Summary. Oxidative stress, inflammation and theaggregation of oxidized, misfolded or aberrant proteinsin the brain induces deregulations in programmed celldeath: apoptosis and autophagy. Apoptosis is one ofprocesses implicated in aging and neurodegenerativepathologies, and for the last decade, has been one of themost studied processes due to its essential role, not onlyin aging, but also in many neurodegenerative diseases,including Parkinson’s, Alzheimer’s and Huntington’s.However, autophagy being the major intracellularpathway for the degradation and recycling of long-liveproteins and organelles is widely involved in thepathogenesis or prevention of many age-related diseases,including neurodegenerative conditions. Recently,autophagy activation has been considered as part of thecellular responses to elevated oxidative stress,eliminating unwanted, damaged and oxidative structures;thus favouring, in this way, the key anti-agingmechanism associated with the caloric restriction.Longevity factors, such as sirtuins, and redox-sensitivetranscriptional factors, such as NF-κB and p53, can alsoregulate basal autophagy in cells, with a direct impact onlongevity and the development of inflammation andneurodegeneration. Here, we reviewed the criticalchanges of autophagy in the aging and neuro-degenerative brain and the role of key regulators ofautophagy, which are directly related to oxidative stress,inflammation and longevity pathways.

Key-words: Neurodegeneration, Autophagy, Sirtuins,NF-κB, p53

Introduction to oxidative stress and inflammation inthe aging brain

Aging is defined as a complex, irreversible andmultifactorial process that leads to changes over time,affecting multiple biological functions, with a gradualdeterioration in the adaptability of the organisms toenvironmental changes and stressful conditions. Thesechanges are detected at all levels, molecular, cellular,tissular levels and organismal (Yu and Chung, 2006),leading to functional systemic disorders related to theaging process and a higher risk of succumbing to age-related pathologies, such as neurodegenerative disease,diabetes, autoimmune and inflammatory diseases andcancer. Initially, aging was proposed as the major riskfactor in most neurodegenerative disorders (Floyd andHensley, 2002). The incidence of neurodegenerativediseases, such as Alzheimer’s disease (AD) andParkinson’s disease (PD) increases significantly with age(Wilson et al., 2007). Given that the ratio of elderlypeople is increasing, it is crucial that research uncoversthe mechanisms associated with senescence andimplicated in the transition from benign aging todegenerative disease to prevent the development of theage-related pathologies and in particular, the cognitivedecline associated with aging. In the central nervoussystem, the neuroendocrine changes observed duringaging appear to be more related to disorders of therelationship between neural and hormonal signals, ratherthan alterations of specific structures (Ferrari and Magri,2008; Ferrari et al., 2008).

However, in a review of aging, it is essential discussthe previously well-known processes underlying theaging phenomenon, such as oxidative stress and itssubsequent inflammation. Oxidative stress is the maincausal factor of aging and the development of variousdiseases, including age-related sporadic degenerative

Review

An insight into the role of autophagy in cell responses in the aging and neurodegenerative brainB. Caballero1 and A. Coto-Montes21Department of Molecular Pharmacology, The Bruce Rappaport Faculty of Medicine, Israel Institute of Technology-Technion, Haifa,Israel and 2Department of Morphology and Cell Biology, Faculty of Medicine, University of Oviedo, Oviedo, Spain

Histol Histopathol (2012) 27: 263-275

Offprint requests to: Dr. Ana Coto-Montes, Departamento de Morfologíay Biología Celular , Facultad de Medicina, C/ Julián Clavería s/n, 33006,Oviedo, Spain. e-mail: acoto@uniovi.es

http://www.hh.um.esHistology andHistopathology

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diseases (e.g., AD, atherosclerosis and diabetes)(Dubinina and Pustygina, 2007). In fact, theenhancement of oxidative stress resistance is consideredto be the mechanism underlying the extended longevityof genetic variants of non-mammalian and mammalianorganisms (Agarwal and Sohal, 1996; Holzenberger etal., 2003; Madsen et al., 2004; Ayyadevara et al., 2005,2008). The free radical theory of aging proposes a slowand progressive generation of reactive oxygen species(ROS), an unavoidable consequence of life in an aerobicenvironment, resulting in the accumulation of defectivecellular components (Harman, 1956, 1992). Oxidativestress can be defined within the context of a subtlechanged redox status (Yoon et al., 2002). In this regard,the age-related oxidative stress in mammalian cells isgenerated by a redox deregulation (Humphries et al.,2006) as a consequence of prominent enzymatic defects,leading to an increased production of free radicals,including ROS, reactive nitrogen species (RNS) andother oxidant agents, together with an importantdecrease in antioxidant levels and impairment of therepair of oxidative damages. Hence, aging occurs as theresult of accumulative and unrepaired damage in thecellular constituents, best exemplified by products oflipid peroxidation, protein oxidation and toxicaggregates, such as lipofuscin, toxic proteins (α-synuclein, phosphorylated Tau), the glycoxidation ofmacromolecules and oxidative modifications in nuclearand mitochondrial DNA (Stadtman, 1992; Beckman andAmes, 1998; Sohal, 2002) (Fig. 1). Oxidatively alteredstructures and functions are detected and accumulated at

