Neurobiology of Aging 26 (2005) 9951000

Caloric restriction attenuates A-deposition in Alzheimer transgenic models

Nilay V. Patela,1 , Marcia N. Gordonb , Karen E. Connorb , Robert A. Goodc , Robert W. Engelmanc , Jerimiah Masonb , David G. Morganb , Todd E. Morgana,,2 , Caleb E. Fincha,2
a

Andrus Gerontology Center and Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089-0191, USA b Alzheimers Research Laboratory, Department of Pharmacology, University of South Florida, Tampa, FL 33162, USA c Alzheimers Research Laboratory, Department of Pediatrics, University of South Florida, Tampa, FL 33162, USA Received 13 May 2004; received in revised form 13 August 2004; accepted 20 September 2004

Abstract Dietary inuences on Alzheimer disease (AD) are gaining recognition. Because many aging processes are attenuated in laboratory mammals by caloric restriction (CR), we examined the effects of short-term CR in two AD-transgenic mice, APPswe/ind (J20) and APPswe + PS1M146L (APP + PS1). CR substantially decreased the accumulation of A-plaques in both lines: by 40% in APPswe/ind (CR, 6 weeks), and by 55% in APP + PS1 (CR, 14 weeks). CR also decreased astrocytic activation (GFAP immunoreactivity). These inuences of CR on AD-transgenic mice are consistent with epidemiological reports that show that high caloric diets associate with the risk of AD, and suggest that dietary interventions in adult life might slow disease progression. 2004 Elsevier Inc. All rights reserved.
Keywords: Caloric restriction; AD transgenics; Amyloid deposition; Astrocyte activation

1. Introduction Diet strongly inuences the incidence and outcome in major age-related diseases including diabetes, obesity, and vascular disease. Recent ndings extend inuences of diet to Alzheimer disease (AD) [8,13,16,18]. Obesity is also a risk factor for AD [12,26,28]. Animal models support these associations: brain amyloid is increased by cholesterol-rich diets to AD-transgenic mice [29,32], dogs [10], and rabbits [33]. An opposite paradigm is caloric restriction (CR) which reduces body fat and increases life span while slowing many aging processes [14], including normal aging changes in the brain [17,19,20,23]. CR in laboratory animals diminishes
Corresponding author. Tel.: +1 213 740 4083; fax: +1 213 740 0853. E-mail address: temorgan@usc.edu (T.E. Morgan). 1 Present address: Department of Gene Expression and Drug Discovery, City of Hope National Cancer Center, Duarte, CA 91010, USA. 2 T.E.M. and C.E.F. contributed equally to this work. 0197-4580/$ see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.neurobiolaging.2004.09.014

the apparent escalation of neuroinammation and oxidative stress during aging by decreasing both glial activation and the production of reactive oxygen species [17,20,24]. Moreover, CR shows remarkable neuroprotective effects in young rodent models of neurodegenerative disease [4,19]. For example, as little as 2 months of CR is neuroprotective for excitotoxic and metabolic insults [4]. Since pathogenesis of AD involves activated glia, increased oxidative damage and reduced neuroprotection, we tested whether CR of AD-transgenic mice can attenuate AD-like pathology, with a focus on A deposits and glial activation. In AD, most A-plaques are surrounded by activated astrocytes and microglia, which are thought to secrete numerous cytokines, complement factors and other inammation-related proteins and which may contribute to neuroinammatory and oxidative processes in AD pathology [1]. However, glial activation also occurs during normal aging in humans [21,23,31]. Glial brillary acidic protein (GFAP), a marker of astrocytic activation, increases relatively early in adult life with progressive increases during aging [20,23].

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To avoid confounds of normal aging processes with ADprocesses, we chose two transgenic models with early onset amyloid by age of 6 months: APPswe/ind (J20 line) mice with the double Swedish and Indiana APP mutations [22] and the doubly transgenic APP + PS1 mice [15].

