Progressive cognitive decline is a hallmark of normal and degenerative brain aging and is characterized by deficits in learning, memory, and executive function (Roberts and Knopman, 2013). It is estimated that more than 20 % of individuals 65 and older experience cognitive impairment without meeting the pathophysiological criteria of Alzheimer’s (AD) or other neurodegenerative dementias (Rajan et al., 2021). Such normal brain aging differs from neurodegenerative diseases like AD, in that neurons are not lost and therefore cannot explain the cognitive deficits of normal aging (Freeman et al., 2008, Morrison and Hof, 1997, Peters et al., 1998). Instead, magnetic resonance imaging (MRI) and histological studies in healthy humans and monkeys have shown that subcortical white matter volume is reduced with age and this loss correlates with cognitive decline (Guttmann et al., 1998, Marner et al., 2003, Peters and Rosene, 2003, Wisco et al., 2008). This reduction begins in middle age but does not occur uniformly across the brain. Interestingly, diffusion tensor imaging (DTI) and other MRI studies have shown that frontal white matter is the first to experience microstructural and volumetric changes associated with aging and these changes are associated with worse cognitive performance (Coelho et al., 2021, Fujita et al., 2023, Ouyang et al., 2021, Yoon et al., 2008, Ziegler et al., 2010). Electron microscopy has revealed that breakdown of the myelin sheath is the etiological factor that underlies age-related white matter loss (Bowley et al., 2010, Kohama et al., 2012). Ultrastructural studies of myelin in aged rhesus monkeys have shown myelin sheath pathology, including splitting within the sheath, intracytoplasmic inclusions, and in some cases axon degeneration (Bowley et al., 2010). When the myelin sheath is damaged, effective neural communication is disrupted which contributes to reduced processing speeds and cognitive impairment (Chen et al., 2021, Lu et al., 2013, Wang et al., 2020).

White matter is particularly vulnerable to oxidative damage given the abundance of polyunsaturated fatty acids and the presence of post-mitotic oligodendrocytes that are very sensitive to alterations in redox state. Reactive oxygen species (ROS), such as O2- and OH, are highly reactive free radical molecules that form as a byproduct of metabolic reactions, with roughly 90 % of ROS produced in the mitochondria during ATP production (Balaban et al., 2005). During aging, mitochondria show a reduction in protein quality control, fission, and turnover which results in elevated ROS levels that, in combination with reduced antioxidant buffering mechanisms, result in pathogenic cellular levels of ROS. These molecules can directly damage myelin through peroxidation of the lipids composing the myelin sheath, which compromises sheath integrity and creates myelin debris that requires clearance and turnover (Chia et al., 1983, Konat and Wiggins, 1985, van der Goes et al., 1998). These byproducts, in addition to ROS itself, directly attack DNA and RNA of surrounding oligodendrocytes and microglia, creating strand breaks or adducts which require repair by DNA damage repair enzymes (Esterbauer et al., 1990). Therefore, ROS not only chemically perturbs myelin, but also disrupts the genomic integrity of surrounding glia which are responsible for myelin maintenance and regeneration. Additionally, nuclear and mitochondrial DNA damage repair pathways become less effective with age (Gorbunova et al., 2007, Gredilla et al., 2010). As a result, accumulation of damaged DNA leads to disruptions in transcription and genome replication, threatening cellular viability and even inducing apoptosis (Carusillo and Mussolino, 2020, Jackson and Bartek, 2009). Oligodendrocytes, being terminally differentiated cells that lack strong antioxidant mechanisms, are especially vulnerable to DNA damage. It is suggested that the genomic instability of oligodendrocytes and their vulnerability to ROS are main contributors to loss of effective remyelination with age (Tse and Herrup, 2017). In addition, microglia, the resident immune cell of the brain, are responsible for phagocytosing myelin debris and other extracellular aggregates to allow for effective remyelination. Chronic elevations in ROS are sufficient to initiate inflammatory pathways within microglia that result in cellular dysfunction, including the inability to effectively clear myelin debris, disrupting remyelination (Li et al., 2022, Lin et al., 2022).

Interventions that reduce oxidative stress and metabolic dysfunction associated with aging are of interest in combating the accumulation of cellular damage. Calorie restriction (CR) has long been recognized as one of the most effective interventions to delay the onset of age-associated diseases and has been shown to extend lifespan in organisms ranging from yeast to non-human primates (Colman et al., 2009, Fontana and Klein, 2007, McCay et al., 1989). CR is a simple intervention involving a 25–40 % reduction in total calories consumed daily relative to baseline. The mechanisms underlying the benefit of CR are proposed to be mediated through activation of energy sensor AMP-activated protein kinase (AMPK), a heterotrimeric serine/threonine kinase that acts as a master regulator of mitochondrial biogenesis and metabolism (Cantó et al., 2009). Some of the benefits of CR include improving cellular metabolism and mitochondrial efficiency, reducing oxidative stress, and attenuating inflammation (Martin et al., 2006, Mattson and Arumugam, 2018). The effects of CR have been studied extensively using rodent models of aging, AD, traumatic brain injury, and multiple sclerosis (MS)(de Carvalho, 2022; Lobo et al., 2022). Recent studies investigating mouse models of MS have shown that short-term CR (on the order of weeks to months) attenuated the expression of reactive microglia, reduced oligodendrocyte apoptosis, promoted remyelination, and led to functional improvements in behavior (Mojaverrostami et al., 2020, Zarini et al., 2021).

However, very little is known about the effects of long-term CR in the brains of primates. Monkeys show similar, age-related vulnerability in frontal white matter as humans (Kohama et al., 2012, Marner et al., 2003). Moreover, compared to rodents where the brain is composed of about 10% white matter, the monkey brain is composed of 40 % white matter which is comparable to the 50 % white matter of the human brain (Krafft et al., 2012). This makes the monkey an ideal species to study white matter pathology. Furthermore, rhesus monkeys are spared from AD and other neurodegenerative diseases which allows white matter to be studied without the confounding effects of neurodegeneration (Herndon et al., 1997, Peters et al., 1998). To date, only two studies have investigated the effects of long-term CR in rhesus monkeys and its potential to promote healthy aging and improve survival outcomes - one at the NIA Intramural Research Program and the other at the University of Wisconsin (UW) National Primate Research Center (Colman et al., 2009, Colman et al., 2014, Mattison et al., 2017, Mattison et al., 2012). Consistent with literature in rodents, CR demonstrated significant improvements in health (e.g., reduced incidence of cancer and of glucoregulatory dysfunction) in both the NIA and UW cohorts. However, while the UW cohort showed that CR increased survival relative to controls, this was not observed in the NIA cohort. (Colman et al., 2009, Colman et al., 2014). The discrepancy in survival effect is likely due to differences in study design that resulted in longer fasting times and greater differences in body weight and adiposity between control and CR monkeys in the UW cohort than was seen in the NIA cohort (Mattison et al., 2017). In terms of brain health, a DTI study of the Wisconsin cohort found that old monkeys subject to long-term CR showed reduced age-related loss of frontal white matter (Bendlin et al., 2011). However, the cellular mechanisms underlying this CR-induced white matter preservation are unknown.

To determine if CR can provide a neuroprotective effect in the white matter of the aging brain by reducing oxidative damage within the glia supporting the myelin sheath, we examined whether long-term CR would reduce levels of oxidative DNA damage within oligodendrocytes and microglia residing in frontal white matter tracts.