1:1Department of Biochemistry and Molecular Biology, Universitat Autònoma Barcelona, Bellaterra, Spain; 2:Institut de Neurociènces (INc), Universitat Autònoma Barcelona, Bellaterra, Spain; 3:Pharmacology Section, Department of Pharmacology, Toxicology and Therapeutic Chemistry, Faculty of Pharmacy and Food Sciences, Universitat de Barcelona, Barcelona, Spain; 4:Institute of Neuroscience, Universitat de Barcelona (NeuroUB), Barcelona, Spain; 5:Centro de Investigación Biomédica en Red sobre enfermedades Neurodegenerativas (CIBERNED), Instituto Carlos III, Madrid, Spain; 6:Vall d'Hebron Institut de Recerca (VHIR), Research Group on Gene Therapy at Nervous System, Passeig de la Vall d'Hebron, Barcelona, Spain; 7:Unitat producció de Vectors (UPV), Universitat Autònoma Barcelona, Bellaterra, Spain; 8:Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain

Senescence represents a stage in life associated with elevated incidence of morbidity and increased risk of mortality due to the accumulation of molecular alterations and tissue dysfunction, promoting a decrease in the organism’s protective systems. Thus, aging presents molecular and biological hallmarks, which include chronic inflammation, epigenetic alterations, neuronal dysfunction and worsening of physical status. αKlotho (KL) has been described as a powerful neuroprotector factor that delays aging consequences. It is mainly expressed in the kidneys and the choroid plexus in central nervous system (CNS) and presents two major splicing variants, a transmembrane isoform (m-KL) and a shorter secreted one (s-KL). Interestingly, Klotho expression decreases during non-pathological aging, which causes deregulation of key metabolic pathways and a decrease in the neuroprotection that this protein confers. In this context, we explored the AAV9-mediated expression of the two main isoforms of the aging-protective factor Klotho (KL) as a strategy to prevent these general age-related features using the senescence- accelerated mouse prone 8 (SAMP8) model. Our principal findings demonstrated that KL expression improved physical condition and cognitive performance without changes in age-related behaviors in treated SAMP8 mice. KL treatment rescues global epigenetic landscape and improves histological and biochemical markers related with CNS inflammation and cellular senescence. Moreover, at the bone level, KL treatment restores structural changes observed in SAMP8 tibia. In summary, our in vivo results showed, after a single treatment with AAVs-expressing KL in SAMP8 mice, an improvement of age-related deficits and relevant molecular aging-hallmarks such as epigenetic, inflammatory and bone alterations.