2. Methods 2.1. APPswe/ind and caloric restriction The APPswe/ind (J20 line) transgenic mice express human APP with the K670N/M671L Swedish and the V717F Indiana mutations under the PDGF promoter[22]. Male virgin mice carrying the transgene were separated into two groupsad libitum (AL) and calorie-restricted (CR). Starting at 1415 weeks of age, the CR group was gradually ramped down over a 4-week period from 5 to 3 g/day (60% of AL consumption) without micronutrient deciency (NIA31 fortied diet). The mice were maintained in a specic pathogen-free environment (NIH Guidelines for the care and use of laboratory animals). After 2 weeks of 40% CR, mice were anesthetized with isouorane and perfused transcardially with saline. Mice weighed 25.7 2.2 (AL) and 24.1 2.5 (CR) at sacrice. Brain regions were dissected from one hemisphere and frozen on dry ice. The other hemisphere was immersion-xed in 4% fresh-buffered paraformaldehyde (PFA; pH 7.4) for 24 h, cryoprotected in a sucrose series and saggitally sectioned (10 m) on a cryostat. Animal housing and all procedures were performed at USC. 2.2. APP + PS1 and caloric restriction The doubly transgenic APP + PS1 mice were generated by breeding carriers of the M146L mutation of PS1 (driven by the PDGF promoter, line5.1) with those carrying the Swedish mutation in APP (driven by the prion protein promoter) (Tg2576) [15]. Male virgin mice carrying both transgenes and their non-transgenic littermates were randomly assigned to receive semi-puried diets [5] either AL or 60% of AL (CR) starting at 9 weeks of age. At 24 weeks of age (15 weeks on diet), mice were anesthetized with pentobarbital (50 mg/kg) and perfused with saline. Mice weighed 41.4 3.2 g (AL) and 27.4 3.0 g (CR) at sacrice (P < 0.01). Horizontal sections (25 m) were cut on a sliding microtome. Animal housing and all procedures were performed at USF. 2.3. APP RNA quantication Quantitative real-time PCR as described previously[7]. 2.4. Immuno- and histochemistry Brain sections were analyzed for immunohistochemistry [20], with the exception of pretreating the sections processed

for A immunostaining with 88% formic acid for 10 min. The APPswe/ind brain sections were processed for A deposits (1:500; biotinylated anti-4G8, Signet, Dedham, MA), astrocytic GFAP (1:1000 DAKO, Carpentaria, CA), and microglial CD68 (1:250, Serotec, Oxford, UK). Some sections were also analyzed for double-labeling for A deposits, and GFAP or CD68. Single- and double-chromagenic reactions were visualized with DAB and Vector Blue (Vector Laboratories). The APP + PS1 brain sections were processed for A deposits as described [11]. To detect brillar plaques, sections were stained with Congo Red or Thioavin S [11]. 2.5. Image analysis Light microscope images were acquired using a CCD camera at 200 or 400 magnication. For plaque size determination, individual plaques were selected as region of interest and the number of pixels (converted to micrometer) exceeding the predetermined gray-scale value was quantied (IPlab, Scantronics Inc.). GFAP and CD68 immunoreactivity around the plaque was assessed in concentric rings drawn by IPlab. The %A immunoreactive area and Congo Red area was determined as described [11]. 2.6. Statistical analysis Statistical analysis (Statview 5.0; SAS, Cary, NC) was used to compare CR and AL, by one-way ANOVA and Scheffes test for post hoc analysis. Scholl analysis (GFAP, distance from the plaque) used Prism software (GraphPad, San Diego, CA).

