Here, to first demonstrate the effects of dietary Met supplementation on cognitive function in subacute aging mice, several behavioral experiments were conducted. Furthermore, plasma metabolite levels, redox and inflammatory states, and H2S- and GSH-generating pathways in the hippocampi were evaluated. Our findings indicate that appropriate dietary Met supplementation increased flux via the TSP, which may improve cognitive impairment in subacute aging mice.
Dietary Met supplementation improves cognitive dysfunction in subacute aging mice. Clinically, older patients with dementia frequently show a cluster of behavioral disturbances, including learning disorders, amnesia, anxiety, and neuropathic pain.39 Anxiety is a common problem of aging and is inextricably linked with cognitive disorders.40 The OFT and EPMT were used to assess the anxiety state of mice under different environmental stressors. In this study, under litter environmental pressure, there were no differences in the duration in the center during the OFT between the groups (Fig. 2), suggesting that the anxiety states of mice in the different groups were similar under mild environmental stress. Under greater environmental pressure, subacute aging mice exhibited anxiety-like behaviors (manifested as less access to the open arms), whereas dietary Met supplementation ameliorated the anxiety in these mice (Fig. 2). The different anxiolytic properties of the OFT and EPMT have been elucidated, and the test results may vary.41,42 Furthermore, the NORT, YMT, and MWMT were preformed to evaluate declarative memory, working memory, and spatial memory in mice, respectively. These tests necessitate interactions among various brain regions, especially between the hippocampi and prefrontal cortex. By analyzing the behavior of mice in the NORT and YMT, subacute aging mice exhibited impaired non-spatial declarative memory (manifested as significantly decreased DI values) and working memory (manifested as a significant decrease in spontaneous alternation rate), whereas dietary Met supplementation effectively improved these cognitive impairments (Fig. 3). Moreover, cognitive dysfunction manifested as longer latency period to find the hidden platform and decreased duration and distance spent moving around the platform after its removal, was detected during the MWMT in subacute aging mice. Conversely, dietary Met supplementation effectively improved spatial memory (Fig. 3). Dietary Met supplementation also increased the levels of a variety of cognitive-function-related metabolites in the plasma, including betaine, creatine, myo-inositol, choline, glutamine, acetate, propionate, and isobutyrate, all of which showed decreased levels in subacute aging mice. These metabolites play critical roles in maintaining intracellular osmotic pressure (betaine),43 energy metabolism (glutamine, creatine, acetate, propionate, and isobutyrate),44–46 and signal transduction between neurons (myo-inositol and choline) in the brain.47,48 Additional supplementation with these metabolites has shown beneficial effects on learning and memory function.43–48 Collectively, these findings demonstrate that dietary Met supplementation improved cognitive dysfunction in subacute aging mice.
Dietary Met supplementation, which reduces oxidative stress and inflammation, contributes to improvements in cognitive function. Oxidative stress and neuroinflammation are critical factors for the occurrence and development of aging and many types of neurological dysfunction.49 Based on the mechanism of aging, excess D-Gal can be metabolized to produce aldoses and hydrogen peroxide to stimulate ROS production to construct a model very similar to normal aging.50 Excess ROS production leads to the occurrence of oxidative stress-related stimuli, additionally causing an inflammatory response, mitochondrial damage, and apoptosis in the CNS, which are hallmarks of aging.51 In this study, dietary Met supplementation was found to significantly reduce ROS levels and enhance the resistance to oxidative stress (T-SOD and T-AOC) in the plasma and hippocampi (Fig. 6). Excess ROS or weakened resistance to oxidative stress leads to cellular redox disequilibrium, eventually promoting the production of harmful products, such as MDA and AOPPs in the hippocampi of subacute aging mice, whereas dietary Met supplementation effectively improved the redox disequilibrium in the hippocampi (Fig. 6). There is a mutually reinforcing relationship between the redox disequilibrium and inflammatory response. Redox disequilibrium activates inflammatory responses, which in turn increase the degree and duration of redox disequilibrium, forming a vicious circle.52 Moreover, the two interact with each other and are involved in aging and the development of related diseases, such as