all levels along the aging phenomenon (Yu and Chung,2006). An increase in protein carbonyl levels has beendemonstrated for various brain regions including thehippocampus (Siqueira et al., 2005), a key area of brainimplicated in learning and memory functions. Theeffects of aging on energy production or changes in ROSproduction could be particularly detrimental in non-proliferating neuronal tissues. In fact, damage by ROS ismore exacerbated in the brain, because it is highlyvulnerable to free radical damage due to its higheroxygen utilization, high concentrations ofpolyunsaturated fatty acids and transition metals, such asiron, and low concentration of cytosolic antioxidants(Reiter, 1995).

Inflammation is another important factor that affectsthe normal brain and, in particular, the aging of thebrain. The age-related chronic inflammatory state createsan activated immune response that includes the acutephase protein response, cytokines (interferon andinterleukins), macrophages, lymphocytes, and otherimmune system cells. Nitric oxide (NO) and nitritelevels (NO•) accurately reflect the nitrosative stressstatus that is caused by inflammation. The increase ofnitrite levels is particularly relevant because it is welldocumented that NO• and its toxic metabolite,peroxynitrite, can inhibit components of themitochondrial respiratory chain, leading to a cellularenergy deficiency and, eventually, to cell death (Cassinaet al., 2000; Brown, 2001). Within the brain, neurons, incontrast to astrocytes, appear particularly vulnerable tothe effects of nitrosative stress. Mammalian

264Autopaghy in the aging brain

Fig 1. Oxidative damages associated withaging and neurodegeneration. The increase ofreactive oxygen species (ROS) during aging inthe brain leads to signif icant damage tomolecules that are key for cell survival. Theoxidative modifications of l ipids (l ipidperoxidation) from the cellular membrane leadto changes in cell membrane fluidity and canalso favor oxidative modifications of proteins,leading to the formation of toxic proteinaggregates inside cells and the alteration ofseveral enzymatic activities. The oxidativemodifications of nuclear DNA can favormutations. Other important oxidative alterationsthat can compromise cell survival includealterations of the cytoskeleton, by the oxidationof structural and microtubule-associatedproteins that stabil ize the cytoskeleton.Transcription factors can be activated underoxidative stress, such as p53 and NF-κB, thelatter promotes the expression of several pro-inflammatory genes. In addition, the alterationof several organelles, such as mitochondria,results in the generation of less cellular ATPand the production of even more ROS, furthercontributing to oxidative damage. Thedysfunction of the endoplasmic reticulum (ER)

alters calcium homeostasis and multiple calcium-dependent signaling pathways. The oxidative modifications of lysosomal membranes releaseproteolytic enzymes (such as cathepsins) to the cytosol, which can be pro-apoptotic signals.

inflammation in the aging brain is also associated withthe activation of the NF-κB transcription factor system(Caballero et al., 2008) and the chronic activation of NF-κB signaling has the capacity to induce the senescentphenotype associated with aging (Salminen andKaarniranta, 2009a). The NF-κB system is an ancienthost defense system concerned with immune responsesand different external and internal dangers, such asoxidative and genotoxic stress. In addition to being themaster regulator of inflammatory responses, NF-κBsignaling can also regulate several homeostaticresponses through its anti-apoptotic effects (Michiels etal., 2002) and antioxidant functions; NF-κB can increasethe expression of antioxidant enzymes in responses toelevated oxidative stress (Tomas-Zapico and Coto-Montes, 2005). Therefore, an increased rate of freeradical generation, inflammation and the inefficiency ofthe reparative/recycling mechanisms are factors thatprimarily contribute to the age-related deteriorationduring the aging brain, what implies that an antioxidanttreatment can be highly beneficial against theseprocesses (Gutierrez-Cuesta et al., 2007; Caballero et al.,2008, 2009; Garcia et al., 2010).Autophagy, oxidative stress and neurodegeneration

In postmitotic cells, such as neurons, which cannotbecome senescent because they are already terminallydifferentiated, autophagy (self-eating) is a majorhomeostatic mechanism to cope with stress. Thedamaged organelles, long-lived or aberrant proteins andsuperfluous or aged portions of the cytoplasm areeliminated by the autophagy-lysosomal system that doesnot compromise cellular functions and tissuehomeostasis. The best characterized form of autophagy,macroautophagy (mainly referenced in this review),involves the rearrangement of sub-cellular membranes tosequester parts of cytoplasm and organelles in double-membrane vesicles, called autophagosomes, for deliveryto lysosomes where the sequestered cargo is degradedand recycled within autophagolysosomes (Cuervo et al.,2005; Klionsky, 2005; Cuervo, 2008). This sequestrationprocess is controlled by the mammalian target ofrapamycin (mTOR) kinase pathway, the major negativeregulator of macroautophagy, which is regulated byinsulin via the phosphoinositol 3 kinase/serine-threonineprotein kinase (PI3K/AKT) pathway and by specificamino acids via AMP kinases (Petiot et al., 2000).Upstream of mTOR, macroautophagy can be inhibitedby the insulin/IGF-1 (insulin-like growth factor-1)receptor pathway (Levine and Kroemer, 2008). Incontrast, microautophagy sequesters the cytosolicmaterials through direct invagination of the lysosomemembrane in a constitutive mechanism. Lastly,chaperone-mediated autophagy (CMA) is responsible forthe selective lysosomal degradation of cytosolic proteinswith a particular pentapeptide motif (KERFQ), aftertargeting them with a cytosolic chaperone complex andby their selective translocation after binding to the

lysosome-membrane associated protein type 2a(Lamp2a) (Cuervo et al., 2005).

