In this work, we aimed to characterise the spatio-temporal metabolic profile changes during the onset of AD-like pathology in the TgF344-AD rat brain. Longitudinal MRS studies were performed in the same cohort of WT and TgF344-AD animals. Four time points, from 9 to 18 months, were evaluated to capture the onset of metabolic changes: three points before the manifestation of gross pathological changes, as described in this model [27]; and another one later on. To account for specific regional differences in the progression of the disease, MRS data was acquired from four voxel positions, whose accurate location was evaluated based on automatic segmentation of structural MRI.
Based on a strict quality control and processing pipeline, the longitudinal metabolic profiles demonstrated progressive changes in TgF344-AD rats compared to WT, consistent with the major findings reported in clinical AD, such as decreased N-acetylaspartate and glutamate. Specifically, decreased NAA and/or tNAA in the cortex, hippocampus and thalamus, and decreased Glu in the thalamus and striatum were detected in TgF344-AD compared to WT animals, further confirming the suitability of this model for translational studies on AD progression. Additional analysis revealed strong intra-regional couplings of cortical NAA, tNAA and tCr with myo-inositol (either Ins or Ins+Gly) in WT, but with Tau in transgenics rats; whereas in the hippocampus, Tau showed strong correlations with Ins+Gly and tCho only in the WT group. Moreover, divergent inter-regional metabolic couplings in TgF344-AD and WT animals were observed. Specific crosstalks between cortex, hippocampus and striatum were found for Tau in the WT group and for myo-inositol in the TgF344-AD rats. This analysis sheds light into the complex spatio-temporal metabolic rewiring in AD. The different associations of Ins and Tau with other metabolites in WT and TgF344-AD rats, suggest a relevant role for these two metabolites during the course of the pathology.
Importantly, the age-dependent decrease of cortical and thalamic creatine and its strong correlation with metabolites such as NAA or myo-inositol, reveals its inadequacy as an internal reference for relative quantification methods routinely used in the clinic (e.g. Ins/Cr), highlighting the need for careful interpretation of such results in the context of AD.
Objective quality control was used to assure the reliability of the absolute metabolite concentrations estimated from each MRS dataset, including spectral selection based on SNR and FWHM, and fitting performance based on Cramér-Rao Lower Bounds assessment. Moreover, the study groups were assessed for potential outliers (based on interquartile range), controlled physiological condition throughout the acquisition (breathing and rectal temperature), and similar percentage of the region of interest covered by the MRS voxel. The slightly lower rectal and brain-estimated temperatures noticed in the cortex and thalamus of the transgenic group are consistent with previous findings in AD patients suggesting limited thermoregulation functions which have been associated with reduced number of suprachiasmatic nucleus neurotensin neurons [42,43]. Importantly, these differences, although significant, were not associated with the metabolic changes detected.
Temporal metabolic profile changes in the TgF344-AD rat brain
Metabolic profile analysis showed different longitudinal evolutions of NAA and/or tNAA in the cortex, hippocampus and thalamus of WT and TgF344-AD rats. Specifically, these metabolites decreased during ageing only in transgenic animals. NAA is a marker of neuronal density and its decrease has been associated with neuronal loss or dysfunction [4]. Consistently, decreased NAA is one of the most reproducible findings in the cortex and hippocampus of AD patients [4], as well as in rat [28,32] and mouse models [15]. Progressive neuronal damage has been described in the TgF344-AD model [27], aligned with decreased NAA also reported in the hippocampus and thalamus [32]. Since NAA/Cr has also been negatively correlated with Aβ burden [5,20], decreased NAA at the latest time points in TgF344-AD animals could be also associated with the presence of Aβ plaques, observed at 16 months of age [27].
