Our results provide support for widespread altered metabolomics in AD conditions, associating metabolomic shifts across brain tissue and blood plasma.
4.1 Alterations in glucose metabolism
Under healthy conditions, glucose is the sole source of energy for the brain. Abnormalities in its metabolism are key indicators of declining brain health, contributing to neuroinflammation, neurofibrillary plaques and tangles, oxidative stress, and malfunctions in other related systems [13].
Normally, lactic acid is produced during glycolysis via pyruvate reduction; however, abnormal mitochondrial function and/or lactic acid clearance can disrupt lactic acid homeostasis, thereby affecting physiological brain functions [14–17]. Numerous studies report a prominent role for lactate in regulating brain function, providing energy fuel for neurons, and cellular signaling [16–20]. However, alterations in lactate homeostasis can result in neurological dysfunction and further the development of neuropsychiatric disorders [17–19, 21]. We detected significantly increased levels of lactic acid in both cortex and hippocampus and non-significant increases in blood plasma, consistent with prior findings [17, 22]. Increased lactate secretion by astrocytes was shown to be present in neurodegenerative diseases, favoring Aβ plaque deposition, while increased lactate uptake by neurons may cause reduced mitochondrial activity, cytoplasmic acidification, and neuronal death [17, 23]. Furthermore, reduced activity of mitochondrial pyruvate carriers has been reported in neurodegenerative states, leading to excess cytoplasmic pyruvate and aberrant anaerobic glycolysis, thus aggravating lactate accumulation and neuroinflammation [24]. We observed increases in pyruvic acid across all tissues and biofluids, corroborating these results.
Glucose-6-phosphate was decreased in plasma, but no significant differences were observed in brain tissue. This suggests its conversion to pyruvate via glycolysis but may also indicate downregulation of the pentose phosphate pathway, which generates metabolites that alleviate redox stress for neuronal protection [25, 26]
Levels of trans-aconitic acid, a Krebs cycle metabolite, were increased in cortex and hippocampal tissue but decreased in blood plasma. Similarly, cis-aconitic acid, an intermediate metabolite in the isomerization from citrate to isocitrate, was seen to decrease in plasma, which aligns with our observed increase in citrate levels in plasma. In a previous study of Parkinson’s disease, decreases in cis-aconitic acid were noted in patient blood samples, alluding to its role in neurodegeneration through downregulation of Krebs Cycle [25]. As a competitive inhibitor for aconitase (a Krebs Cycle enzyme), increased trans-aconitic acid in brain tissue may suggest further inhibition of the Krebs Cycle at these sites. On the other hand, decreased citrate levels in the hippocampus may indicate its conversion to pyruvic acid (increasing across all samples) and reduced anti-inflammatory activity in the brain [27].
4.2 Oxidative stress leading to brain dysfunction
Abnormal energy metabolism, particularly via mitochondrial dysfunction, has long been associated with AD pathophysiology through oxidative stress in neurodegeneration and tau hyperphosphorylation [28, 29]. For instance, galactitol, a reduced product of galactose, has been documented to accumulate in neurons and generate free radicals by impacting the electron transport chain, resulting in oxidative stress and mitochondrial dysfunction [30–33]. Here, a decrease in galactitol in the cortex (significant) and hippocampus (non-significant) was observed. Decreased levels of galactitol may result from its oxidation into aldehyde and hydrogen peroxide [34], further exacerbating oxidative stress in cells. Interestingly, Tsakiris et al. found that supplementation of sulfhydryl-containing antioxidants, such as L-cysteine and glutathione, reverses the oxidative stress impact of galactitol by binding to free radicals [32]. Our findings of significant decreases in cortex L-cysteine are corroborated by previous studies noting its 3-fold decrease in AD versus healthy rodent brains [31].
Moreover, we found an increase in propionate (which has been associated with an increased risk of cognitive decline in human subjects [35]) in both brain tissue and blood plasma, complementing results from prior studies using saliva samples [36]. MMA and propionic acidemia, characterized by increased concentrations of free MMA and propionate, can hinder mitochondrial function by disrupting Krebs Cycle and electron transport chain, producing reactive oxygen species (ROS) that lead to neurodegeneration and AD-related pathophysiology [37–39].
Finally, we observed significantly decreased betaine (trimethylglycine) levels in plasma. Betaine was demonstrated to have a positive impact in alleviating neurodegenerative cognitive deficits, attenuating tau phosphorylation, and decreasing Aβ accumulation [40]. By donating methyl groups, betaine can convert homocysteine to methionine, a potential antioxidant [41]. Decreased betaine could explain AD pathological alterations; however, the observed increasing trends in brain tissues (non-significant) conflict with previously reported results [42, 43]. In our study, increased betaine may be in response to oxidative stress; however, further testing is required to verify this finding and elucidate its potential neuroprotective properties [44].