The primary roles of autophagy are the baselineturnover of intracellular proteins and organelles, theproduction of amino acids in nutrient emergency, and theregression of retired tissues (Bergamini et al., 2007).But, in recent works, autophagy has started to beconsidered as a cytoprotective response during stressconditions (Cuervo, 2004; Moore, 2008) to remove toxicor altered components and unwanted or unnecessaryorganelles (mitochondrias, peroxisomes, etc...),recycling the components for reuse (Kim and Klionsky,2000). These actions are a quality control mechanism fororganelles, particularly important for neuron survival,since these might, otherwise, can lead to cell death byapoptosis (Erlich et al., 2006; Li et al., 2006; Cao andKlionsky, 2007). In this way, ROS induce cytoprotectionsince they are essentials to stimulate autophagy byboosting the activity of autophagic protein 4 (ATG4)(Scherz-Shouval et al., 2007). During intracellular stress,including the aggregation of misfolded proteins (Qin etal., 2003), the accumulation of altered organelles(Klionsky and Ohsumi, 1999; Klionsky and Emr, 2000)and during starvation and hypoxia conditions (Yen andKlionsky, 2008), the degradation by basal autophagy isincreased to allow cell survival. Recently, the differenttypes of macroautophagy have been characterized by thestimuli that mediate their activation or by the molecularmechanisms involved in the activation and execution ofautophagy; basal in-bulk macroautophagy andstarvation-induced autophagy are at the extremes of thisscale, whereas quality-control autophagy and autophagyinduced by protein aggregates, organelle stress orpatho¬gen invasion are located in the middle levels ofthis classification (Wong and Cuervo, 2010) along withthe essential properties of the cellular stress responses.Moreover, in non-physiological situations, autophagiccell death, known as type II non-apoptotic programmedcell death, which has been also reported in neurons(Larsen and Sulzer, 2002), shows a negative feedback onapoptosis; autophagy can lead to cell death whenapoptosis is inhibited (Shimizu et al., 2004) andconsequently, if autophagy is inhibited under nutrientstarvation conditions, cell death by apoptosis isaccelerated (Maiuri et al., 2007).

It is well-known that a decline of autophagicdegradation in older tissues (Cuervo and Dice, 1998,2000) impairs the cellular housekeeping process ofaberrant and dysfunctional molecules, organelles andprotein aggregates. Defective autophagy has beenextensively linked to aging and the development of age-related neurodegeneration (review in Wong and Cuervo,2010). At first sight, macroautophagy is altered duringaging as a consequence of impaired autophagosomesformation or maduration to autophagolysosomes(Terman, 1995). In the brain of mice prone to acceleratedsenescence, control quality by autophagy is severelyaltered contributing to the accumulation of toxic proteinaggregates that are already observed at early ages

265Autopaghy in the aging brain

(Caballero et al., 2008, 2009). In addition, it is well-known that defects in autophagic activity and the loss ofthe basal autophagy level causes neurodegeneration(Hara et al., 2006; Komatsu et al., 2006) such as occursin AD and Huntington’s diseases (HD) (Levine andKroemer, 2008). Reduced autophagy induction,enhanced in the repression of autophagy, altered cargorecognition, inefficient autophagosome/lysosome fusionor inefficient degradation of the autophagic cargo inlysosomes are all potential defects that could influencethe malfunctioning of macroautophagy in differentneurodegenerative disorders (Wong and Cuervo, 2010).Indeed, Pickford et al. (2008) reported that cellularlevels of autophagy-related protein beclin1 were oftencorrelated with autophagic activity, and that theheterozygous deletion of beclin-1 leads toneurodegeneration (Pickford et al., 2008). Therefore, theelimination of basal neuronal autophagy is sufficient tocause neurodegeneration in the absence of other insults(Hara et al., 2006; Komatsu et al., 2006). Thus, it seemsthat increased autophagic activity might help to clearaggregates of toxic proteins (such as mutant α-synucleinand Huntingtin), which are associated with pathologiessuch as Parkinson’s and Huntington’s disease (Lee andGao, 2008). However, autophagy functions may notalways be beneficial (Hashimoto et al., 2009). Forinstance, Yu et al. (2005) demonstrated that inducedneuronal macroautophagy in the presenilin (PS)/Aßprecursor protein (APP)-mouse model of ß-amyloidosiswas impaired causing the profuse accumulation ofautophagic vacuoles (AVs) in dystrophic dendrites due toan impaired maturation of AVs to lysosomes (Yu et al.,2005). An extensive basal activation of autophagy, ratherthan the characteristic decline occurred during normalaging, could contribute to the systemic degeneration andpremature aging observed in progeroid mouse models(Mariño et al., 2008). Therefore, the dual role ofautophagy, in cytoprotection and cell death as well as itsimpact on longevity is one of the most fascinatingfeatures of this process, which clearly has a direct impacton age-related development of neurodegeneration (Table1). Autophagy and longevity: role of key regulators ofcellular stress responses

