Functional Metabolic Mapping Reveals Highly Active Branched-Chain Amino Acid Metabolism in Human Astrocytes, Which Is Impaired in iPSC-Derived Astrocytes in Alzheimer's Disease

Abstract

The branched-chain amino acids (BCAAs) leucine, isoleucine, and valine are important nitrogen donors for synthesis of glutamate, the main excitatory neurotransmitter in the brain. The glutamate carbon skeleton originates from the tricarboxylic acid (TCA) cycle intermediate α-ketoglutarate, while the amino group is derived from nitrogen donors such as the BCAAs. Disturbances in neurotransmitter homeostasis, mainly of glutamate, are strongly implicated in the pathophysiology of Alzheimer's disease (AD). The divergent BCAA metabolism in different cell types of the human brain is poorly understood, and so is the involvement of astrocytic and neuronal BCAA metabolism in AD. The goal of this study is to provide the first functional characterization of BCAA metabolism in human brain tissue and to investigate BCAA metabolism in AD pathophysiology using astrocytes and neurons derived from human-induced pluripotent stem cells (hiPSCs). Mapping of BCAA metabolism was performed using mass spectrometry and enriched [¹⁵N] and [¹³C] isotopes of leucine, isoleucine, and valine in acutely isolated slices of surgically resected cerebral cortical tissue from human brain and in hiPSC-derived brain cells carrying mutations in either amyloid precursor protein (APP) or presenilin-1 (PSEN-1). We revealed that both human astrocytes of acutely isolated cerebral cortical slices and hiPSC-derived astrocytes were capable of oxidatively metabolizing the carbon skeleton of BCAAs, particularly to support glutamine synthesis. Interestingly, hiPSC-derived astrocytes with APP and PSEN-1 mutations exhibited decreased amino acid synthesis of glutamate, glutamine, and aspartate derived from leucine metabolism. These results clearly demonstrate that there is an active BCAA metabolism in human astrocytes, and that leucine metabolism is selectively impaired in astrocytes derived from the hiPSC models of AD. This impairment in astrocytic BCAA metabolism may contribute to neurotransmitter and energetic imbalances in the AD brain.

Keywords: AD; BCAA; astrocytes; energy metabolism; glutamate; glutamine; induced pluripotent stem cell; neuron.

Figure 1

Primary ¹³C-labeling patterns from [U-¹³C]branched-chain amino acid (BCAA) metabolism. (A) Metabolism of [U-¹³C]leucine and [U-¹³C]isoleucine (M+6) will result in acetyl coenzyme A (CoA), a process initiated by branched-chain aminotransferase (BCAT) catalyzing BCAA transamination and hereby producing branched-chain α-keto acids (BCKAs), α-ketoisocaproate (KIC), and α-keto-β-methylvalerate (KMV), respectively. Subsequently, irreversible oxidative decarboxylation of the BCKAs occurs by the branched-chain α-keto acid dehydrogenase (BCKDH) enzyme complex and through multiple enzymatic reactions entering the TCA cycle as double-labeled acetyl CoA (M+2). Filled circles (orange) represent labeled ¹³C and empty circles unlabeled ¹²C. (B) Valine and isoleucine can also enter into the TCA cycle via succinyl CoA. [U-¹³C]valine (M+5) and [U-¹³C]isoleucine (M+6) will be transaminated, resulting in BCKAs, α-ketoisovaleric (KIV), and KMV, respectively, and then decarboxylated, entering TCA cycle metabolism, after numerous reactions, as succinate (M+3). Filled circles (yellow) represent labeled ¹³C and empty circles unlabeled ¹²C. BCATs, branched-chain aminotransferase; BCKDH, branched-chain α-keto acid dehydrogenase; α-KG, α-ketoglutarate; KIC, α-ketoisocaproate; KMV, α-keto-β-methylvalerate; KIV, α-ketoisovaleric; β-MC CoA, β-methylcrotonyl CoA; MGC CoA, β-methylglutaconyl CoA; HIB CoA, β-hydroxyisobutyryl CoA; MHB CoA, α-methyl-β-hydroxyisobutyryl CoA; HMG CoA, β-hydroxy-β-methylglutaryl CoA; MAA CoA, α-methylacetoacetyl CoA; HIB, β-hydroxyisobutyrate; MMSA, methylmalonate semialdehyde; MAA CoA, α-methylacetoacetyl CoA.