neurodegeneration.53 Nrf2 and NF-κB are referred to as the "master regulators" of antioxidant and inflammatory responses, and they can also modulate cognitive impairment and addiction.54,55 We found that dietary Met supplementation significantly decreased the expression levels of Nrf2 and NF-κB, increased the levels of downstream antioxidant enzymes HO-1 and NQO-1 in the hippocampi, normalized the levels of inflammatory cytokines in the plasma and hippocampi (IL-1β, IL-6, TNF-α, and IL-10), and further decreased the expression levels of apoptosis-related genes, including Caspase 3 and Bax (Figs. 7 and 8). Further insights into the effects of dietary Met supplementation on systemic oxidative stress and inflammatory responses can be obtained by analyzing blood metabolites. Allantoin, which is formed during the non-enzymatic oxidation of uric acid by ROS and nitrogen species,56 is a promising biomarker of systemic oxidative stress. Taurine is a product of Met metabolism that has several roles in protecting against oxidative stress as a fundamental regulator of homeostasis.57 N-Acetyl glycoprotein and O-Acetyl glycoprotein are typical inflammatory markers.58 In this study, dietary Met supplementation regulated plasma allantoin, taurine, N-Acetyl glycoprotein, and O-Acetyl glycoprotein levels, thus further supporting the notion that Met supplementation ameliorated oxidative stress and inflammation. Oxidative stress is usually accompanied by disturbances in energy metabolism, and normal energy metabolism is essential for energy-intensive brain functions including cognitive function.59,60 We found that dietary Met supplementation up-regulated the levels of citrate and fumarate, and down-regulated the levels of 3-hydroxybutyrate and pyruvate. These results indirectly support the idea that dietary Met supplementation ameliorates oxidative stress and inflammation. Taken together, these findings demonstrate that dietary Met supplementation abolished oxidative stress and inflammatory responses in subacute aging mice.
Reducing oxidative stress and inflammation by dietary Met supplementation alleviated cognitive decline by regulating the BDNF-TrkB pathway, improving neuronal signal transmission by regulating the N-methyl-D-aspartate receptor (NMDAR) and NMDA-transmitted Ca2+ influx, improving synaptic plasticity and synaptic structure, and reducing the accumulation of toxins such as Aβ (a characteristic feature of patients with AD). First, dietary Met supplementation significantly increased the expression levels of BDNF and its receptor TrkB in the hippocampi (Fig. 8). BDNF is a widely characterized neurotrophin in mammals, mainly signaling through the TrkB receptor, It plays a critical role in the growth, development, differentiation, synaptic connectivity, and post-injury regenerative repair and protection (especially in the prefrontal and hippocampal areas, which are related to cognitive function) of neurons.61 BDNF levels and BDNF-mediated signaling pathways are particularly adversely affected by oxidative stress and inflammation.62 Second, NMDAR is one of the important targets for BDNF modulation,63 and hippocampal long-term potentiation (LTP) requires the activation of postsynaptic Ca2+ channels and NMDAR,64 constituting cellular substrates of learning and memory.65 A previous study demonstrated that a redox imbalance influences NMDAR activity in a way corresponding to age-related cognitive impairment.66 Aging changes the intracellular redox state into a state of oxidative stress, leading to a reduction in NMDAR activity via the redox regulation of CaMKII, and GSH can rescue this effect.66 We found that dietary Met supplementation significantly increased the expression levels of Nr2b and the CaMKII subunits, including CaMK2A, CaMK2B, and CaMK2D (Fig. 8). Meanwhile, dietary Met supplementation significantly up-regulated RC3, Gap-43, PSD-95, and SYNAPO in the hippocampi (Fig. 8). These genes encode synaptic-plasticity-related proteins (neurogranin and neuromodulin) and neurotransmission-regulating synaptic proteins (postsynaptic density protein 95 and synaptopodin), which are involved in learning and memory functions and are down-regulated in response to oxidative stress and inflammation.67,68 Finally, dietary Met supplementation reduced the levels of aging-related toxins, including AGEs, lipofuscin, and Aβ-40, in the hippocampi of subacute aging mice (Fig. 8). Excess AGEs and lipofuscin are produced in the aging brain,69,70 causing the aggregation of proteins, the formation of Aβ, and subsequent cognitive impairment.71–73 Thus, dietary Met supplementation alleviated aging-related cognitive decline, which was attributed to enhanced BDNF-TrkB signaling and neuronal signal transmission mediated by the NMDAR pathway, as well as the reduced accumulation of toxins.