The accumulation of cellular damage is the majorhallmark of the aged cell; oxidized, misfolded, cross-linked or aggregated macromolecules and damagedorganelles cannot function properly and can activelycompromise cellular functions. However, the overall rateat which damage is accumulated is influenced byconserved longevity pathways and redox-sensitivetranscriptional factors, which have key roles in cellresponses to stress conditions. Autophagy activity isessential for life-span extension and cytoprotectiveresponses during cellular stress because it can eliminateunwanted or damaged intracellular materials; thisactivity is, therefore, regulated by longevity proteins and

redox-sensitive factors, thus making autophagy a centralregulatory mechanism for aging (Salminen andKaarniranta, 2009b). Thereby, we review the regulatoryfeatures of sirtuins, p53 proteins and the NF-κB systemin autophagy activity with special emphasis on theirimpact in the aging brain or under neurodegeneration.Autophagy and NF-κκB activation

The NF-κB/Rel DNA-binding complexes containthe Rel family components, RelA/p65, c-Rel, and RelB,as well as the NF-κB components p50 (p105) and p52(p100). The inhibitory IκB components α, ß, γ, ε andBcl-3, the IKK kinases complex proteins IKKα andIKKß, and the regulatory NEMO protein can trigger NF-κB activation. The NF-κB complexes are normallylocated in the cytoplasm because they are bound to theinhibitory IκB proteins. Multiple stressors, such asoxidative stress, DNA damage and death receptoractivation, induces the phosphorylation of the IκBproteins that are subsequently ubiquitinated anddegraded via the proteasome. After their release from theIκB proteins, NF-κB complexes can translocate to nucleiand activate the transcription of a number of specifictarget genes, especially those of inflammatory genes thatare up-regulated during aging (Haddad, 2002; Salminenand Kaarniranta, 2009a). NF-κB signaling is the masterregulator of inflammatory and immune responses (Qinget al., 2006) and plays a key role in the cellularresponses to oxidative stress (Michiels et al., 2002) byits antioxidant and anti-apoptotic functions (Tomas-Zapico and Coto-Montes, 2005). Remarkably, the NF-κB system is key in aging regulation since a reducedlongevity could be due to the constitutive activation ofNF-κB factor by ROS, which can lead to cancer,inflammation and others diseases related to aging (Libertet al., 2006). For this reason, the DNA-binding activityof the NF-κB complex is significantly increased inseveral rat and mouse tissues during aging (Salminenand Kaarniranta, 2009a). Moreover, NF-κB has beenconsidered as a new therapeutic target againstinflammatory damages associated with neuro-degenerative diseases (Camandola and Mattson, 2007).Inflammation is a potent inhibitor of autophagy(Salminen and Kaarniranta, 2009b), and remarkably, theactivation of NF-κB system can suppress autophagyfunctions, thus contributing to neurodegeneration(Caballero et al., 2008, 2009). Other studies have alsoreported the reciprocal inhibition between autophagy andNF-κB activation (Djavaheri-Mergny et al., 2007; Zhu etal., 2011), therefore, NF-κB signaling might beconsidered as a potent inhibitor of autophagocytosis(Lee et al., 2007; Dan and Baldwin, 2008; Salminen andKaarniranta, 2009b). Similarly, autophagy negativelyregulates NF-κB through the autophagocytosis-mediateddegradation of the NF-κB-inducing kinase (NIK) andIKK kinases (Qing et al., 2006, 2007). Notably, IKK canalso promote the autophagic pathway in an NF-κB -independent manner (Criollo et al., 2010; Comb et al.,

266Autopaghy in the aging brain

2011). All these observations suggest that, theinteraction between oxidative stress-induced NF-κBactivation and autophagy activity is important in theregulation of cellular responses in the aged brain. Aging-related chronic inflammation by NF-κB activation cancontribute to neurodegeneration by two ways; thedefective/altered autophagy in neurons (Caballero et al.,2008, 2009) and cell death and inflammatory damagesdue to astrocytes activation (Hwang et al., 2010). Thus,it is reasonable that signaling via longevity factors, suchas FoxOs and sirtuins, can inhibit the NF-κB system andsimultaneously protect against chronic inflammationduration the aging process (Salminen et al., 2008) toimprove longevity. Sirtuins and autophagy

