi: 10.1016/j.eplepsyres.2011.12.002
35. Trougakos IP, Lourda M, Antonelou MH, Kletsas D, Gorgoulis VG, Papassideri IS, et al. Intracellular clusterin inhibits mitochondrial apoptosis by suppressing p53-activating stress signals and stabilizing the cytosolic Ku70-Bax protein complex. Clin Cancer Res 2009; 15(1): 48–59. doi: 10.1158/1078-0432.CCR-08-1805
36. Zhang H, Kim JK, Edwards CA, Xu Z, Taichman R, Wang CY. Clusterin inhibits apoptosis by interacting with activated Bax. Nat Cell Biol 2005; 7(9): 909–15. doi: 10.1038/ncb1291
37. Lizarraga-Mollinedo E, Alvarez C, Fernandez-Millan E, Escriva F, Gonzalez-Martin C, Salas E, et al. Undernutrition upregulates fumarate hydratase in the rat nucleus accumbens. Metab Brain Dis 2013; 28(1): 111–5. doi: 10.1007/s11011-012-9358-y
38. Moyse E, Arsenault M, Gaudreau P, Ferland G, Ramassamy C. Brain region-specific effects of long-term caloric restriction on redox balance of the aging rat. Mech Ageing Dev 2019; 179: 51–9. doi: 10.1016/j.mad.2019.01.002
39. Morgan TE, Xie Z, Goldsmith S, Yoshida T, Lanzrein AS, Stone D, et al. The mosaic of brain glial hyperactivity during normal ageing and its attenuation by food restriction. Neuroscience 1999; 89(3): 687–99. doi: 10.1016/S0306-4522(98)00334-0