bims-medebr 2023-11-19 papers

bims-medebr

Biomed News

on Metabolism of the developing brain

Issue of 2023–11–19
fiveteen papers selected by
Regina F. Fernández, Johns Hopkins University

Neurons regulate the esterification of bioactive lipid mediators in the brain of acid sphingomyelinase deficient mice.
Non-invasive in vivo measurements of metabolic alterations in the type 2 diabetic brain by 1 H magnetic resonance spectroscopy.
Astrocyte and L-lactate in the anterior cingulate cortex modulate schema memory and neuronal mitochondrial biogenesis.
Short-term consumption of highly processed diets varying in macronutrient content impair the sense of smell and brain metabolism in mice.
Mitochondrial dysfunction in Parkinson's disease - a key disease hallmark with therapeutic potential.
Heptanoate Improves Compensatory Mechanism of Glucose Homeostasis in Mitochondrial Long-Chain Fatty Acid Oxidation Defect.
Increased cerebral lactate levels in adults with autism spectrum disorders compared to non-autistic controls: a magnetic resonance spectroscopy study.
Review of Eukaryote Cellular Membrane Lipid Composition, with Special Attention to the Fatty Acids.
Ketone body β-hydroxybutyrate (BHB) preserves mitochondrial bioenergetics.
Neurodegenerative Etiology of Aromatic L-Amino Acid Decarboxylase Deficiency: a Novel Concept for Expanding Treatment Strategies.
Developmental and epileptic encephalopathy 82 (DEE82) with novel compound heterozygous mutations of GOT2 gene.
Glyceryl triacetate promotes blood-brain barrier recovery after ischemic stroke through lipogenesis-mediated IL-33 in mice.
Identification of Acetylation Sites of Fatty Acid Synthase (FASN) by Mass Spectrometry and FASN Activity Assay.
Branched-Chain Amino Acid Accumulation Fuels the Senescence-Associated Secretory Phenotype.
Mitochondrial aminoacyl-tRNA synthetases trigger unique compensatory mechanisms in neurons.

Prog Neuropsychopharmacol Biol Psychiatry. 2023 Nov 11. pii: S0278-5846(23)00182-3. [Epub ahead of print] 110896

Neurons regulate the esterification of bioactive lipid mediators in the brain of acid sphingomyelinase deficient mice.

Ameer Y Taha, Ángel Gaudioso, Maria Moran-Garrido, Sandra M Camunas-Alberca, Jaime Bachiller-Hernández, Jorge Sáiz, Maria Dolores Ledesma, Coral Barbas.

  Acid sphingomyelinase deficiency is a neurodegenerative lysosomal storage disorder caused by mutations in the sphingomyelin-degrading enzyme acid sphingomyelinase (ASM) gene. Upregulated neuroinflammation has been well-characterized in an ASM knockout mouse model of acid sphingomyelinase deficiency disease, but lipid mediator pathways involved in 'mediating' inflammation and inflammation-resolution have yet to be characterized. In this study, we 1) measured free (bioactive) and esterified (inactive) lipid mediators involved in inflammation and inflammation resolution in cerebellum and neuronal cultures of ASM knockout (ASMko) mice and wildtype (WT) controls, and 2) tested the incorporation of labeled pro-resolving free d11-14(15)-epoxyeicosatrienoic acid into culture neurons from ASMko and WT mice. We found elevated concentrations of esterified pro-resolving lipid mediators and hydroxyeicosatrienoic acids typically destined for pro-resolving lipid mediator synthesis (e.g. lipoxins) in the cerebellum and neurons of ASMko mice compared to controls. Free d11-14(15)-epoxyeicosatrienoic acid incorporation into neurons of ASMko mice was significantly elevated compared to WT. Our findings show evidence of increased inactivation of free pro-resolving lipid mediators through esterification, suggesting impaired resolution as a new pathway underlying ASM deficiency pathogenesis.

Keywords:  Bound oxylipins; Free oxylipins; Neurodegeneration; Neuroinflammation; Niemann pick disease

DOI:  https://doi.org/10.1016/j.pnpbp.2023.110896
J Neurochem. 2023 Nov 15.

Non-invasive in vivo measurements of metabolic alterations in the type 2 diabetic brain by 1 H magnetic resonance spectroscopy.