Dietary Met supplementation potentially induces the abovementioned beneficial effects by increasing flux via the TSP. Met primarily participates in protein synthesis and a variety of metabolic pathways such as the TSP, Met cycle, and salvage cycle. The TSP is increasingly recognized as a "master regulator" of redox balance and integrates stress responses via the generation of several sulfur-containing metabolites, such as H2S and GSH.8 H2S is capable of modulating multiple physiological processes, including responses to inflammatory stimuli, oxidative stress, and neuronal signaling pathways.8 In the hippocampi, H2S selectively enhances NMDAR-mediated signaling, facilitates the induction of LTP,74 and regulates Ca2+ in all important brain cell types including neurons. Thus H2S plays important roles in the communication between neurons and glia cells and in regulating synaptic plasticity.75 Furthermore, H2S supplementation has been confirmed to effectively prevent cognitive dysfunction in several models of cognitive deficiency.76–80 In this study, dietary Met supplementation significantly increased H2S levels in the hippocampi and plasma (Figs. 9 and 10). Additionally, CBS and CSE are key enzymes regulating H2S production in the brain through the TSP, and they have been used as important targets to stimulate the TSP, thus reducing oxidative stress and inflammation, and improving brain function.8,81 The absence of CBS and CSE can result in oxidative stress, aberrant stress responses, and cognitive impairment.82–84 In this study, dietary Met supplementation increased the expression levels of CBS and CSE (Figs. 9 and 10), suggesting that the increase in H2S levels was attributable to the increased levels of key H2S-producing enzymes. Just as striking as the increase in H2S levels is the increased levels of GSH as well as GS in the hippocampi after dietary Met supplementation. GSH is one of the most important antioxidants in the brain.85 The redox state of the brain, largely controlled by GSH, regulates NMDAR activity, and GSH is highly relevant to aging and aging-induced learning and memory dysfunction.66 A deficiency in GSH during brain development can lead to cognitive deficiencies and schizophrenia-like behavior.86–89 In addition, dietary Met supplementation led to a decreased SAM/SAH ratio in the TSP (Figs. 9 and 10), which is otherwise increased in AD mice.90 Moreover, MTHFR, MS, and BHMT are rate-limiting enzymes for the remethylation of Hcy to synthesize Met. In this study, dietary Met supplementation significantly increased the expression levels of MTHFR and BHMT and decreased the expression level of MS in the hippocampi (Figs. 9 and 10), implying that dietary Met supplementation activates the Met cycle, thus reducing excessive Hcy levels. Although Hcy levels in the plasma and hippocampi were higher in the D + MS group than in the ND group, increased flux via the TSP may balance the adverse effects of Hcy. Of note, a previous study showed that H2S supplementation inhibits glial activation and inflammatory responses induced by Hcy.91 Amino acids are substrates for the synthesis of proteins and are essential for the renewal and repair of brain tissue.92,93 Dietary Met supplementation increased the levels of multiple amino acids, including Met, tyrosine, isoleucine, glycine, threonine, and phenylalanine, and decreased the level of urea, the end-product of amino acid metabolism (urea) (Fig. 4). Increased utilization of Met by the body may result in the conservation of other amino acids, which, could then facilitate the brain damage repair and improve cognitive function. For example, glycine is a substrate for GSH synthesis.94 Taking these findings together, the dietary Met supplementation-mediated prevention of cognitive dysfunction potentially occurs by increasing flux via the TSP.