Lifespan extension seems to depend on the efficientmaintenance of autophagic degradation (Hars et al.,2007; Jia and Levine, 2007; Cavallini et al., 2008; Vellaiet al., 2009). Therefore, there is an increased interest in

studying the longevity signaling pathways that canregulate autophagy. Considering that acetylation is animportant post-translational modification, whichregulates autophagosome formation (Lee et al., 2008),activity of sirtuins becomes more interesting forunderstanding the aging process. Sirtuins are NAD+-dependent histone/protein deacetylases that arehomologous to the yeast protein Sir2 (silent informationregulator 2) (Sinclair et al., 1998). The mammaliansirtuins (SIRT1-SIRT7) have important functions in theregulation of metabolism, growth and differentiation,inflammation, cellular survival and aging (review inSalminen and Kaarniranta, 2009c). Oxidative stress,mitochondrial dysfunction, inflammation and defectiveautophagy are hallmarks of the aging process, andtherefore, it is reasonable to think that the regulation ofsirtuins in these processes is essential for longevitycontrol. Moreover, sirtuins play a role in mitochondrialROS production (Nakagawa and Guarente, 2011) andcan also mediate peroxisome proliferator-activatedreceptor γ coactivator-1 α (PGC-1α) effects to regulate

267Autopaghy in the aging brain

Table 1. An insight into the impact of changes in autophagy on neurodegeneration and longevity.

Positive or Negative effects of Autophagy in Neurodegeneration/Longevity References

Life span-extending effect of the p53 orthologue CEP-1 mutation by increasing baseline autophagy in C.elegans Tavernarakis et al. 2008Promoting levels of autophagy in the nervous system enhances longevity and oxidant resistance in adult Drosophila melanogaster. Simonsen et al., 2008

Autophagy is required for lifespan extension in various long-lived mutant organisms Cuervo, 2008; Reviewed in Vellai et al., 2009

Autophagy is required for dietary restriction-mediated life span extension in C.elegans Jia and Levine, 2007Caloric restriction and resveratrol prolong longevity via the Sirt-1 dependent induction of autophagy Morselli et al., 2010Impairment of the ubiquitin-proteasome system or the autophagy-lysosome pathway predispose individuals toneurodegenerative disorders such as Parkinson's disease Matsuda and Tanaka, 2010

Autophagy may also function to restrict lifespan in C.elegans Hashimoto et al., 2009Cytoprotective function of autophagy-lysosome pathway by disruption of the synthesis, transport, folding or glycosylation of ER-targeted in Drosophila Arsham and Neufeld, 2009

Defective autophagy has been linked to age-related neurodegeneration developmentCao et al., 2006; Komatsu et

al., 2006; Hara et al., 2006;Pickford et al., 2008

ß-amyloid production by accumulated autophagy vacuoles in dystrophic dendrites of the presenilin (PS)/A‚ precursor protein (APP) mice model Yu et al., 2005

Autophagy activity helps to clear aggregated-prone proteins from the cytosol Williams et al., 2006

Autophagy alterations in the brain of senescence accelerated mouse prone 8 (SAMP8) Caballero et al., 2009; Ma et al., 2011

Increased autophagy clearance toxic proteins associated with pathologies such Parkinson’s and Huntington’s disease Ferrucci et al., 2008; Lee and Gao, 2008

Autophagy has anti-aging effects being beneficial toward retardation of aging and prevention of age-related disease in humans Bergamini et al., 2007Autophagy induction in the systemic metabolic response associated with premature aging Mariño et al., 2008TOR-mediated autophagy suppresses cell death in Drosophila model of Huntington’s disease Wang et al., 2009aLoss of PINK1 or Parkin results in failure of mitophagy and may contribute to the pathogenesis of Parkinson’s disease Geisler et al., 2010Contribution of the autophagy-lysosomal genes deficits to Alzheimer and Parkinson diseases and potential involvement in tuberous sclerosis, neuronal ceroid-lipofuscinoses, sepsis and neoplasms Jegga et al., 2011