3. Results 3.1. CR reduces amyloid plaque number and size We tested whether CR would attenuate AD-like amyloid accumulation in two established AD-transgenic mouse models: APPswe/ind [22] and APPswe + PS1M146L (APP + PS1)[15]. Both lines display an early onset of plaque deposition, with all the mice showing at least some Adeposition by the young age of 21 and 25 weeks, respectively (Fig. 1(a) and (b)). The A deposits of this sample were consistent with previous reports for these transgenic mice of these ages fed AL [11,22]. In the APP + PS1 mice, the A load (area of A-immunoreactivity) in anterior cortex was 55% less (P < 0.005) in CR than in AL (Fig. 1(c)). A similar trend for less A load was observed in the CA1 region of hippocampus (not signicant). However, the A load in the dentate gyrus, which showed the lowest levels of the regions measured, was not altered by diet. In the APPswe/ind mice, the total number of A-plaques in cortex and hippocampus was reduced 40% by CR (Fig. 1(d), P < 0.05). The plaque size was also reduced by CR: unlike the AL mice, CR mice did not have any large plaques > 45 m2

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Fig. 1. Caloric restriction (CR) reduces A load relative to ad libitum (AL) feeding; mean S.E.M. APP + PS1 mice (N = 45): (a) A immunostaining in the cortex showing fewer plaques in CR vs. AL, size bar = 250 m. (c) A immunoreactive area was lower in cortex (CX) (*** P < 0.005) with trends in dentate gyrus (DG) and hippocampal CA1. (e) Fibrillar amyloid (Congo Red staining) was reduced in CX and CA1 (** P < 0.01). APPswe/ind (N = 78): (b) A immunostaining in the hippocampus showing fewer plaques in CR vs. AL, size bar = 100 m. (d) Plaque size and total A-plaques were reduced by CR (* P < 0.05). (f) APP mRNA was not altered by CR.

(Fig. 1(d)). The number of smaller plaques (1530 m2 ) was reduced by CR (Fig. 1(d), P < 0.05). Immunostaining for A with 4G8 was selective for the transgene and was not detected in the non-transgenic controls (not shown). Fibrillar amyloid deposits in the APP + PS1 mice (Congo Red staining) were reduced by CR in both anterior cortex and hippocampus (Fig. 1(e), P < 0.01). The amyloid deposits in the APPswe/ind mice stained too weakly with Congo Red or Thioavin S for measurement (not shown). By quantitative real-time PCR, APP mRNA did not differ between the two groups.

3.2. CR reduces A-plaque induced astrocyte activation Immunostaining for GFAP showed a major increase in the APP + PS1 transgenic mice relative to the non-transgenic controls (ANOVA; P < 0.001). CR reduced this GFAP immunoreactivity in this line, however, it was not significantly different from AL (Fig. 2(a)). Similarly, GFAPimmunostained astrocytes were associated with plaques in the APPswe/ind mice. The levels of GFAP associated with plaques were lower than those observed for the APP + PS1 mice and did not differ between the two diet groups at the

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Fig. 2. Caloric restriction (CR) reduces A-associated astrocyte activation. (a) APP + PS1: GFAP immunostaining is increased in transgenics relative to wild-type (P < 0.001). (b) APPswe/ind : Sholl analysis of concentric rings around A-plaques (inset) showed reduced GFAP immunoreactivity nearest to plaques in CR vs. AL (* P < 0.05).

tissue level. GFAP was quantied in concentric rings around the A-immunostained plaques (Sholl analysis) (Fig. 2(b), inset). Astrocytic activation within the 20 m ring outside of the plaque boundary was lower in the CR APPswe/ind mice relative to AL (P < 0.05). GFAP immunoreactivity progressively decreased as a function of distance from the plaque, with the signal intensity returning to baseline by 60 m (Fig. 2(b)). 3.3. A-plaques and microglial markers Activated microglia were also associated with Aimmunostained plaques in the APPswe/ind mice, but stained less intensely than astrocytes. We were unable to quantify A-plaque associated CD11b- and F4/80- immunopositive microglia because these antibodies were not effective on formic-acid xed sections. Double-labeling with CD68 (macrosialin), another marker for activated microglia/macrophages, and 4G8 showed activated microglia in association with A-plaques. However, CD68 immunostaining was not reduced by CR, although CR plaques were significantly smaller in these double-labeled sections (not shown).