A decrease of Glu was observed in the thalamus of TgF344-AD rats during ageing, not observed in WT, while, in the striatum, TgF344-AD showed stable lower levels of Glu than WT rats over the study. Glu is one of the main neurotransmitters in the brain, essential for dendrite and synapse formation. Lower Glu concentration has been associated with neuronal loss and correlated with poor cognitive outcome [44]. Therefore, the observed changes are consistent with the neuronal loss described in the transgenic model [27]. Indeed, we observed significantly lower Glu levels at 15 months of age in TgF344-AD with respect to WT animals, when clear cognitive impairment has been described [27]. In the same line, the early lower levels of striatal Glu observed in transgenic rats could be related to the early learning deficits previously described in these rats [38], as Glu neurotransmission is crucial for learning and memory function [45]. In mouse models of AD, decreased Glu or Glu/Cr was found in the cortex and hippocampus [13,18,46,47], correlating with Aβ plaque deposition in mouse models [20]. In human cohorts, decreased cortical levels of Glx or Glu have been reported in AD, MCI and at-risk populations [4,44], as well as non-significant decreases of Glx/Cr in the striatum and thalamus of AD patients [48].
The time-course profiles of cortical Cr and thalamic tCr also showed an age-dependent decrease in TgF344-AD animals, not observed in the WT group. In the thalamus, significant lower tCr concentration was observed in transgenic with respect to WT animals at 12, 15 and 18 months of age. These changes might be associated with cell energetics disturbances occurring in neurodegenerative pathologies [49], consistent with a neuroprotective effect of Cr against Aβ toxicity reported in the McGill-R-Thy1-APP rat model of AD [50]. Previous studies in AD or MCI patients reporting lower tCr levels in areas such as the hippocampus [51,52] support our findings. In the same line, ex-vivo analysis revealed lower tCr levels in the hippocampus and frontal cortex in a transgenic mouse model of AD [53]. However, increased tCr levels were reported in the cortex of McGill-R-Thy1 rats [28] and in the hippocampus and thalamic regions of TASTPM mice [21]. These discrepancies may be related to the particular features mimicked by each model and the different time window selected in each study. Importantly, these results warn that metabolite to tCr ratios should be analysed with caution, since tCr is not a constant reference in AD and therefore it can lead to conflicting conclusions.
Regarding myo-inositol, a metabolite commonly related to human AD [4], the MRS literature typically quantify it as mixed Ins+Gly levels, since the metabolic profiles of both metabolites significantly overlap. Thus, Ins and Gly were both included in our basis set for their individual quantification. Although Gly was not further evaluated after quality control assessment, it improved the estimations of Ins+Gly compared to Ins alone, as expected. Accordingly, we detected significant differences between the age-dependent trajectories of Ins+Gly levels in the thalamus and striatum of TgF344-AD and WT animals, characterized by their stronger increase with ageing in WT rats. Although myo-inositol has been considered a glial marker, with increased levels suggesting gliosis, its role in neuroinflammation or glial activation remains to be discerned [16,54]. In this line, while the TgF344-AD model presents early gliosis, detected at 6 months of age [27], we and others have not detected increased Ins in this model compared to WT [32]. Notwithstanding, striatal and thalamic median Ins+Gly levels tended to be higher in transgenic than in control animals at early stages, but lower at later ages (Figure 3). These observations strengthen the importance of accounting for the timing of the disease when comparing literature findings, which might explain some of the discrepancies reported in human cohorts and animal models. For instance, even though Ins increases have been commonly reported in AD patients [4], significant decreases of Ins/Cr have been also reported in the striatum, hippocampus and other regions in AD or dementia with Lewy’s body, compared to healthy controls [48]. In the APP/PS1 mouse, both decreased Ins/Cr [15] and increased Ins/Cr were reported in the hippocampus [13,46], while no changes were found in Ins absolute concentration in the cortex [18]. In the McGill-R-Thy1 rat model of AD, Ins decreased at 3 months of age in the hippocampus, while increased at 9 months of age in the hippocampus and 12 months of age in the frontal cortex [28].