In-Young Choi, Wen-Tung Wang, Bhumsoo Kim, Junguk Hur, David C Robbins, Dae-Gyu Jang, Masha G Savelieff, Eva L Feldman, Phil Lee.

  Type 2 diabetes (T2D) is a complex chronic metabolic disorder characterized by hyperglycemia because of insulin resistance. Diabetes with chronic hyperglycemia may alter brain metabolism, including brain glucose and neurotransmitter levels; however, detailed, longitudinal studies of metabolic alterations in T2D are lacking. To shed insight, here, we characterized the consequences of poorly controlled hyperglycemia on neurochemical profiles that reflect metabolic alterations of the brain in both humans and animal models of T2D. Using in vivo 1 H magnetic resonance spectroscopy, we quantified 12 metabolites cross-sectionally in T2D patients and 20 metabolites longitudinally in T2D db/db mice versus db+ controls. We found significantly elevated brain glucose (91%, p < 0.001), taurine (22%, p = 0.02), glucose+taurine (56%, p < 0.001), myo-inositol (12%, p = 0.02), and choline-containing compounds (10%, p = 0.01) in T2D patients versus age- and sex-matched controls, findings consistent with measures in T2D db/db versus control db+ littermates. In mice, hippocampal and striatal neurochemical alterations in brain glucose, ascorbate, creatine, phosphocreatine, γ-aminobutyric acid, glutamate, glutamine, glutathione, glycerophosphoryl-choline, lactate, myo-inositol, and taurine persisted in db/db mice with chronic disease progression from 16 to 48 weeks of age, which were distinct from control db+ mice. Overall, our study demonstrates the utility of 1 H magnetic resonance spectroscopy as a non-invasive tool for characterizing and monitoring brain metabolic changes with T2D progression.

Keywords:  brain metabolism; human brain; magnetic resonance spectroscopy; mouse; neurochemical profile; type 2 diabetes

DOI:  https://doi.org/10.1111/jnc.15996
Elife. 2023 11 14. pii: e85751. [Epub ahead of print]12

Astrocyte and L-lactate in the anterior cingulate cortex modulate schema memory and neuronal mitochondrial biogenesis.

Mastura Akter, Mahadi Hasan, Aruna Surendran Ramkrishnan, Zafar Iqbal, Xianlin Zheng, Zhongqi Fu, Zhuogui Lei, Anwarul Karim, Ying Li.

  Astrocyte-derived L-lactate was shown to confer beneficial effects on synaptic plasticity and cognitive functions. However, how astrocytic Gi signaling in the anterior cingulate cortex (ACC) modulates L-lactate levels and schema memory is not clear. Here, using chemogenetic approach and well-established behavioral paradigm, we demonstrate that astrocytic Gi pathway activation in the ACC causes significant impairments in flavor-place paired associates (PAs) learning, schema formation, and PA memory retrieval in rats. It also impairs new PA learning even if a prior associative schema exists. These impairments are mediated by decreased L-lactate in the ACC due to astrocytic Gi activation. Concurrent exogenous L-lactate administration bilaterally into the ACC rescues these impairments. Furthermore, we show that the impaired schema memory formation is associated with a decreased neuronal mitochondrial biogenesis caused by decreased L-lactate level in the ACC upon astrocytic Gi activation. Our study also reveals that L-lactate-mediated mitochondrial biogenesis is dependent on monocarboxylate transporter 2 (MCT2) and NMDA receptor activity - discovering a previously unrecognized signaling role of L-lactate. These findings expand our understanding of the role of astrocytes and L-lactate in the brain functions.

Keywords:  DREADD; anterior cingulate cortex; astrocyte; lactate; mitochondrial biogenesis; neuroscience; rat; schema

DOI:  https://doi.org/10.7554/eLife.85751
Mol Metab. 2023 Nov 15. pii: S2212-8778(23)00171-0. [Epub ahead of print] 101837

Short-term consumption of highly processed diets varying in macronutrient content impair the sense of smell and brain metabolism in mice.

Melanie Makhlouf, Débora G Souza, Smija Kurian, Bruna Bellaver, Hillary Ellis, Akihito Kuboki, Asma Al-Naama, Reem Hasnah, Gianina Teribele Venturin, Jaderson Costa da Costa, Neethu Venugopal, Diogo Manoel, Julie Mennella, Johannes Reisert, Michael G Tordoff, Eduardo R Zimmer, Luis R Saraiva.

