The cystine/glutamate antiporter system xc- and healthy (brain) aging

We all age and as the average life expectancy is steeply increasing, a lot of researchers focused their work on the mechanisms underlying age-related dysfunctions and, by extension, healthy aging. Not only to better understand why we age, but also to find interventions that could delay or prevent the aging process in humans. The rapid advances in the field of aging research revealed
that aging is a consequence of the interplay of many different mechanisms, and that it affects the human being in its entirety. As such, when studying the potential benefit of an intervention to prevent age-related deterioration, it is important to consider multiple pathways and different aspects involved in the process of aging.
The cystine/glutamate antiporter, system xc-, has been identified as a potential therapeutic target in mouse models for age-related neurological disorders, such as Parkinson’s disease. System xc- is involved in regulating glutamatergic neurotransmission, redox signaling and the immune response.
As the aging process is characterized by dysregulation of the glutamatergic system as well as by the presence of inflammation and oxidative stress, the protective effects of system xc-- deficiency in age-related neurological disorders might arise from a beneficial effect on the aging process itself.
However, when mice lacking xCT, the specific subunit of system xc- (xCT-/- mice), were first characterized by Sato and colleagues in 2005, an oxidative shift in the plasma cystine/cysteine ratio of these mice was seen. This led to the hypothesis that the aging process would be accelerated in the absence of system xc-. As this would question the potential of targeting system xc- as a treatment
strategy in an aged population, we aimed at investigating the role of system xc-
in the process of physiological aging.
In the first part of this thesis, we focused on the peripheral aging process of xCT-/- mice. Our findings revealed that absence of xCT induced an oxidative shift in the plasma cystine/cysteine ratio of aged mice, compared to wildtype littermates (xCT+/+ mice), confirming previous findings in adult mice. The age-related changes in other parameters reflecting different aspects of the redox
homeostasis, were similar between xCT-/- and xCT+/+ mice. Contrary to the expectations, we observed an increased lifespan in xCT-/- mice compared to xCT+/+ mice and we showed that these additional months of lifetime are not spent in poor health, as the level of frailty at the end of their life is similar to xCT+/+ mice. When evaluating general health parameters such as body temperature, body weight, organ weights, muscular strength and plasma chemistry, we observed age-related differences that were mostly unaffected by xCT deletion. As such, all these results indicate beneficial effects of xCT deficiency on both life- and healthspan of mice and support the recent
hypothesis stating that mild levels of oxidative stress are beneficial for life- and healthspan.
As system xc- - deficiency has been shown to modulate the inflammatory process and both the onset of frailty and the occurrence of age-related disorders are associated with age-related changes in the immune system, in the second part of this thesis we investigated the effects of aging in the absence
of system xc- on the immune system. We did not observe any genotype effect on the occurrence of T-cell senescence or on the age-induced changes in splenic immune cell populations. However, our findings revealed that aged xCT-/- mice show an attenuated response to a peripheral inflammatory
stimulus. This was illustrated by decreased sickness behavior as well as an attenuated cytokine response induced by a low dose of lipopolysaccharide (LPS), reflecting reduced priming of the peripheral innate immune system of aged xCT-/- mice. Moreover, while neither aging nor genotype affected the proliferation and morphology of microglia, the macrophages of the central nervous system, absence of system xc- seemed to favor a more anti-inflammatory cytokine profile in the aged brain. These results are in line with our previous observations showing that LPS-injected adult xCT-/- mice encounter reduced signs of both peripheral and central inflammation compared to their
wildtype littermates and support a role for system xc- in driving (neuro)inflammaging. Moreover, these results indicate that inhibition of system xc- can be beneficial in (age-related) disorderscharacterized by chronic inflammation.
The hippocampus is a brain structure that undergoes profound age-related functional changes.
These changes are linked to dysfunctional glutamatergic neurotransmission and are responsible for the occurrence of cognitive decline, and thus have evastating impact on the quality-of-life of the elder. As glutamate released by system xc- can modulate glutamatergic neurotransmission, we investigated the effects of the absence of system xc- on typical age-related changes in hippocampal function in the third chapter of this thesis. Even though oxidative stress and inflammation, typically present in the aging brain, have been shown to enhance the functionality of system xc-, we could not detect changes in xCT expression and distribution nor in system xc- activity in the aged compared to the adult hippocampus. However, we showed that the morphology and functionality
of hippocampal neurons was clearly affected in aged mice. Interestingly, these changes were attenuated in xCT-/- mice and these mice were protected against age-induced impairment of hippocampus-dependent memory. These results, together with the positive effects on both life-and healthspan, suggest that inhibition of system xc- is an effective strategy to retain quality-of-life in the aging population. Moreover, it can be considered a safe strategy that has the potential to relieve comorbid cognitive impairments in the elderly.
In the final chapter of this thesis, we focused on sulfasalazine (SAS), the most commonly used inhibitor of system xc-. While this molecule is EMA- and FDA-approved for the use as an antiinflammatory drug for the treatment of ulcerative colitis and Crohn’s disease, side effects have been reported and some of these have been attributed to system xc- inhibition. However, these could also be linked to off-target effects of SAS such as inhibition of nuclear factor-κB (NFkB) and blocking of NMDA receptors. As these adverse effects resulted in concerns about the safety of targeting system xc-, we aimed at identifying the effects of SAS that are mediated by inhibition of system xc-, by chronically administering SAS to xCT-/- and xCT+/+ mice. Even though SAS-induced chronic inhibition of system xc- was reported to induce motor problems resulting from myelin
degeneration in the white matter of the spinal cord, we were unable to replicate this. Moreover, in our hands, SAS did not induce anxiety- or depressive-like behavior or other long-term behavioral deficits. Over the course of the treatment, all SAS-treated mice experienced weight loss as well as loss of motivation to explore a novel environment, and chronic SAS treatment influenced body temperature regulation. All these effects were independent of its action as an inhibitor of system xc-, but most probably linked to toxic off-target effects of the increased plasma levels of intact SAS (or its metabolites) that are needed to allow inhibition of system xc-. As such, further research on
the development of new molecules that selectively act on system xc- is needed to allow using this strategy in a clinical setting.