Figure 2

BCAA nitrogen metabolism of human and mouse brain slices. After incubation with [¹⁵N]BCAA (valine, leucine, or isoleucine), M+1 labeling was observed in glutamate, glutamine, gamma-aminobutyric acid (GABA), alanine, and aspartate, indicating significant nitrogen incorporation into amino acids supported by BCAA metabolism. ¹⁵N-enrichment in intracellular amino acids was determined by gas chromatography–mass spectrometry (GC-MS). Values represent mean (±) standard error of the mean (SEM) (n = 6), ^*p < 0.05, ^**p < 0.01 (when compared with human cortical slices) analyzed by Student's paired t-test. BCAA, branched-chain amino acid; BCKA, branched-chain α-keto acids; BCATs, branched-chain aminotransferase; GAD, glutamate decarboxylase; GDH, glutamate dehydrogenase; ALAT, alanine aminotransferase; AAT, aspartate aminotransferase; GS, glutamine synthetase.

Figure 3

BCAA oxidative metabolism in human and mouse brain slices. ¹³C-enrichment of tricarboxylic acid (TCA) cycle metabolites and amino acids in human and mouse cerebral cortical slices after incubation with [U-¹³C]isoleucine, [U-¹³C]leucine, or [U-¹³C]valine. M+2 and M+3 ¹³C-enrichment in (A) TCA cycle intermediates. Increased M+2 ¹³C-enrichment in (B) glutamate, glutamine, and GABA after incubation with [U-¹³C]leucine in mouse cortical slices when compared with human cortical slices. ¹³C-enrichment in intracellular metabolites was determined by GC-MS. Values represent mean (±) SEM (n = 6), ^*p < 0.05 analyzed by two-way ANOVA. N/D, not detectable.

Figure 4

BCAA oxidative metabolism in human induced pluripotent stem cell (hiPSC)-derived astrocytes. hiPSC-derived astrocytes were incubated with [U-¹³C]isoleucine, [U-¹³C]leucine, or [U-¹³C]valine. Labeling in TCA cycle intermediates and amino acids represented as molecular carbon labeling (MCL) confirms hiPSC-derived astrocytes capability to oxidatively metabolize the BCAA isoleucine, leucine, and, to a lesser extent, valine by incorporating them in the TCA cycle. Values represent mean ± SEM (n = 3) from different hiPSC-derived astrocyte set-ups individually depicted as circles.

Figure 5

Reduced oxidative BCAA metabolism entering as acetyl CoA in Alzheimer's disease (AD) astrocytes. (A) Increased glutamate and citrate M+2 labeling but decreased aspartate and malate ¹³C-enrichment after incubation with [U-¹³C]leucine and (B) [U-¹³C]isoleucine in human AD astrocytes when entering via acetyl CoA. (C) Increased TCA cycle metabolites M+3 labeling after incubation with [U-¹³C]valine but (D) decreased aspartate ¹³C-enrichment after incubation with [U-¹³C]isoleucine in human AD astrocytes when entering via succinyl CoA. Values represent mean (±) SEM (n = 3), ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^#p < 0.0001, analyzed by two-way ANOVA. WT, wild type; APP, amyloid precursor protein; PSEN-1, presenilin-1.

Figure 6

Decreased aspartate synthesis from oxidative isoleucine metabolism in AD neurons. (A) No significant differences were found in AD neurons after incubation with [U-¹³C]leucine. (B) Decreased glutamate M+2 and aspartate M+2 labeling after incubation with [U-¹³C]isoleucine in AD neurons. (C) No significant differences were found in AD neurons after incubation with [U-¹³C]valine in AD neurons. (D) Decreased aspartate M+3 labeling after incubation with [U-¹³C]isoleucine in AD neurons when compared with control. No significant differences were found in TCA cycle intermediates. Values represent mean (±) SEM (n = 3), ^*p < 0.05, ^#p < 0.0001 analyzed by two-way ANOVA. WT, wild type; APP, amyloid precursor protein; PSEN-1, presenilin-1.