4. Discussion Short-term CR in early adulthood attenuated A-plaque deposits in two transgenic mouse models of AD with early on-

set amyloid deposits. We detected A-plaques in all the transgenic mice by age of 2125 weeks, as expected [15,22], and observed lower frequency in all CR groups. In the APPswe/ind mice, CR for only 6 weeks decreased the number of plaques and plaque-size by 40%; in the APP + PS1, 15 weeks of CR attenuated the A load by 55%. Since CR reduces plaques in these two different familial AD-transgenic lines, we suggest that CR acts on a fundamental pathway required for A-plaque genesis. The efcacy of CR does not appear to depend on the ratio of A42 :A40 in the deposits, which differs between the strains (A42 :A40 , APP + PS1, 0.36 [15] APPswe/ind line, 0.12 [22]). Glial activation was selectively modied by CR. GFAP is well known as a rapidly induced astrocytic activation marker that remains elevated during chronic neurodegeneration and inammation. GFAP was attenuated by CR around the plaque. On one hand, the attenuation of GFAP by CR in these AD transgenics is consistent with effects of CR in attenuating age-related, GFAP increases which regularly occur in the absence of amyloid deposits in aging non-transgenic rodents [20,23]. This implied reduction in astrocytic activation may have inuenced plaque genesis by attenuating expression of apoE, apoJ, or s100 [20,21], which have major roles in AD pathogenesis. For example, in PDAPP transgenics, A-deposition was increased by the absence of both genes [6]. The age-related increase in apoE and apoJ expression, like GFAP, is attenuated by CR [20]. We are considering the hypothesis that attenuation of astrocytic activation in the CR transgenic mice near the sites of plaque genesis attenuated A-deposition by reducing the expression of apoE or apoJ. Both apoE and apoJ were detected in A-plaques in these APPswe/ind mice (data not shown). On the other hand, WyssCoray et al. [36], showed that astrocytes can phagocytose levels A. Thus, GFAP levels may not be a useful marker for phagocytic activities of astrocytes. CD68 (macrosialin) immunostaining was examined because of its occurrence in microglia near senile plaques of AD brains in the Golgi organellar system [3] which may mediate A phagocytosis [30]. CD68 was also associated with A-plaques in APPswe/ind (present data) and APP23 transgenic mice [3]. However, we did not detect an effect of CR on the plaque-associated CD68. Other markers of microglial activation are needed to dene the effects of CR on microglia in the CR transgenic mice. Several mechanisms may be at work in CR. At the systemic level, CR lowers blood insulin (50%), glucose (15%), and cholesterol (15%), but increases corticosteroids (75%) [25]. CR also attenuates the usual post-pubertal increase of body fat. Recent data suggests that blood insulin, which is transported to the brain, inuences A clearance from the brain. Insulin and A are substrates for IDE (insulin-degrading enzyme, or insulysin), which cleaves insulin preferentially. Gene deletion of IDE increases brain A [9]. Moreover, transient hyperinsulinemia from insulin infusion rapidly increases A in the cerebrospinal uid [35]. Thus, the 50% decrease of blood insulin in CR may enhance

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clearance of brain A by reducing brain insulin as a competing substrate. Other mechanisms could include the lower cholesterol [19,27] and higher glucocorticoids [34] in CR mice, each of which can independently inuence A levels in the CNS. The present studies were not designed to obtain 24 h proles of plasma glucose, insulin or glucocorticoids, which are needed to assess CR effects in a new genotype. Overall, these ndings are consistent with diabetes and obesity as risk factors in AD. How might these ndings apply to humans? In rodents, CR of 1040% proportionally restricts weight gain and extends lifespan. These benecial effects of CR on life span are also observed in rodent models of several diseases and mice carrying various human disease genes. In rhesus monkeys, CR improves insulin regulation and reduces cardiovascular disease in association with maintenance of post-pubertal body weight [2]. Our current ndings broadly imply that CR reduces A-plaque-deposition by attenuating the progression of disease pathology. If so, maintaining a healthy bodyweight may decrease the risk of AD, even in subjects with genetic predisposition. However, the ideal lifelong body mass index to optimize both cognition and longevity remains to be dened.