mitochondrial biogenesis and the supply of newmitochondrias (Aquilano et al., 2010; Kong et al., 2010).Remarkably, SIRT1 is induced by calorie restrictionincreasing organism longevity in yeast, worms, flies andmammals (Guarente and Picard, 2005; Vellai et al.,2009); SIRT1 can be directly associated with thesirtuins-dependent induction of autophagy (Morselli etal., 2010) for the clearance of old and damagedorganelles. In this regard, SIRT1 activity can regulateautophagocytosis by the direct deacetylation ofautophagic proteins, such as ATG5, ATG7 and ATG8,thus activating the basal level of recycling autophagyactivity (Lee et al., 2008). Likewise, SIRT1 activity caninhibit NF-κB-mediated transcription by thedeacetylation of the RelA/p65 subunit of the NF-κBcomplex, protecting against age-related inflammation(Salminen et al., 2008) and hence also favoringautophagy functions. However, it should be noted thatthe activation of sirtuins, per se, induces the autophagyrequired for the lifespan-prolonging effects of caloricrestriction and pharmacological Sirtuin-1 activators(Morselli et al., 2010). Sirtuins regulate not only severalphysiologic conditions (embryogenesis, glucosemetabolism, apoptosis, autophagy, chromatin integrity,and transcriptional state) but also pathologic (diabetes,cancer, cardiovascular disorders, and neurodegeneration)conditions. Driven by all these considerations, sirtuinshave been considered as novel therapeutic targets to treatage-associated diseases (Lavu et al., 2008). Underneurodegenerative conditions, SIRT1 appears to protectagainst certain forms of neuronal degeneration (Kim etal., 2007). In fact, SIRT1 activation by the naturalphytocompound resveratrol, proved beneficial forreducing amyloid-beta protein accumulation in both invitro and in vivo models of AD (Albani et al., 2009).Resveratrol and caloric restriction also inducedneuroprotective actions by SIRT1 activation inParkinson’s disease, Huntington’s disease and epilepsy(Qin et al., 2006; Albani et al., 2010). In this regard,several neurodegenerative conditions and other age-related pathologies can be benefit from the induction ofbasal autophagy though an increase of sirtuins activity(Lee et al., 2008). Remarkably, SIRT1 expressiondecreases with age in the senescent andneurodegenerative brain (Gutierrez-Cuesta et al., 2008),and autophagy is altered (Caballero et al., 2009; Wongand Cuervo, 2010). Therefore, the decreased SIRT1expression in the aging brain might have two importantconsequences: to have a negative and direct effect onautophagy or to indirectly favor age-relatedinflammation through NF-κB activation, which alsointerferes in autophagy. Sirtuins likely play a key role inbrain susceptibility to neurodegeneration during agingthrough its well-established effect on the regulation ofautophagy in physiological and pathological conditions. The p53 and autophagy

Considered the “guardian of the genome” and maintumor suppressor, the p53 transcriptional factor responds

to a wide variety of stress signals, including DNAdamage, hypoxia, heat/cold shock, nutrition starvationand oncogene activation, to maintain genomic stabilityby limiting the error frequency of cell growth anddivision. The p53-mediated cellular responses, such ascell cycle arrest, DNA repair and apoptosis, depend oncell type, environmental context and degree of stress(Feng, 2010). The relationship between p53 and agingappears to be complex. The p53 functions decline withaging, increasing tumor incidence in older organs (Fenget al., 2007). Remarkably, there is functional antagonismbetween p53 and NF-κB signaling; the aging-associateddecline in p53 efficiency favors the NF-κB-mediatedsenescence and inflammation (Salminen andKaarniranta, 2011). It is well-known that aberrant ornon-regulated p53 activity could also accelerate aging(Serrano and Blasco, 2007). Indeed, mice modelsshowing over-expressing of p53 and increased p53activity present shortened life-spans (Tyner et al., 2002;Maier et al., 2004). However, mouse models withcontrolled, constitutive p53 activity are resistant tocancer and display a normal life span and aging (Garcia-Cao et al., 2002, 2006). In short, inappropriate p53activity promotes aging, whereas the normal androbustly regulated p53 response provides protectionfrom the aging process (Vigneron and Vousden, 2010).Furthermore, p53 can regulate aging by autophagy(Tavernarakis et al., 2008). Recently, it has beensuggested a regulation of senescence by p53 due to itsability to promote or inhibit oxidative stress andautophagy according its level of acetylation, which willhave contrary effects on longevity and aging (Vigneronand Vousden, 2010). In this regard, it is important tonote that nuclear p53 can induce autophagy through itstranscriptional effects, while cytoplasmic p53 acts as amaster repressor of autophagy (Tasdemir et al., 2008;Green and Kroemer, 2009). Thus, loss of cytoplasmicp53 can induce autophagy in humans, mice andnematode cells (Tasdemir et al., 2008), and this effecthas been linked to longevity in nematodes (Tavernarakiset al., 2008). The mechanism through which p53 canactivate autophagy includes the down-regulation of IGF-1/AKT-1/mTOR pathways and the up-regulation of thetranscription of autophagy proteins, such as the damage-regulated autophagy modulator (DRAM) and Sestrin2(Green and Kroemer, 2009; Feng, 2010). However, aswith aging, inappropriate p53 activity can contribute toneurodegeneration by inducing apoptotic and/orautophagic cell death (Wang et al., 2008, 2009b; Pehar etal., 2010). Interestingly, p53 was the first discoverednon-histone target of SIRT1 (Luo et al., 2001). Thedeacetylation of p53 by SIRT1 leads to the inactivationof p53-mediated transcription (Vaziri et al., 2001; Luo etal., 2001), which is important in neuronal survival(Hasegawa and Yoshikawa, 2008). In fact, SIRT1 canregulate both types of known p53-mediated apoptoticpathways, transcriptional dependent and independentmechanisms (Yi and Luo, 2010) and, even, block thenuclear translocation of p53 induced by oxidative stressvia deacetylation (Han et al., 2008). As a potent tumor