With regards to Tau, our results showed significant age-dependent decreases in the cortex of TgF344-AD rats, not observed in the WT group. Indeed, significant lower Tau concentrations were detected in the transgenic group with respect to WT were detected at 15 and 18 months. These results are in line with previous findings including decreased Tau/Cr ratio in AD patients [55] or aged animal models [56]. However, a recent study with the TgF344-AD model depicted increased cortical Tau levels at 18 months of age [32]. This discrepancy may be related to differences in the MRS acquisition protocol (such as its ~70% longer echo time, leading to mixed J-coupling and T2 saturation effects, reflected in the detection and quantification of Tau), or different voxel location in the cortex, which in our study was more frontal. In this line, it has been shown that cortical amyloid deposition starts in frontal and temporal areas, affecting medial and posterior cingulate cortex only later on [57]. In fact, our results suggest a trend towards increased Tau levels in the cortex of transgenic animals before 12 months, but decreased levels at later time points, compared to controls. Thus, the differences could also be associated with regional specific timings of the pathological changes. Tau plays modulatory and regulatory roles in different physiological processes. It has been reported as a neuroprotector in neurodegenerative diseases such as AD and used to ameliorate several neurological disorders [58]. Indeed, Tau supplementation improved cognition in the APP/PS1 mouse model of AD [59] and enhanced adult neurogenesis under both in vitro and in vivo conditions [60].
Finally, cortical tCho revealed different age-dependent evolution in TgF344-AD rats compared to WT, showing (non-significant) increased level at earlier stages which decreased later on. The genotype had a significant effect in the hippocampus with increased tCho values in theTgF344-AD rats, although both groups followed a similar trajectory. Accordingly, tCho age-effect differences between TgF344-AD and control rats in the hypothalamus and hippocampus were also reported in [32]. Choline compounds are found in myelin sheets and cell membranes and variations in tCho have been related to white matter integrity and membrane turnover, although opposite findings have been reported in the cortex of AD subjects [4]. Indeed, decreased tCho/Cr was detected in the prefrontal cortex and thalamus [48], while increased levels were reported in the posterior cingulate cortex in the posterior cingulate cortex [61]. Moreover, increased tCho has been reported with ageing in both humans and animal models [54,62,63], associated with demyelination, inflammation, and functional changes including cognitive decline [64]. As for myo-inositol, higher cortical tCho in young transgenic animals might suggest early damage, being demyelination and inflammation processes less evident at more advanced ages. On the other hand, decreased tCho at later time points could also be associated with neuronal damage and subsequent cognitive impairment, as described in aged TgF344AD rats [27]; also aligned with decreased hippocampal tCho observed in a drug-induced memory deficit model [65]. This strengthens again the importance of accounting for the timing of the disease in metabolic profile investigations.
Intra-regional correlations of brain metabolic profiles
We further investigated the associations between different metabolites in each brain regions, from a systems biology perspective. Thus, we found different metabolic correlation patterns in transgenic and wild-type groups.
As expected, there were significant correlations between NAA and tNAA, Glu and Glx and Ins and Ins+Gly, regardless of the brain region and group; except for Ins and Ins+Gly in the thalamus. The latter could be related to the significant age effect observed in this region for Ins+Gly, but undetected in Ins. Although Gly could not be evaluated independently in our study, its neuroprotective properties in neurodegeneration and memory impairment [66], merit further investigation with more sensitive technique in the context of AD.
The strong correlations between creatine with other metabolites altered in TgF344-AD rats represent one of the main findings of this study. In the cortex, tCr was highly correlated with NAA and tNAA in both TgF344-AD and WT groups, whereas correlations with Glu and Glx were specific to TgF344-AD, and with Ins+Gly and Ins were only found in WT animals. In the hippocampus, tCr correlated strongly with NAA in both groups; but with Ins, Ins+Gly and Glx only in TgF344-AD rats, and with Glu only in WT animals. Interestingly, in the thalamus and hippocampus, tCr only correlated with tNAA in the transgenic group. Altogether, these results suggest an important role for Cr in AD-like processes, since it was highly correlated with metabolites showing pathology specific time-course changes. This supports the idea that the commonly considered metabolic ratios to tCr, such as decreased NAA/tCr and increased Ins/tCr [4,53], might not be appropriate in the case of AD-like pathologies, given the potential confounding effect of altered tCr levels.