Acknowledgment Supported by grants to C.E.F and T.E.M: AG13499 and Alzheimer Association Temple Award; D.M. and M.G.: AG 15490 and AG 18478; N.V.P: AG05901.

References
[1] Akiyama H, Barger S, Barnum S, Bradt B, Bauer J, Cole GM, et al. Inammation and Alzheimers disease. Neurobiol Aging 2000;21:383421. [2] Bodkin NL, Alexander TM, Ortmeyer HK, Johnson E, Hansen BC. Mortality and morbidity in laboratory-maintained rhesus monkeys and effects of long-term dietary restriction. J Gerontol A Biol Sci Med Sci 2003;58:2129. [3] Bornemann KD, Wiederhold KH, Pauli C, Ermini F, Stalder M, Schnell L, et al. Abeta-induced inammatory processes in microglia cells of APP23 transgenic mice. Am J Pathol 2001;158:6373. [4] Bruce-Keller AJ, Umberger G, McFall R, Mattson MP. Food restriction reduces brain damage and improves behavioral outcome following excitotoxic and metabolic insults. Ann Neurol 1999;45:815. [5] Cuenca AG, Cress WD, Good RA, Marikar Y, Engelman RW. Calorie restriction inuences cell cycle protein expression and DNA synthesis during liver regeneration. Exp Biol Med (Maywood) 2001;226:10617. [6] DeMattos RB, Cirrito JR, Parsadanian M, May PC, ODell MA, Taylor JW, et al. ApoE and clusterin cooperatively suppress Abeta levels and deposition. Evidence that apoE regulates extracellular abeta metabolism in vivo. Neuron 2004;41:193202. [7] Dickey CA, Loring JF, Montgomery J, Gordon MN, Eastman PS, Morgan D. Selectively reduced expression of synaptic plasticityrelated genes in amyloid precursor protein + presenilin-1 transgenic mice. J Neurosci 2003;23:521926.