Figure 7

Selective disruptions of amino acid amounts in AD astrocytes with APP and PSEN-1 mutations following incubation with [U-¹³C]leucine. (A) nmol/mg protein in M+2 (via acetyl CoA) labeling and total amounts after incubation with [U-¹³C]leucine. (B) nmol/mg protein in M+2 (via acetyl CoA) and M+3 (via succinyl CoA) labeling and total amounts after incubation with [U-¹³C]isoleucine. (C) nmol/mg protein in M+3 (via succinyl CoA) labeling and total amounts after incubation with [U-¹³C]valine in hiPSC-derived astrocytes. (D) nmol/mg protein in M+2 (via acetyl CoA) labeling and total amounts after incubation with [U-¹³C]leucine. (E) nmol/mg protein in M+2 (via acetyl CoA) and M+3 (via succinyl CoA) labeling and total amounts after incubation with [U-¹³C]isoleucine. (F) nmol/mg protein in M+3 (via succinyl CoA) labeling and total amounts after incubation with [U-¹³C]valine in hiPSC-derived neurons. Values represent mean (±) SEM (n = 3), ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 when compared with control, analyzed by one-way ANOVA. No significant differences were found in M+3 labeling amounts in AD astrocytes. WT, wild type; APP, amyloid precursor protein; PSEN-1, presenilin-1.

Figure 8

BCAA oxidative metabolism and nitrogen transfer in human astrocytes and potential blood brain barrier-astrocyte-neuron axis. BCAAs are taken up by the blood brain barrier (BBB) endothelial cells, and then BCAAs can undergo transamination by the action of BCATm, forming glutamate and the branched-chain α-keto acids (BCKAs), KIC, KIV, and KVM. In the reverse reaction when α-ketoglutarate is formed from glutamate, the BCAT/GDH metabolon in the endothelial cells may release ammonia, and BCAAs can be released and taken up by neurons or astrocytes. Alternatively, BCKAs from endothelial cells can be released or metabolized by the BCKDH/BCATm metabolon. Likewise, in neurons, BCAAs can also be transaminated, producing BCKAs and glutamate. BCKAs can be further metabolized by BCKDH, and glutamate can be used for neurotransmission. Glutamate from neurons in the synaptic cleft can be taken up by astrocytes. Astrocytes synthesize glutamine from glutamate and ammonia which is catalyzed by glutamine synthetase (GS) and is released for neuronal uptake to replenish the glutamate pool, in the so-called glutamate-glutamine cycle. The data indicate that in astrocytes, BCAAs may be transaminated by the action of BCAT, and that the BCAA carbon skeleton can be oxidized by BCKDH activity. This set of metabolic reactions give rise to acetyl CoA or succinyl CoA, replenishing TCA cycle intermediates and amino acids in the astrocytes. Furthermore, we present evidence of reduced aspartate, glutamate, and glutamine total and labeling amounts after incubation with [U-¹³C]leucine in AD astrocytes (represented within the dotted box). This altered leucine metabolism in AD astrocytes may contribute to the disrupted neurotransmitter homeostasis observed in the AD brain. BBB, blood brain barrier; BCATm, mitochondrial branched-chain aminotransferase; Glu, glutamate; α-KG, α-ketoglutarate, GDH, glutamate dehydrogenase, ${\text{NH}}_4^{+}$, ammonium; BCKA, branched-chain α-keto acids; KIC, α-ketoisocaproate; KIV, α-ketoisovaleric; KMV, α-keto-β-methylvalerate; BCAT, branched-chain aminotransferase; BCKDH, branched-chain α-keto acid dehydrogenase; Gln, glutamine; GS, glutamine synthetase; BCATc, cytosolic branched-chain aminotransferase; PAG, phosphate-activated glutaminase.