Blanca I. Aldana

Patient iPSC-Derived Neurons for Disease Modeling of Frontotemporal Dementia with Mutation in CHMP2B

Summary The truncated mutant form of the charged multivesicular body protein 2B (CHMP2B) is causative for frontotemporal dementia linked to chromosome 3 (FTD3). CHMP2B is a constituent of the endosomal sorting complex required for transport (ESCRT) and, when mutated, disrupts endosome-to-lysosome trafficking and substrate degradation. To understand the underlying molecular pathology, FTD3 patient induced pluripotent stem cells (iPSCs) were differentiated into forebrain-type cortical neurons. FTD3 neurons exhibited abnormal endosomes, as previously shown in patients. Moreover, mitochondria of FTD3 neurons displayed defective cristae formation, accompanied by deficiencies in mitochondrial respiration and increased levels of reactive oxygen. In addition, we provide evidence for perturbed iron homeostasis, presenting an in vitro patient-specific model to study the effects of iron accumulation in neurodegenerative diseases. All phenotypes observed in FTD3 neurons were rescued in CRISPR/Cas9-edited isogenic controls. These findings illustrate the relevance of our patient-specific in vitro models and open up possibilities for drug target development.

Alterations in Cerebral Cortical Glucose and Glutamine Metabolism Precedes Amyloid Plaques in the APPswe/PSEN1dE9 Mouse Model of Alzheimer’s Disease

Alterations in brain energy metabolism have been suggested to be of fundamental importance for the development of Alzheimer’s disease (AD). However, specific changes in brain energetics in the early stages of AD are poorly known. The aim of this study was to investigate cerebral energy metabolism in the APPswe/PSEN1dE9 mouse prior to amyloid plaque formation. Acutely isolated cerebral cortical and hippocampal slices of 3-month-old APPswe/PSEN1dE9 and wild-type control mice were incubated in media containing [U-13C]glucose, [1,2-13C]acetate or [U-13C]glutamine, and tissue extracts were analyzed by mass spectrometry. The ATP synthesis rate of isolated whole-brain mitochondria was assessed by an on-line luciferin-luciferase assay. Significantly increased 13C labeling of intracellular lactate and alanine and decreased tricarboxylic acid (TCA) cycle activity were observed from cerebral cortical slices of APPswe/PSEN1dE9 mice incubated in media containing [U-13C]glucose. No changes in glial [1,2-13C]acetate metabolism were observed. Cerebral cortical slices from APPswe/PSEN1dE9 mice exhibited a reduced capacity for uptake and oxidative metabolism of glutamine. Furthermore, the ATP synthesis rate tended to be decreased in isolated whole-brain mitochondria of APPswe/PSEN1dE9 mice. Thus, several cerebral metabolic changes are evident in the APPswe/PSEN1dE9 mouse prior to amyloid plaque deposition, including altered glucose metabolism, hampered glutamine processing and mitochondrial dysfunctions.

Characterization of energy and neurotransmitter metabolism in cortical glutamatergic neurons derived from human induced pluripotent stem cells: A novel approach to study metabolism in human neurons

ABSTRACT Alterations in the cellular metabolic machinery of the brain are associated with neurodegenerative disorders such as Alzheimers disease. Novel human cellular disease models are essential in order to study underlying disease mechanisms. In the present study, we characterized major metabolic pathways in neurons derived from human induced pluripotent stem cells (hiPSC). With this aim, cultures of hiPSC‐derived neurons were incubated with [U‐13C]glucose, [U‐13C]glutamate or [U‐13C]glutamine. Isotopic labeling in metabolites was determined using gas chromatography coupled to mass spectrometry, and cellular amino acid content was quantified by high‐performance liquid chromatography. Additionally, we evaluated mitochondrial function using real‐time assessment of oxygen consumption via the Seahorse XFe96 Analyzer. Moreover, in order to validate the hiPSC‐derived neurons as a model system, a metabolic profiling was performed in parallel in primary neuronal cultures of mouse cerebral cortex and cerebellum. These serve as well‐established models of GABAergic and glutamatergic neurons, respectively. The hiPSC‐derived neurons were previously characterized as being forebrain‐specific cortical glutamatergic neurons. However, a comparable preparation of predominantly mouse cortical glutamatergic neurons is not available. We found a higher glycolytic capacity in hiPSC‐derived neurons compared to mouse neurons and a substantial oxidative metabolism through the mitochondrial tricarboxylic acid (TCA) cycle. This finding is supported by the extracellular acidification and oxygen consumption rates measured in the cultured human neurons. [U‐13C]Glutamate and [U‐13C]glutamine were found to be efficient energy substrates for the neuronal cultures originating from both mice and humans. Interestingly, isotopic labeling in metabolites from [U‐13C]glutamate was higher than that from [U‐13C]glutamine. Although the metabolic profile of hiPSC‐derived neurons in vitro was particularly similar to the profile of mouse cortical neurons, important differences between the metabolic profile of human and mouse neurons were observed. The results of the present investigation establish hallmarks of cellular metabolism in human neurons derived from iPSC. HIGHLIGHTSMetabolic pathways can be mapped in cultured hiPSC‐derived neurons using 13C‐labeled substrates and mass spectrometry.hiPSC‐derived neurons are metabolically highly active cells.hiPSC‐derived neurons avidly metabolize glucose via glycolysis and TCA cycle.Glutamate is a preferred substrate over glutamine in cultured hiPSC‐derived neurons.Cultured hiPSC‐derived neurons can be used as a valuable model to study cellular metabolism in health and disease.