Moreover, the correlations between taurine and myo-inositol with other metabolites also differed between TgF344-AD and WT animals. Several studies have suggested an important role of either Tau or Ins in AD pathology [4,15,18,28,46,48,55], highlighting the need to better understand their changes and apparent discrepancies reported in the literature. Beyond concentration differences in specific regions, intra-regional metabolite correlation analysis provided new insights into this issue. Thus, cortical levels of Tau in TgF344-AD rats were highly correlated with NAA, tNAA and tCr, suggesting a link between Tau decreases and neuronal dysfunction; while in WT animals, cortical NAA, tNAA and Cr correlated with Ins+Gly and Ins. Due to the minimal sample size criteria defined, Tau concentration was not evaluated in the thalamus and, therefore, no correlations could be investigated in this case. Such metabolic correlation patterns could reflect compensatory effects between neuroinflammation (Ins) [4,54] and neurotrophic (Tau) [58–60] processes associated with normal ageing, potentially mitigated in transgenic animals. In TgF344-AD, impaired neurotrophic processes could be related to neuronal dysfunction, manifested by decreased NAA.
Further correlations were found in the hippocampus of WT animals, between myo-inositol and NAA and tNAA and between Tau and both Ins+Gly and tCho. While the metabolic interplay between Ins, tCho and Tau remains to be unravelled, these results suggest a role for Tau in the normal ageing process, similar to Ins and tCho in inflammation and degeneration during normal ageing, as described in humans and animal models [62–64]. Tau has a modulatory and regulatory function in physiological processes and its neuroprotective role has been reported in neurodegenerative diseases [58]. Therefore, Tau changes in transgenic animals leading to loss of its correlation with Ins and tCho, might reflect an underlying dysfunction diverging from the normal ageing trajectory.
Correlations between Glu (or Glx) and NAA, tNAA and/or tCr were detected in the cortex and hippocampus, as well as between Glx and NAA in the striatum were observed only WT rats. In this region, TgF344-AD rats showed lower levels of Glu compared to WT, potentially reflecting Gln-Glu cycle changes, associated with the neuronal loss described in this model [27].
Inter-regional correlations of brain metabolic profiles
Finally, we investigated the correlation between metabolite concentration across different brain regions. In WT animals, Tau levels were strongly correlated between the cortex, striatum and hippocampus, and with Ins+Gly in the hippocampus. In turn, TgF344-AD rats exhibited the same inter-regional metabolic crosstalk between cortex, striatum and hippocampus for Ins, instead of Tau. This could be related to the specific correlations of cortical NAA (and tNAA) with Tau found in transgenic animals, but with Ins (or Ins+Glyc) in the cortex and hippocampus of WT animals. Such metabolic couplings could reflect a whole brain imbalance in Tau levels associated with region-specific onsets of neuronal dysfunction [27,57]. Altogether, this would support the hypothesis of a balance between neuroinflammation (Ins) and neurotrophic (Tau) processes during normal ageing impaired in TgF344-AD rats.
Interestingly, while no significant group or interaction effects were found for GSH or Gln, highly significant correlation of striatal GSH with cortical NAA in the WT group and with Gln in transgenic animals were observed. GSH is the most prevalent anti-oxidant in the brain [54,67] and decreased levels have been reported in MCI and AD patients [67] and in the APPTg2576 model of AD [18]. Assuming that the GSH cycle compensates for decreased excitatory neurotransmission during Glu-Gln shuttle inhibition [68], the correlation between Gln and GSH in TgF4344-AD rats could be related to alterations in the normal Glu-Gln cycle, consistent with the decreased Glu levels observed in this group.