   BACKGROUND: Food processing greatly contributed to increased food safety, diversity, and accessibility. However, the prevalence of highly palatable and highly processed food in our modern diet has exacerbated obesity rates and contributed to a global health crisis. While accumulating evidence suggests that chronic consumption of such foods is detrimental to sensory and neural physiology, it is unclear whether its short-term intake has adverse effects.
METHODS: We assessed how short-term consumption (<2 months) of three diets varying in composition and macronutrient content influence olfaction and brain metabolism in mice. The diets tested included a grain-based standard chow diet (CHOW; 54% carbohydrate, 32% protein, 14% fat; #8604 Teklad Rodent diet, Envigo Inc.), a highly processed control diet (hpCTR; 70% carbohydrate, 20% protein, 10% fat; #D12450B, Research Diets Inc.), and a highly processed high-fat diet (hpHFD; 20% carbohydrate, 20% protein, 60% fat; #D12492, Research Diets Inc.). We performed behavioral and metabolic phenotyping, electro-olfactogram (EOG) recordings, brain glucose metabolism imaging, and mitochondrial respirometry in different brain regions. We also performed RNA-sequencing (RNA-seq) in the nose and across several brain regions, and conducted differential expression analysis, gene ontology, and network analysis.
RESULTS: We show that short-term consumption of the two highly processed diets, but not the grain-based diet, regardless of macronutrient content, adversely affects odor-guided behaviors, physiological responses to odorants, transcriptional profiles in the olfactory mucosa and brain regions, and brain glucose metabolism and mitochondrial respiration.
CONCLUSIONS: Even short periods of highly processed food consumption are sufficient to cause early olfactory and brain abnormalities, which has the potential to alter food choices and influence the risk of developing metabolic disease.

Keywords:  Highly processed food; diet; metabolism; obesity; olfaction

DOI:  https://doi.org/10.1016/j.molmet.2023.101837
Mol Neurodegener. 2023 Nov 11. 18(1): 83

Mitochondrial dysfunction in Parkinson's disease - a key disease hallmark with therapeutic potential.

Martin T Henrich, Wolfgang H Oertel, D James Surmeier, Fanni F Geibl.

  Mitochondrial dysfunction is strongly implicated in the etiology of idiopathic and genetic Parkinson's disease (PD). However, strategies aimed at ameliorating mitochondrial dysfunction, including antioxidants, antidiabetic drugs, and iron chelators, have failed in disease-modification clinical trials. In this review, we summarize the cellular determinants of mitochondrial dysfunction, including impairment of electron transport chain complex 1, increased oxidative stress, disturbed mitochondrial quality control mechanisms, and cellular bioenergetic deficiency. In addition, we outline mitochondrial pathways to neurodegeneration in the current context of PD pathogenesis, and review past and current treatment strategies in an attempt to better understand why translational efforts thus far have been unsuccessful.

Keywords:  Antioxidants; Electron transport chain; MPTP; Mitochondria; Mitochondrial dysfunction; Neuroprotective therapies; Parkinson’s disease; Synuclein

DOI:  https://doi.org/10.1186/s13024-023-00676-7
Nutrients. 2023 Nov 05. pii: 4689. [Epub ahead of print]15(21):

Heptanoate Improves Compensatory Mechanism of Glucose Homeostasis in Mitochondrial Long-Chain Fatty Acid Oxidation Defect.

Siti Nurjanah, Albert Gerding, Marcel A Vieira-Lara, Bernard Evers, Miriam Langelaar-Makkinje, Ute Spiekerkoetter, Barbara M Bakker, Sara Tucci.

  Defects in mitochondrial fatty acid β-oxidation (FAO) impair metabolic flexibility, which is an essential process for energy homeostasis. Very-long-chain acyl-CoA dehydrogenase (VLCADD; OMIM 609575) deficiency is the most common long-chain mitochondrial FAO disorder presenting with hypoglycemia as a common clinical manifestation. To prevent hypoglycemia, triheptanoin-a triglyceride composed of three heptanoates (C7) esterified with a glycerol backbone-can be used as a dietary treatment, since it is metabolized into precursors for gluconeogenesis. However, studies investigating the effect of triheptanoin on glucose homeostasis are limited. To understand the role of gluconeogenesis in the pathophysiology of long-chain mitochondrial FAO defects, we injected VLCAD-deficient (VLCAD-/-) mice with 13C3-glycerol in the presence and absence of heptanoate (C7). The incorporation of 13C3-glycerol into blood glucose was higher in VLCAD-/- mice than in WT mice, whereas the difference disappeared in the presence of C7. The result correlates with 13C enrichment of liver metabolites in VLCAD-/- mice. In contrast, the C7 bolus significantly decreased the 13C enrichment. These data suggest that the increased contribution of gluconeogenesis to the overall glucose production in VLCAD-/- mice increases the need for gluconeogenesis substrate, thereby avoiding hypoglycemia. Heptanoate is a suitable substrate to induce glucose production in mitochondrial FAO defect.