Characterization of the L-glutamate clearance pathways across the blood–brain barrier and the effect of astrocytes in an in vitro blood–brain barrier model:

The aim was to characterize the clearance pathways for L-glutamate from the brain interstitial fluid across the blood–brain barrier using a primary in vitro bovine endothelial/rat astrocyte co-culture. Transporter profiling was performed using uptake studies of radiolabeled L-glutamate with co-application of transporter inhibitors and competing amino acids. Endothelial abluminal L-glutamate uptake was almost abolished by co-application of an EAAT-1 specific inhibitor, whereas luminal uptake was inhibited by L-glutamate and L-aspartate (1 mM). L-glutamate uptake followed Michaelis–Menten-like kinetics with high and low affinity at the abluminal and luminal membrane, respectively. This indicated that L-glutamate is taken up via EAAT-1 at the abluminal membrane and exits at the luminal membrane via a low affinity glutamate/aspartate transporter. Metabolism of L-glutamate and transport of metabolites was examined using [U-13C] L-glutamate. Intact L-glutamate and metabolites derived from oxidative metabolism were transported through the endothelial cells. High amounts of L-glutamate-derived lactate in the luminal medium indicated cataplerosis via malic enzyme. Thus, L-glutamate can be transported intact from brain to blood via the concerted action of abluminal and luminal transport proteins, but the total brain clearance is highly dependent on metabolism in astrocytes and endothelial cells followed by transport of metabolites.

Integrative Characterization of the R6/2 Mouse Model of Huntington’s Disease Reveals Dysfunctional Astrocyte Metabolism

Huntingtons disease is a fatal neurodegenerative disease, where dysfunction and loss of striatal and cortical neurons are central to the pathogenesis of the disease. Here, we integrated quantitative studies to investigate the underlying mechanisms behind HD pathology in a systems-wide manner. To this end, we used state-of-the-art mass spectrometry to establish a spatial brain proteome from late-stage R6/2 mice and compared this with wild-type littermates. We observed altered expression of proteins in pathways related to energy metabolism, synapse function, and neurotransmitter homeostasis. To support these findings, metabolic 13C labeling studies confirmed a compromised astrocytic metabolism and regulation of glutamate-GABA-glutamine cycling, resulting in impaired release of glutamine and GABA synthesis. In recent years, increasing attention has been focused on the role of astrocytes in HD, and our data support that therapeutic strategies to improve astrocytic glutamine homeostasis may help ameliorate symptoms in HD.

The novel anticonvulsant neuropeptide and galanin analogue, NAX‐5055, does not alter energy and amino acid metabolism in cultured brain cells

A large body of evidence suggests that the neuropeptide galanin plays an important role in seizure control. In line with this, it was demonstrated that the galanin analogue, NAX‐5055, exerts a potent anticonvulsant activity in animal seizure models. We recently found that the NAX‐5055‐mediated anticonvulsant action involves modulation of both excitatory and inhibitory neurotransmission. Since homeostasis of neurotransmitters and cerebral energy metabolism are intimately linked, it was investigated whether the effects of NAX‐5055 on neurotransmission involve changes in energy metabolism and in particular glucose‐ and amino acid metabolism. With this aim, cultured neurons from mouse brain were incubated with [U‐13C]glucose in absence or presence of NAX‐5055. Since effects of NAX‐5055 on neurotransmission were detected during repetitive stimulation, we tested potential metabolic effects while mimicking repetitive bursts of neurotransmitter release as occurring in the intact brain. The metabolic pathways were mapped using gas‐chromatography coupled to mass‐spectrometry. We found that NAX‐5055 does not modify glucose metabolism in glutamatergic and GABAergic neurons. Furthermore, the effect of NAX‐5055 on astrocyte‐neuron metabolic interactions was investigated by incubating co‐cultures of astrocytes and either glutamatergic or GABAergic neurons with [U‐13C]glucose or the glial‐selective substrate [1,2‐13C]acetate, with or without NAX‐5055. In the presence of NAX‐5055, no changes in the metabolic landscape were traced. The findings suggest that the anticonvulsant action of NAX‐5055 and the accompanying changes in neurotransmission do not involve alterations in energy and amino acid metabolism. Hence, NAX‐5055 appears to be an anti‐seizure drug candidate displaying no unwanted side effects concerning brain energy and amino acid homeostasis.