268Autopaghy in the aging brain

suppressor, SIRT1 can negatively regulate various tumorsuppressors, including p53, ß-catenin and survivin (Yiand Luo, 2010) and SIRT1 interactions with p53 can alsoregulate both autophagic degradation and lifespanextension (Salminen and Kaarniranta, 2009c). Therefore,considering these observations, the regulation of p53responses could favor autophagy and neuronal survivalduring the aging brain with an important positive impacton longevity and against neurodegeneration. Autophagy in animal models of accelerated aging

SAMP8 mice are non-genetically modified mice thatshow a shortened life-span with important learning andmemory deficits (Miyamoto et al., 1986, 1992;Miyamoto, 1997), which are also well-known age-related signs and symptoms of human aging.Interestingly, for the actual aging research, the SAMP8mouse appears to be an excellent model for studying themechanism of age-related cognitive dysfunction andneurodegeneration (Alvarez-Garcia et al., 2006;Caballero et al., 2008, 2009; Zhang et al., 2009),displaying degenerative changes caused by theimpairment of oxidative metabolism (Zhang et al.,2009), resembling those observed in brain affected withAD (Diez-Vives et al., 2009). Indeed, oxidative-stressrelated alterations in SAMP8 mice are observed at earlyages not only in the brain, but also in various key organs,such as the liver and spleen (Alvarez-Garcia et al., 2006;Lardone et al., 2006; Caballero et al., 2008, 2009). Inaddition, increased levels of protein carbonyl and severalneurodegenerative markers, such as phosphorylated Tauin the neurofibrillary tangles, α synuclein (Alvarez-Garcia et al., 2006; Caballero et al., 2008) and ß-amyloidaggregates (Morley, 2002; Gutierrez-Cuesta et al., 2008),are described in the SAMP8 brain. The toxic proteinaggregates from the aged brain of SAMP8 mice werealso associated with deficits in specific lysosomal andcytosolic proteolytic activities (Caballero et al., 2009),although without an important neuronal loss byapoptosis (Takeuchi et al., 2000). Neurotransmission inthe SAMP8 brain is also altered by a decrease in levelsof NMDA (N-methyl-d-aspartic acid) (Tomobe andNomura, 2009), MT-1 (high-affinity G-protein-coupledmelatonin receptor) and ROR-α (Retinoic acid receptor-related orphan receptor alpha) receptors (Caballero et al.,2008) together with an early loss in adenosine receptors(Castillo et al., 2009). Inflammatory processes weredriven by the strong activation of NF-κB in the SAMP8brain (Caballero et al., 2008; Gutierrez-Cuesta et al.,2008). It is remarkable that, although our previousresearch did not show the activation of autophagy in thebrain of 5 and 10-month-old SAMP8 mice (Caballero etal., 2009), more recent work have described autophagicmarkers in the 7-month-old SAMP8 brain, especially inthe cortex and hippocampus, which decrease at 12-months of age, displaying autophagic vacuolesaccumulation in the axons and cytoplasm in both areas,similar to late-onset AD (Ma et al., 2011). Therefore, it

must be emphasized that the age-related autophagyalterations in the brains of SAMP8 mice can beassociated with their susceptibility to age-relatedneurodegeneration and early cognitive decline.

Thus, taken together, there are several factors thatmight impair autophagy in the SAMP8 brain. Higheroxidative stress-induced NF-κB signaling in the SAMP8brain (Caballero et al., 2008) would lead to age-relatedpro-inflammation (Rodriguez et al., 2007), and bothfactors are well-known autophagy suppressors(Salminen and Kaarniranta, 2009a,b). Notably, p53 wasalso increased with age in the SAMP8 brain (Caballeroet al., 2009), but without a higher activation byacetylation (Gutierrez-Cuesta et al., 2008). Thus, thealteration of both the p53 and NF-κB responses might bealso a key factor in regulating autophagy levels.Remarkably, SIRT1 expression decreased with age in theSAMP8 brain (Gutierrez-Cuesta et al., 2008). Therefore,the loss of this longevity factor, which plays a key rolein autophagy induction (Lee et al., 2008), together withan increased inflammatory process and proteolyticdeficiencies, might play a role in autophagy impairmentsin SAMP8. Moreover, the SAMP8 brain is also a usefulmodel for glucose hypometabolism, which is alsoobserved in the aged brain and with dementias(Kurokawa et al., 1996; Ohta et al., 1996). A diminishedglucose metabolisms has been shown to induce thehyperphosphorylation of Tau (Planel et al., 2004) andincreased production of the ß-amyloid peptide (Gabuzdaet al., 1994) and both markers are also observed in theSAMP8 brain (Gutierrez-Cuesta et al., 2008; Caballeroet al., 2009). More recent studies have revealed thatthese mice have low glucose levels in serum ascompared with their control SAMR1 mice (Jiang et al.,2008). The energy production (ATP) in the centralnervous system is based almost exclusively upon theoxidation of glucose, and in that way, diminished energyproduction in the brain down-stream impairs ATP-dependent processes, such as synaptic functions,ubiquitin-proteasome system degradation and, therefore,also autophagy degradation. Likewise, there is anincreased glucose transport to the brain by increasedGLUT3 expression in zones, such as the cortex, of theSAMP8 brain (Sato et al., 1994). Glucose, considered apro-aging factor, could activate the insulin receptorsignaling pathway (Kassi and Papavassiliou, 2008),leading to the subsequent activation of the mTORcomplex, which inhibits several steps in autophagosomeformation (Kamada et al., 2000). In this way, theincreased glucose transport to the SAMP8 brain,together with a decreased glucose metabolism, couldalso negatively affect autophagy in these mice. Takentogether, these results suggest that there are severallongevity factors and oxidative/nitrosative stress-relatedsignaling pathways that can disrupt cellular responses toincrease the susceptibility of the SAMP8 brain to earlyneurodegenerative changes.

