Age-related memory loss affects approximately 40 % of the world’s population after the age of 65 and is a significant risk factor for the development of dementia and Alzheimer’s disease. The hippocampus is crucial for consolidating new memories and is highly susceptible to aging, with its deterioration linked to cognitive decline and neurodegenerative disorders (Geinisman et al., 1995). Studies have shown changes at the cellular level in the hippocampus with aging, such as atrophy that results in age-related decline in memory (Mu et al., 1999, Schuff et al., 1999, Rosenzweig and Barnes, 2003, Raz et al., 2004, Walhovd et al., 2005, Driscoll et al., 2006, Persson et al., 2012), which has been found in a number of mammals including rats (Kuhn et al., 1996, Rosenzweig and Barnes, 2003, Rao et al., 2006, Epp et al., 2009, Koehl et al., 2009, Olesen et al., 2020, Crown et al., 2022), dogs (Siwak-Tapp et al., 2007), macaques (Jabès et al., 2010, Thomé et al., 2016), humans (Golomb et al., 1993, Knoth et al., 2010, Ferrarini et al., 2014), marmosets (Leuner et al., 2007), lesser hedgehog tenrecs (Alpár et al., 2010), mice (Kronenberg et al., 2006, Morgenstern et al., 2008, Rodríguez et al., 2008, Ben Abdallah et al., 2010, Lazic, 2012, Gil-Mohapel et al., 2013), tree shrews (Simon et al., 2005) and red foxes (Amrein & Slomianka, 2010). Further, with aging there are functional, vascular, inflammatory, epigenetic and molecular changes in the hippocampus which, in many cases, correlate with reduced memory performance seen in aged animals (Johnson, 2023, Iadecola et al., 2019, Saw and Tang, 2020).

The ubiquitin proteasome system (UPS) is responsible for ∼ 90 % of all protein degradation in eukaryotic cells and plays a role in memory formation (Patrick et al., 2023). In this system ubiquitin marks proteins for various fates with the most common being degradation by the proteasome, a process essential for regulating various cellular functions. Ubiquitination involves tagging a target protein with ubiquitin, a small of the 76 amino acid protein, via the actions of ubiquitin activating (E1) and conjugating (E2) enzymes and specific ubiquitin ligases (E3). The E3 ligases provide target-specificity in this pathway as they can attach ubiquitin to one or many proteins. Proteins can acquire one or several ubiquitin, the latter forming polyubiquitin chains for which there are 8 possible linkage sites on the ubiquitin protein (M1, K6, K11, K27, K29, K33, K48, K63). Of these linkage sites, only one does not occur at a lysine (K) but instead a methionine (M) – the M1 site, also known as linear polyubiquitination (Patrick et al., 2023). Linear polyubiquitin chains are conjugated by the linear ubiquitin chain assembly complex (LUBAC), which is a trimeric complex composed of heme-oxidized IRP2 ubiquitin ligase 1 (HOIL-1), HOIL-1-Interacting Protein (HOIP, also known as RNF31) and SHANK-associated RH domain interacting protein (SHARPIN) (Kirisako et al., 2006, Gerlach et al., 2011, Ikeda et al., 2011). HOIL-1 and RNF31 are both E3 ubiquitin ligases, however, the specific linear ubiquitin-ligating activity is enacted by RNF31 (Smit et al., 2012), making it an essential regulator of linear polyubiquitination. Importantly, M1 polyubiquitination is independent of the proteasome-mediated protein degradation process. Previously we found that M1 polyubiquitination in the amygdala plays a critical role in contextual fear memory formation in a sex-specific manner as both males and females had impaired contextual fear memory following inhibition of the M1 polyubiquitin ligase Rnf31 in the amygdala (Musaus et al., 2021). However, surprisingly, males and females targeted vastly different proteins with this ubiquitin modification following fear conditioning. This suggests unique sex differences in how males and females use M1 polyubiquitination in the amygdala to form the same memory. This remains the only examination of M1 polyubiquitination in the brain and it is unknown if this non-lysine polyubiquitin modification regulates memory formation in other brain regions. Additionally, though recent research in Drosophilia melanogaster has suggested that linear polyubiquitin E3 ligase (LUBEL), a RNF31 ortholog, could be used to alter the effects of aging (Choi et al., 2022), it remains unknown if linear polyubiquitination levels change in the aged brain and contribute to memory formation in advanced age.

Here, we tested the role of linear polyubiquitination in memory formation in the young and aged hippocampus of male rats. Our results suggest that aging results in dramatic increases in M1 polyubiquitination in the hippocampus. Further while learning in young adults typically increases M1 polyubiquitination in the hippocampus, in advanced age it results in robust decreases in this non-lysine polyubiquitin modification. CRISPR-dCas9 mediated increases in M1 polyubiquitination in the hippocampus enhances memory in young adult, but not aged, rats. Together, these data suggest that while hippocampal M1 polyubiquitination is a critical regulator of memory formation, increasing it late in life does not improve memory.