Graded perturbations of metabolism in multiple regions of human brain in Alzheimer's disease: Snapshot of a pervasive metabolic disorder

Alzheimer's disease (AD) is an age-related neurodegenerative disorder that displays pathological characteristics including senile plaques and neurofibrillary tangles. Metabolic defects are also present in AD-brain: for example, signs of deficient cerebral glucose uptake may occur decades before onset of cognitive dysfunction and tissue damage. There have been few systematic studies of the metabolite content of AD human brain, possibly due to scarcity of high-quality brain tissue and/or lack of reliable experimental methodologies. Here we sought to: 1) elucidate the molecular basis of metabolic defects in human AD-brain; and 2) identify endogenous metabolites that might guide new approaches for therapeutic intervention, diagnosis or monitoring of AD. Brains were obtained from nine cases with confirmed clinical/neuropathological AD and nine controls matched for age, sex and post-mortem delay. Metabolite levels were measured in post-mortem tissue from seven regions: three that undergo severe neuronal damage (hippocampus, entorhinal cortex and middle-temporal gyrus); three less severely affected (cingulate gyrus, sensory cortex and motor cortex); and one (cerebellum) that is relatively spared. We report a total of 55 metabolites that were altered in at least one AD-brain region, with different regions showing alterations in between 16 and 33 metabolites. Overall, we detected prominent global alterations in metabolites from several pathways involved in glucose clearance/utilization, the urea cycle, and amino-acid metabolism. The finding that potentially toxigenic molecular perturbations are widespread throughout all brain regions including the cerebellum is consistent with a global brain disease process rather than a localized effect of AD on regional brain metabolism.

[18], and also determined the neuropathological severity by assigning 153 a Braak stage [16] to each brain (Supplementary Table 1).   icant change in abundance in AD-brain.

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This study compared results from cases and controls with compara-294 ble age, sex, and post-mortem delay between study-groups. Median 295 brain weight was~16% lower in AD: median (range) brain weight was  Table 1). PCA of GC-MS data revealed: 1) excellent class separation in 298 all brain regions between AD and control samples; 2) greater biological 299 than technical variation; and 3) absence of run-order effects (Fig. 2).

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One control sample (green in Fig. 2 Untargeted GC-MS analysis enabled us to categorize 69 metabolite 308 features per brain region (Table 2 and Supplementary Table 3

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Specific findings of this study identify metabolites from several key 320 biological pathways, including glucose utilization/clearance and brain 321 energetics, and urea and amino-acid metabolism (Table 2).

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To cope with its high energy demands, the brain is capable of 332 switching to alternative fuel sources, including butanediol, β-333 hydroxybutyrate and lactic acid. These three metabolites were also 334 significantly elevated in AD-brain, consistent with alternative fuel 335 use. We also found increased levels of certain sugars (threitol, xylitol, 336 and disaccharide not-otherwise-specified) and derivatives (N-337 acetylglucosamine, myo-inositol, and myo-inositol-1P). Glycerol 338 levels were decreased, whereas the phosphate derivative, glycerol-339 3P, was increased (Table 2). Increases in fuels other than carbohy-340 drates were less apparent in SCX and MCX. We found increased 341 levels of two TCA cycle intermediates, citric acid and malic acid, in 342 the heavily-affected regions of AD-brain. While the levels of urea 343 were dramatically elevated in all brain regions (Fig. 4), the urea 344 cycle metabolites ornithine and N-acetylglutamic acid were appar-345 ently decreased (Table 2).

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Amino acids comprised the largest group of metabolites identified 347 by this study to be altered in AD compared to control brain. There  naling molecules with regulatory functions in biological systems [19]. ing approach is also unprecedented in the field of AD research.

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Here, the close case-control matching and short post-mortem delays 378 have contributed to the quality of our data ( Table 1). The observed de-379 crease in brain weight in AD is generally consistent with histological se-380 verity [16]. The included cases all had 'classical' or 'usual' AD as  Table 1) that appeared as outliers in the PCA plots (Fig. 1).

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However, no legitimate reasons for exclusion of the corresponding  Defective energy metabolism is a core component of AD pathology 392 [25] and impaired brain-glucose uptake, known to manifest decades be-393 fore the onset of clinical symptoms of AD, is believed to lie at the centre 394 of this defect. AD-brains show impaired glucose uptake [26] and region-395 al impairment of cerebral perfusion [15], which are thought to be con-396 sistent with low brain-glucose levels and cerebral hypometabolism 397 being responsible for cognitive decline in AD [27].

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Here, by contrast, we found robust evidence for marked, pan-399 cerebral elevation of free glucose in the AD-brain (Table 2)  PCA-score biplots for seven brain regions showing class separations between control (blue) and AD (red) cases as demonstrated for each brain region. One control patient (green) had premanifest disease (Braak Stage II). Tight QC-clustering (crosses) in each brain region confirms low levels of technical variation throughout these measurements.    We conclude that the potential presence of urea cycle activity in brain 471 tissue seems not to have been systematically excluded to date and 472 that, accordingly, the potential exists that a functional urea cycle, capa-473 ble of generating urea from suitable substrates, could be operative in 474 brain tissue, for example in astrocytes. Further direct experimental evi-475 dence for the potential presence of the urea cycle in brain tissue and in 476 astrocytes, will need to be sought in future experiments.

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Here, one of the most striking changes observed in AD-brain was the 478 marked elevation of urea levels across all regions examined (Table 2, 479 Fig. 4). By contrast, systemic over-production of urea, leading to elevat-480 ed urea levels, for example, in the plasma, is not known to occur in AD.

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Urea is generally regarded as a detoxification product formed from am-482 monia/ammonium ion and/or amine-nitrogen moieties. However, urea 483 itself can also be toxic at sufficiently elevated levels, according to sys-484 tematic studies of the impact of elevated urea levels in cell-culture 485 and in vivo rodent models [37]. Our current findings are consistent 486 with impaired local urea regulation in brain in AD, by up-regulation of 487 its synthesis and/or defective clearance. 488 We hypothesize that defective urea metabolism could play a sub-