Regulation of the P62 Promoter by Oxidative Damage in Neurodegenerative Disease and Aging

Absence of the p62 gene in mouse brain leads to biochemical and cognitivedeficits that resemble Alzheimer’s disease (AD). In this context, the objective of thisstudy was to examine the age-associated oxidative damage to the p62 promoter in AD.Increased 8-OHdG staining, a marker of oxidative stress, was observed in brain sectionsfrom mice deficient in the p62 gene compared to control. Treatment of MEF cellsdeficient in p62 withH2O2 resulted in decreased cell survival and an absence of Nrf2nuclear translocation. The mouse p62 promoter exhibited elevated oxidative damage withincreasing age and the degree of p62 promoter damage was also age-correlated in humanbrain samples. In human subjects, the expression of p62 was decreased in AD brainrelative to age-matched controls, and likewise decreased p62 expression correlated withoxidative damage to the promoter. Treatment of HEK cells withH2O2 resulted indecreased p62 expression concomitant with increased promoter damage. Consistent withthese findings, a transgenic AD mouse model also exhibited increased p62 promoter* Published in Free Radic. Biol. Med. 46, 492-501, 2009. 95damage and reduced p62 levels in brain. Altogether, our results reveal that oxidativedamage to the p62 promoter correlates with decreased expression of p62 and maycontribute to age-associated neurodegenerative disease such as AD and others. INTRODUCTIONOxidative stress is the result of imbalance between productions of reactive oxygenspecies (ROS) and a biological system's ability to detoxify the reactive intermediates orrepair the oxidative damage. Low levels of ROS can act as signaling molecules in variousintracellular processes (Scherz-Shouval and Elazar, 2007). However, when the ROS leveloverwhelms the antioxidant and repair system, cells might be damaged by thesemolecules. Progressive and irreversible accumulation of oxidative damage contributes tothe aging process, and is thus implicated in development of several age-associateddiseases, such as neurodegenerative diseases (Filipcik et al., 2006). Oxidative damage toDNA is particularly harmful since it may cause mutations that can be inherited by thenext generation, leading to genome instability. Among the five nucleobases, guanine isthe most susceptible to oxidation because of its high electron density (Steenken, 1989).The predominant DNA oxidative adduct is 8-hydroxydeoxyguanosine (8-OHdG),which serves as a common biomarker of DNA oxidative damage (Steenken, 1989; Kasai,1997). Most 8-OHdG lesions are repaired by a base excision repair (BER) pathway. It hasbeen reported that when the ability of the repair system declines, cumulative oxidativeDNA damage in the mitochondria and nuclear genomes of neurons may play a criticalrole in brain aging and neurodegenerative disorders such as AD, Parkinson’s disease and 96Amyotrophic Lateral Sclerosis (Markesbery and Lovell, 2006; Nakabeppu et al., 2007;Warita et al., 2001).Relationships have been established among increased DNA damage, defectiveDNA repair, aging, and age-associated neurodegenerative disease (Lovell andMarkesbery, 2007; Kikuchi et al., 2002; Martien and Abbadie, 2007). However, the exactmechanism by which oxidative DNA damage lead to neurodegeneration or neuronal celldeath still remains obscure. Human p62 is oxidatively-induced in human and mouse celllines (Ishii et al., 1997), and the p62 protein has been localized to aggresomes of variousneurodegenerative diseases (Zatloukal et al., 2002). This gene was first identified by Parket al., as the ligand for the p56 (Park et al.,2002), and referred to as Sequestosome1/SQSTM1. In humans, variants of p62/SQSTM1 have been linked to Pagets Disease ofBone (Layfield and Hocking, 2004). In mouse, the gene is related to A170/STAP (SignalTransduction Adapter Protein (Okazaki et al., 1999); whereas in rat, the gene is referredto as ZIP, the zeta interacting partner of the atypical protein kinase C (Puls et al., 1997).The p62 protein contains several interaction motifs that endow the protein withscaffolding abilities (Moscat et al., 2007). At its C-terminal tail p62 possesses anubiquitin associated domain that interacts with K63 polyubiquitin chains and the N-terminus possesses a PB1 domain. A ZZ-finger recruits the atypical PKC and otherproteins, whereas the TRAF6 binding site (TBS) recruits the E3 ubiquitin ligase TRAF6.Functionally, p62 serves to connect signaling pathways associated with two post-translational modifications. An absence of p62 leads to the loss of aggresomes andneuronal cell death (Nakaso et al., 2004). Moreover, p62 has been reported to activate the