4.3 Interactions with other neural pathways
AD pathophysiology is highly complex, integrating diverse metabolic mechanisms, thereby impacting the neurochemistry of learning and memory. Taurine, a well-researched metabolite in AD, is known for its neuroprotective effects [45], ameliorating the behavioral deficiencies of AD [46]. However, proposed mechanisms-of-action have thus far been inconsistent and contradictory. Though taurine has been reported to directly bind to oligomeric Aβ [44], others propose it effects glutamatergic or GABAergic transmission systems (with no impact on Aβ plaque concentrations/formations) to potentially decrease neuronal vulnerability to excitotoxic stress [46, 47]. We found significantly decreased levels of taurine in the cortex and plasma of AD mice, which is consistent with previous data in human AD patients [45]. This may also explain observed increases in hypotaurine, which can be converted to taurine by hypotaurine dehydrogenase, in the same regions, in response to oxidative stress [48]. Contrastingly, the observed upregulation of taurine (non-significant) in the hippocampus may be a response to early AD pathological changes. Furthermore, increased GABA was also observed in the hippocampus, supporting the hypothesis that taurine activates GABA receptors. GABA dysregulation in AD studies have thus far provided contradictory results; while it was previously thought to contribute to neurodegeneration and cognitive decline, recently, research postulates that GABA may alleviate AD pathology, cognitive impairment, and regulate neurogenesis [49, 50].
Moreover, we observed decreased dimethylamine (DMA) in brain tissues. DMA is a breakdown product of asymmetric dimethylarginine (ADMA), an inhibitor of nitic oxide (NO) synthase, via dimethylarginine dimethylaminohydrolase (DDAH). Due to increasing oxidative stress during AD, DDAH activity is inhibited [51, 52], decreasing production of DMA. Interestingly, NO can play a dual role, acting as both a neurotoxin that exacerbates AD and as an unconventional neurotransmitter with protective functions, inducing learning, memory, and neuroplasticity. As such, regulating NO metabolism through DDAH activity may be a potential target for treating AD [53–55].
4.4 Correlating blood plasma and brain tissue metabolites
By observing the metabolic flux in tissue and blood plasma samples, our goal is to investigate the potential relationship between different sample types for early AD detection and prognosis. Several previously discussed metabolites displayed the same trends across all sample types (lactic acid, pyruvic acid, and propionate), and were hypothesized to contribute to AD through glucose oxidation and/or mitochondrial dysfunction. Other metabolites, while not directly related to energy metabolism, also display the same trends across all tissues, namely, methanol and 3-hydroxyisovaleric acid (3-HIVA).
Increased levels of methanol were observed across all samples. Methanol is known to cause neuronal tau phosphorylation and contributes to the deposition of Aβ protein in the hippocampus [56]. Furthermore, its oxidation metabolites, formaldehyde and formic acid, have been shown to cause similar effects in vitro [57, 58]. On the other hand, 3-HIVA levels were decreased in all samples. Previous studies report contradictory results regarding the presence of 3-HIVA in AD [27, 59]. Decreased levels of 3-HIVA, a byproduct of leucine metabolism, may result from reduced leucine levels (plasma, not significant), correlating to severe cognitive decline [60].
However, further research is clearly needed to elucidate the patterns between tissue and blood metabolites, their interrelations, distinctions, and underlying mechanisms. Potential avenues include multidimensional NMR to reduce spectral overlap typically observed in 1D spectroscopy. Future work can also incorporate complementary analytical techniques, such as mass spectrometry to improve the detection of metabolites in low concentrations. We are also interested in investigating the blood and brain metabolomics of mice with other neurological deficits, such as mild cognitive impairment (MCI), to identify potential overlaps between MCI and AD conditions.
In this investigation we also identified two metabolites, syringic acid and 7-methylxanthine, commonly found in food and plants. Their presence warrants further investigation into the diet and handling of the mice, while exploring potential interactions between diet and neuropathy. Moreover, a greater sample size can increase the statistical strength of our findings. By including male mice in the WT condition and a range of ages, we could examine gender and age-sensitive correlations to AD.
In conclusion, we demonstrate that HRMAS NMR can be used to differentiate between AD and WT from blood plasma and brain tissue in AD mouse models, where measured metabolic profiles vary for cortex, hippocampus, and plasma. We report significant alterations in metabolites associated with glucose and energy metabolism, oxidative stress and those interacting with neural pathways. These findings have potential to be implemented into in vivo mice evaluations, leading to new, non-invasive diagnostic methods for AD. The potential of this work can enable clinicians to monitor the effects of prospective future therapies and progress our current understanding of AD metabolic mechanisms.