Keywords:  VLCAD deficiency; fatty acid oxidation disorder; glucose homeostasis; glycerol; heptanoate; stable isotope; very long-chain acyl-CoA dehydrogenase

DOI:  https://doi.org/10.3390/nu15214689
Mol Autism. 2023 Nov 17. 14(1): 44

Increased cerebral lactate levels in adults with autism spectrum disorders compared to non-autistic controls: a magnetic resonance spectroscopy study.

Simon Maier, Kathrin Nickel, Thomas Lange, Georg Oeltzschner, Michael Dacko, Dominique Endres, Kimon Runge, Anke Schumann, Katharina Domschke, Michalis Rousos, Ludger Tebartz van Elst.

   INTRODUCTION: Autism spectrum disorder (ASD) encompasses a heterogeneous group with varied phenotypes and etiologies. Identifying pathogenic subgroups could facilitate targeted treatments. One promising avenue is investigating energy metabolism, as mitochondrial dysfunction has been implicated in a subgroup of ASD. Lactate, an indicator of energy metabolic anomalies, may serve as a potential biomarker for this subgroup. This study aimed to examine cerebral lactate (Lac+) levels in high-functioning adults with ASD, hypothesizing elevated mean Lac+ concentrations in contrast to neurotypical controls (NTCs).
MATERIALS AND METHODS: Magnetic resonance spectroscopy (MRS) was used to study cerebral Lac+ in 71 adults with ASD and NTC, focusing on the posterior cingulate cortex (PCC). After quality control, 64 ASD and 58 NTC participants remained. Lac+ levels two standard deviations above the mean of the control group were considered elevated.
RESULTS: Mean PCC Lac+ levels were significantly higher in the ASD group than in the NTC group (p = 0.028; Cohen's d = 0.404), and 9.4% of the ASD group had elevated levels as compared to 0% of the NTCs (p = 0.029). No significant correlation was found between blood serum lactate levels and MRS-derived Lac+ levels.
LIMITATIONS: A cautious interpretation of our results is warranted due to a p value of 0.028. In addition, a higher than anticipated proportion of data sets had to be excluded due to poor spectral quality.
CONCLUSION: This study confirms the presence of elevated cerebral Lac+ levels in a subgroup of adults with ASD, suggesting the potential of lactate as a biomarker for mitochondrial dysfunction in a subgroup of ASD. The lower-than-expected prevalence (20% was expected) and moderate increase require further investigation to elucidate the underlying mechanisms and relationships with mitochondrial function.

Keywords:  Autism spectrum disorder; Lactate; Magnetic resonance spectroscopy; Mitochondria; Mitochondrial dysfunction; Posterior cingulate cortex

DOI:  https://doi.org/10.1186/s13229-023-00577-y
Int J Mol Sci. 2023 Oct 28. pii: 15693. [Epub ahead of print]24(21):

Review of Eukaryote Cellular Membrane Lipid Composition, with Special Attention to the Fatty Acids.

Omeralfaroug Ali, András Szabó.

  Biological membranes, primarily composed of lipids, envelop each living cell. The intricate composition and organization of membrane lipids, including the variety of fatty acids they encompass, serve a dynamic role in sustaining cellular structural integrity and functionality. Typically, modifications in lipid composition coincide with consequential alterations in universally significant signaling pathways. Exploring the various fatty acids, which serve as the foundational building blocks of membrane lipids, provides crucial insights into the underlying mechanisms governing a myriad of cellular processes, such as membrane fluidity, protein trafficking, signal transduction, intercellular communication, and the etiology of certain metabolic disorders. Furthermore, comprehending how alterations in the lipid composition, especially concerning the fatty acid profile, either contribute to or prevent the onset of pathological conditions stands as a compelling area of research. Hence, this review aims to meticulously introduce the intricacies of membrane lipids and their constituent fatty acids in a healthy organism, thereby illuminating their remarkable diversity and profound influence on cellular function. Furthermore, this review aspires to highlight some potential therapeutic targets for various pathological conditions that may be ameliorated through dietary fatty acid supplements. The initial section of this review expounds on the eukaryotic biomembranes and their complex lipids. Subsequent sections provide insights into the synthesis, membrane incorporation, and distribution of fatty acids across various fractions of membrane lipids. The last section highlights the functional significance of membrane-associated fatty acids and their innate capacity to shape the various cellular physiological responses.