[8] Engelhart MJ, Geerlings MI, Ruitenberg A, Van Swieten JC, Hofman A, Witteman JC, et al. Diet and risk of dementia: does fat matter? The Rotterdam Study. Neurology 2002;59:191521. [9] Farris W, Mansourian S, Chang Y, Lindsley L, Eckman EA, Frosch MP, et al. Insulin-degrading enzyme regulates the levels of insulin, amyloid beta-protein, and the beta-amyloid precursor protein intracellular domain in vivo. Proc Natl Acad Sci USA 2003;100:41627, Epub 2003, March 12. [10] Friedland RP, Shi J, Smith MA, Perry G, Sparks DL. Cholesterol feeding enhances cerebral amyloid beta pathology in beagles. Neurobiol Aging 2004;25:224. [11] Gordon MN, Holcomb LA, Jantzen PT, DiCarlo G, Wilcock D, Boyett KW, et al. Time course of the development of Alzheimer-like pathology in the doubly transgenic PS1 + APP mouse. Exp Neurol 2002;173:18395. [12] Grant WB. Obesity and Alzheimer disease: roles of diet and genetics. Arch Int Med 2004;164:10910, author reply 110. [13] Gustafson D, Rothenberg E, Blennow K, Steen B, Skoog I. An 18year follow-up of overweight and risk of Alzheimer disease. Arch Intern Med 2003;163:15248. [14] Heilbronn LK, Ravussin E. Calorie restriction and aging: review of the literature and implications for studies in humans. Am J Clin Nutr 2003;78:3619. [15] Holcomb L, Gordon MN, McGowan E, Yu X, Benkovic S, Jantzen P, et al. Accelerated Alzheimer-type phenotype in transgenic mice carrying both mutant amyloid precursor protein and presenilin 1 transgenes. Nat Med 1998;4:97100. [16] Laitinen MH, Helkala E-L, Uusitalo U, Nissinen A, Tuomilehto J, Soininen H, et al. Midlife dietary fats and the risk of late-life dementia: a population-based study. Neurobiol Aging 2004;25:399. [17] Lee CK, Weindruch R, Prolla TA. Gene-expression prole of the ageing brain in mice. Nat Genet 2000;25:2947. [18] Luchsinger JA, Tang MX, Shea S, Mayeux R. Caloric intake and the risk of Alzheimer disease. Arch Neurol 2002;59:125863. [19] Mattson MP. Gene-diet interactions in brain aging and neurodegenerative disorders. Ann Intern Med 2003;139:4414. [20] 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;89:68799. [21] Mrak RE, Grifnbc WS. The role of activated astrocytes and of the neurotrophic cytokine S100B in the pathogenesis of Alzheimers disease. Neurobiol Aging 2001;22:91522. [22] Mucke L, Masliah E, Yu GQ, Mallory M, Rockenstein EM, Tatsuno G, et al. High-level neuronal expression of abeta 142 in wild-type human amyloid protein precursor transgenic mice: synaptotoxicity without plaque formation. J Neurosci 2000;20:40508. [23] Nichols NR, Day JR, Laping NJ, Johnson SA, Finch CE. GFAP mRNA increases with age in rat and human brain. Neurobiol Aging 1993;14:4219. [24] Olgun A, Akman S, Serdar MA, Kutluay T. Oxidative phosphorylation enzyme complexes in caloric restriction. Exp Gerontol 2002;37:63945. [25] Patel NV, Finch CE. The glucocorticoid paradox of caloric restriction in slowing brain aging. Neurobiol Aging 2002;23:70717. [26] Petot GJ, Fritsch T, Debanne SM, Traore F, Friedland RP. Obesity during the mid-adult years is a rick factor for AD independent of apoE4 genotype. Neurobiol Aging 2004;25:305. [27] Puglielli L, Tanzi RE, Kovacs DM. Alzheimers disease: the cholesterol connection. Nat Neurosci 2003;6:34551. [28] Razay G, Vreugdenhil A. AD, abdominal obesity and overweight. Neurobiol Aging 2004;25:393. [29] Refolo LM, Malester B, LaFrancois J, Bryant-Thomas T, Wang R, Tint GS, et al. Hypercholesterolemia accelerates the Alzheimers amyloid pathology in a transgenic mouse model. Neurobiol Dis 2000;7:32131.

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N.V. Patel et al. / Neurobiology of Aging 26 (2005) 9951000 cular inammation. Preliminary observations. Ann N Y Acad Sci 2000;903:33544. [34] Tokuda T, Oide T, Tamaoka A, Ishii K, Matsuno S, Ikeda S. Prednisolone (3060 mg/day) for diseases other than AD decreases amyloid beta-peptides in CSF. Neurology 2002;58:1415 8. [35] Watson GS, Peskind ER, Asthana S, Purganan K, Wait C, Chapman D, et al. Insulin increases CSF Abeta42 levels in normal older adults. Neurology 2003;60:1899903. [36] Wyss-Coray T, Loike JD, Brionne TC, Lu E, Anankov R, Yan F, et al. Adult mouse astrocytes degrade amyloid-beta in vitro and in situ. Nat Med 2003;9:4537.

[30] Rogers J, Strohmeyer R, Kovelowski CJ, Li R. Microglia and inammatory mechanisms in the clearance of amyloid beta peptide. Glia 2002;40:2609. [31] Sheng JG, Mrak RE, Grifn WS. Enlarged and phagocytic, but not primed, interleukin-1 alpha-immunoreactive microglia increase with age in normal human brain. Acta Neuropathol (Berl) 1998;95:22934. [32] Shie FS, Jin LW, Cook DG, Leverenz JB, LeBoeuf RC. Diet-induced hypercholesterolemia enhances brain A beta accumulation in transgenic mice. NeuroReport 2002;13:4559. [33] Sparks DL, Kuo YM, Roher A, Martin T, Lukas RJ. Alterations of Alzheimers disease in the cholesterol-fed rabbit, including vas-