Finally, the Zmpste24-deficient mouse is a reliablemodel of human Hutchinson-Gilford progeria, a type of

269Autopaghy in the aging brain

accelerated aging in which autophagy plays an importantrole. These progeroid mice, display defects in nucleararchitecture because Zmpste24 (also called FACE-1) is ametalloproteinase involved in the maturation of lamininA and is an essential component of the nuclear envelope.These nuclear envelope abnormalities are associatedwith premature aging and progeroids syndromes in bothmice and humans (Mariño et al., 2008). Surprisingly,these prematurely-aged mouse models exhibit anextensive basal activation of autophagic degradationinstead of the characteristic decline in this process thatoccurs during normal aging. Therefore, the increase inautophagy in Zmpste24-deficient mice was linked tosevere metabolic alterations in glucose and lipidmetabolism, which lead to elevated liver kinase B1 andthe up-regulation of the AMP-activated protein kinasepathway, which ends with mTOR inhibition (Mariño etal., 2008). The authors noted that these metabolicchanges, including lower insulin and glucose levels inblood, respectively, resemble those occurring undercalorie restriction or in other situations reported toprolong life-span. In this regard, they have described anovel and paradoxical role for autophagic cellulardegradative pathways during pathological agingprocesses (Mariño et al., 2008; Mariño et al., 2010).Remarkably, the chronic activation of autophagy canhave a negative effect on cell death (Maiuri et al., 2007).Hence, the progressive muscle and cardiac deteriorationin Zmpste24-deficient mice is related with uncontrolledautophagy activity (Mariño et al., 2008). In addition, theZmpste24-deficiency mice show a stress signalingpathway associated with a strong hyperactivation of the

tumor suppressor p53 (Varela et al., 2005), reflecting thekey role of a deregulated p53 responses in prematureaging, which might be also linked to autophagyimpairment. Although, there is not relevant informationabout the changes on NF-κB signaling or longevityfactor expression in these progeroid mice, it should benoted that defective NF-κB transcriptional activity hasbeen previously shown in laminin A/C-deficient cells(Lammerding et al., 2004). Therefore, further alterationsin cellular stress responses and longevity pathwaysmight be involved in the up-regulation of autophagy inprogeroid mice. Concluding remarks

Aging and autophagy are two processes that areclearly dependent on increased ROS production, whichmaintain a narrow, though inverse, relationship. It seemsthat when people age, their autophagic capabilitybecomes reduced. This review shows that molecules,such as sirtuins, p53 and NF-κB that are reported bymany articles to be key players in longevity, also have animportant role in autophagic regulation. The functions ofthese molecules in both processes are conflicting and,sometimes, confusing. Some reports show that, whilesirtuins and nuclear p53 induce autophagy, NF-κBabolishes it (Fig. 2).

The relationship between aging and autophagy hasyet to be elucidated. Although some advances have beenmade, results seem contradictory, and while certainaging animal models show that autophagy decreaseswith aging, progeria models present uncontrolled

270Autopaghy in the aging brain

Fig.2. Influence of NF-κB, p53 and sirtuins onautophagy activity. The positive effect ofautophagy on longevity can also be associatedwith its well-known negative regulation ofneurodegenerative damages. Longevity factors,such as sirtuins 1 (SIRT1), can positively affectlongevity by the direct activation of autophagy,which directly impact against neuro-degenerative levels. Likewise, there is anegative regulation of autophagy by oxidativestress-related transcriptional factors, such asNF-κB signaling and p53 activity. In fact,autophagy can be blocked by NF-κB-mediatedinflammation and levels of autophagic activitycan be modified according to the level of p53acetylation.

autophagy increases.Although this connection has huge research

potential, further experiments are required to gain abetter understanding for the complex interactions thatexits among longevity, neurodegeneration andautophagy.Acknowledgements. B. Caballero would like to thanks her post-doctoralfellowship (The Programa Clarin) from FYCIT (Gobierno del Principadode Asturias), Spain. This work was partially performed with funding fromgrants FISS-06-RD06/0013/0011 from the Instituto de Salud Carlos III(Ministerio de Sanidad y Consumo) and BFU2010-20919 from theMinisterio de Ciencia e Innovación and FEDER funds. Both authors aremembers of the INPROTEOLYS network.

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Accepted September 26, 2011

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