Keywords:  bioactive lipids; de novo synthesis; desaturation; fatty acid; membranes; oxygenation; phospholipids; physicochemical; sphingolipids; very long polyunsaturated fatty acids

DOI:  https://doi.org/10.3390/ijms242115693
Sci Rep. 2023 11 11. 13(1): 19664

Ketone body β-hydroxybutyrate (BHB) preserves mitochondrial bioenergetics.

I Llorente-Folch, H Düssmann, O Watters, N M C Connolly, Jochen H M Prehn.

The ketogenic diet is an emerging therapeutic approach for refractory epilepsy, as well as certain rare and neurodegenerative disorders. The main ketone body, β-hydroxybutyrate (BHB), is the primary energy substrate endogenously produced in a ketogenic diet, however, mechanisms of its therapeutic actions remain unknown. Here, we studied the effects of BHB on mitochondrial energetics, both in non-stimulated conditions and during glutamate-mediated hyperexcitation. We found that glutamate-induced hyperexcitation stimulated mitochondrial respiration in cultured cortical neurons, and that this response was greater in cultures supplemented with BHB than with glucose. BHB enabled a stronger and more sustained maximal uncoupled respiration, indicating that BHB enables neurons to respond more efficiently to increased energy demands such as induced during hyperexcitation. We found that cytosolic Ca2+ was required for BHB-mediated enhancement of mitochondrial function, and that this enhancement was independent of the mitochondrial glutamate-aspartate carrier, Aralar/AGC1. Our results suggest that BHB exerts its protective effects against hyperexcitation by enhancing mitochondrial function through a Ca2+-dependent, but Aralar/AGC1-independent stimulation of mitochondrial respiration.

DOI: https://doi.org/10.1038/s41598-023-46776-8
Mol Neurobiol. 2023 Nov 13.

Neurodegenerative Etiology of Aromatic L-Amino Acid Decarboxylase Deficiency: a Novel Concept for Expanding Treatment Strategies.

Zohi Sternberg.

  Aromatic l-amino acid decarboxylase deficiency (AADC-DY) is caused by one or more mutations in the DDC gene, resulting in the deficit in catecholamines and serotonin neurotransmitters. The disease has limited therapeutic options with relatively poor clinical outcomes. Accumulated evidence suggests the involvement of neurodegenerative mechanisms in the etiology of AADC-DY. In the absence of neurotransmitters' neuroprotective effects, the accumulation and the chronic presence of several neurotoxic metabolites including 4-dihydroxy-L-phenylalanine, 3-methyldopa, and homocysteine, in the brain of subjects with AADC-DY, promote oxidative stress and reduce the cellular antioxidant and methylation capacities, leading to glial activation and mitochondrial dysfunction, culminating to neuronal injury and death. These pathophysiological processes have the potential to hinder the clinical efficacy of treatments aimed at increasing neurotransmitters' synthesis and or function. This review describes in detail the mechanisms involved in AADC-DY neurodegenerative etiology, highlighting the close similarities with those involved in other neurodegenerative diseases. We then offer novel strategies for the treatment of the disease with the objective to either reduce the level of the metabolites or counteract their prooxidant and neurotoxic effects. These treatment modalities used singly or in combination, early in the course of the disease, will minimize neuronal injury, preserving the functional integrity of neurons, hence improving the clinical outcomes of both conventional and unconventional interventions in AADC-DY. These modalities may not be limited to AADC-DY but also to other metabolic disorders where a specific mutation leads to the accumulation of prooxidant and neurotoxic metabolites.

