Increased glucose metabolism and impaired glutamate transport in human astrocytes are potential early triggers of abnormal extracellular glutamate accumulation in hiPSC-derived models of Alzheimer's disease.

Glutamate recycling between neurons and astrocytes is essential to maintain neurotransmitter homeostasis. Disturbances in glutamate homeostasis, resulting in excitotoxicity and neuronal death, have been described as a potential mechanism in Alzheimer's disease (AD) pathophysiology. However, glutamate neurotransmitter metabolism in different human brain cells, particularly astrocytes, has been poorly investigated at the early stages of AD. We sought to investigate glucose and glutamate metabolism in AD by employing human induced pluripotent stem cell (hiPSC)-derived astrocytes and neurons carrying mutations in the amyloid precursor protein (APP) or presenilin-1 (PSEN-1) gene as found in familial types of AD (fAD). Methods such as live-cell bioenergetics and metabolic mapping using [13 C]-enriched substrates were used to examine metabolism in the early stages of AD. Our results revealed greater glycolysis and glucose oxidative metabolism in astrocytes and neurons with APP or PSEN-1 mutations, accompanied by an elevated glutamate synthesis compared to control WT cells. Astrocytes with APP or PSEN-1 mutations exhibited reduced expression of the excitatory amino acid transporter 2 (EAAT2), and glutamine uptake increased in mutated neurons, with enhanced glutamate release specifically in neurons with a PSEN-1 mutation. These results demonstrate a hypermetabolic phenotype in astrocytes with fAD mutations possibly linked to toxic glutamate accumulation. Our findings further identify metabolic imbalances that may occur in the early phases of AD pathophysiology.

Brain endothelial cells metabolize glutamate via glutamate dehydrogenase to replenish TCA-intermediates and produce ATP under hypoglycemic conditions.

The endothelial cells of the blood-brain barrier participate in the regulation of glutamate concentrations in the brain interstitial fluid by taking up brain glutamate. However, endothelial glutamate metabolism has not been characterized, nor is its role in brain glutamate homeostasis and endothelial energy production known. The aim of this study was to investigate endothelial glutamate dehydrogenase (GDH) expression and glutamate metabolism and probe its functional significance. The primary brain endothelial cells were isolated from bovine and mouse brains, and human brain endothelial cells were derived from induced pluripotent stem cells. GDH expression on the protein level and GDH function were investigated in the model systems using western blotting, confocal microscopy, 13 C-glutamate metabolism, and Seahorse assay. In this study, it was shown that GDH was expressed in murine and bovine brain capillaries and in cultured primary mouse and bovine brain endothelial cells as well as in human-induced pluripotent stem cell-derived endothelial cells. The endothelial GDH expression was confirmed in brain capillaries from mice carrying a central nervous system-specific GDH knockout. Endothelial cells from all tested species metabolized 13 C-glutamate to α-ketoglutarate, which subsequently entered the tricarboxylic acid (TCA)-cycle. Brain endothelial cells maintained mitochondrial oxygen consumption rates, when supplied with glutamate alone, whereas glutamate supplied in addition to glucose did not lead to additional oxygen consumption. In conclusion, brain endothelial cells directly take up and metabolize glutamate and utilize the resulting α-ketoglutarate in the tricarboxylic acid cycle to ultimately yield ATP if glucose is unavailable.

Downregulation of GABA Transporter 3 (GAT3) is Associated with Deficient Oxidative GABA Metabolism in Human Induced Pluripotent Stem Cell-Derived Astrocytes in Alzheimer's Disease.

Alterations in neurotransmitter homeostasis, primarily of glutamate and GABA, is strongly implicated in the pathophysiology of Alzheimer's disease (AD). Homeostasis at the synapse is maintained by neurotransmitter recycling between neurons and astrocytes. Astrocytes support neuronal transmission through glutamine synthesis, which can be derived from oxidative metabolism of GABA. However, the precise implications of astrocytic GABA metabolism in AD remains elusive. The aim of this study was to investigate astrocytic GABA metabolism in AD pathology implementing human induced pluripotent stem cells derived astrocytes. Metabolic mapping of GABA was performed with [U-13C]GABA stable isotopic labeling using gas chromatography coupled to mass spectrometry (GC-MS). Neurotransmitter and amino acid content was quantified via high performance liquid chromatography (HPLC) and protein expression was investigated by Western blot assay. Cell lines carrying mutations in either amyloid precursor protein (APP) or presenilin1 (PSEN-1) were used as AD models and were compared to a control cell line of the same genetic background. AD astrocytes displayed a reduced oxidative GABA metabolism mediated by a decreased uptake capacity of GABA, as GABA transporter 3 (GAT3) was downregulated in AD astrocytes compared to the controls. Interestingly, the carbon backbone of GABA in AD astrocytes was utilized to a larger extent to support glutamine synthesis compared to control astrocytes. The results strongly indicate alterations in GABA uptake and metabolism in AD astrocytes linked to reduced GABA transporter expression, hereby contributing further to neurotransmitter disturbances.

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.

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.

Astrocytic reactivity triggered by defective autophagy and metabolic failure causes neurotoxicity in frontotemporal dementia type 3.

Frontotemporal dementia type 3 (FTD3), caused by a point mutation in the charged multivesicular body protein 2B (CHMP2B), affects mitochondrial ultrastructure and the endolysosomal pathway in neurons. To dissect the astrocyte-specific impact of mutant CHMP2B expression, we generated astrocytes from human induced pluripotent stem cells (hiPSCs) and confirmed our findings in CHMP2B mutant mice. Our data provide mechanistic insights into how defective autophagy causes perturbed mitochondrial dynamics with impaired glycolysis, increased reactive oxygen species, and elongated mitochondrial morphology, indicating increased mitochondrial fusion in FTD3 astrocytes. This shift in astrocyte homeostasis triggers a reactive astrocyte phenotype and increased release of toxic cytokines, which accumulate in nuclear factor kappa b (NF-κB) pathway activation with increased production of CHF, LCN2, and C3 causing neurodegeneration.

Effects of pre- and neonatal undernutrition on the kyphotic response and c-Fos activity in the caudal periaqueductal gray of primiparous lactating Wistar rats.

In rodents, the most representative component of maternal behavior that meets the purpose of newborn nutrition is the kyphotic posture. During this posture, the mother maintains a unique environment for the protection, thermal regulation and breast-feeding of the progeny. The aim of this study was to investigate possible deficiencies in the kyphotic posture of adult lactating dams with pre- and neonatal undernutrition evoked by their own pups suckling in a home-cage situation. Wistar dams that had been previously exposed to perinatal undernutrition were mated at 90days of age, and pregnancy was confirmed by vaginal smears. Before testing if the perinatal underfed dam affected behavior, pups were removed (4h), and both the maternal response and the kyphotic posture were video-recorded (1h) and analyzed at 4 and 12days of lactation. Pre- and post-test litter weight gain was obtained. To immunostain the caudal periaqueductal gray, the litter was separated from their dams 24h before suckling stimulation. The results showed that underfed dams significantly reduced the duration of high kyphosis by choosing unconventional postures (prone and partial kyphosis). The body weight of the F1 offspring was significantly reduced, and the underfed F0 dams showed reduced c-Fos immunostaining at the caudal periaqueductal gray. The findings showed that early underfed dams have deficiencies in the mechanisms underlying the kyphosis, possibly because the pups' cues to evoke this posture were suboptimal and/or because the dam expressed deficient nursing. The results suggest that the abnormal kyphotic posture may affect the mother-litter bonds and have long-term effects on neonatal brain functions.