Keywords:  Antioxidants; Inborn error of metabolism; Methylation status; Neuroinflammation; Parkinson’s disease; Rare genetic disease; Sulfur-containing compounds

DOI:  https://doi.org/10.1007/s12035-023-03684-2
Seizure. 2023 Nov 09. pii: S1059-1311(23)00290-X. [Epub ahead of print]

Developmental and epileptic encephalopathy 82 (DEE82) with novel compound heterozygous mutations of GOT2 gene.

Özlem Yalçın Çapan, Dilşad Türkdoğan, Sertaç Atalay, Hande S Çağlayan.

   PURPOSE: Developmental and Epileptic Encephalopathies (DEEs) are rare neurological disorders characterized by early-onset medically resistant epileptic seizures, structural brain malformations, and severe developmental delays. These disorders can arise from mutations in genes involved in vital metabolic pathways, including those within the brain. Recent studies have implicated defects in the mitochondrial malate aspartate shuttle (MAS) as potential contributors to the clinical manifestation of infantile epileptic encephalopathy. Although rare, mutations in MDH1, MDH2, AGC1, or GOT2 genes have been reported in patients exhibiting neurological symptoms such as global developmental delay, epilepsy, and progressive microcephaly.
METHOD: In this study, we employed exome data analysis of a patient diagnosed with DEE, focusing on the screening of 1896 epilepsy-related genes listed in the HPO and ClinVar databases. Sanger sequencing was subsequently conducted to validate and assess the inheritance pattern of the identified variants within the family. The evolutionary conservation scores of the mutated residues were evaluated using the ConSurf Database. Furthermore, the impacts of the causative variations on protein stability were analyzed through I-Mutant and MuPro bioinformatic tools. Structural comparisons between wild-type and mutant proteins were performed using PyMOL, and the physicochemical effects of the mutations were assessed using Project Hope.
RESULTS: Exome data analysis unveiled the presence of novel compound heterozygous mutations in the GOT2 gene coding for mitochondrial glutamate aspartate transaminase. Sanger sequencing confirmed the paternal inheritance of the p.Asp257Asn mutation and the maternal inheritance of the p.Arg262Cys mutation. The affected individual exhibited plasma metabolic disturbances, including hyperhomocysteinemia, hyperlactatemia, and reduced levels of methionine and arginine. Detailed bioinformatic analysis indicated that the mutations were located within evolutionarily conserved domains of the enzyme, resulting in disruptions to protein stability and structure.
CONCLUSION: Herein, we describe a case with DEE82 (MIM: # 618721) with pathologic novel biallelic mutations in the GOT2 gene. Early genetic diagnosis of metabolic epilepsies is crucial for long-term neurodevelopmental improvements and seizure control as targeted treatments can be administered based on the affected metabolic pathways.

Keywords:  Aspartate aminotransferase; Developmental and epileptic encephalopathies; GOT2 enzyme; Inborn error of metabolism; Malate-aspartate shuttle; Metabolic epilepsy; Vitamin B6 therapy; Whole exome sequencing

DOI:  https://doi.org/10.1016/j.seizure.2023.11.003
J Neuroinflammation. 2023 Nov 15. 20(1): 264

Glyceryl triacetate promotes blood-brain barrier recovery after ischemic stroke through lipogenesis-mediated IL-33 in mice.

Haidong Wei, Luming Zhen, Shiquan Wang, Liufei Yang, Shuyue Zhang, Yuanyuan Zhang, Pengyu Jia, Tianyue Wang, Kui Wang, Yan Zhang, Lei Ma, Jianrui Lv, Pengbo Zhang.

BACKGROUND: Lipid metabolism has a crucial role in neural repair in neurodegenerative diseases. We recently revealed that lipogenesis-mediated interleukin-33 (IL-33) upregulation lead to blood-brain barrier (BBB) repair after ischemic stroke. However, manipulating the key enzyme fatty acid synthase (FASN) to enhance lipogenesis was very challenging. Glyceryl triacetate (GTA) was used as a donor of acetate and precursor of acetyl coenzyme A, the key substrate for de novo lipogenesis catalyzed by FASN. Therefore, we hypothesized that GTA would promote lipogenesis the peri-infarct after ischemic stroke and contribute to the BBB repair through IL-33.
METHODS: Middle cerebral artery occlusion (MCAO) was performed on C57BL mice and GTA was gavage administrated (4 g/kg) on day 2 and 4 after MCAO. Lipogenesis was evaluated by assessment of the protein level of FASN, lipid droplets, and fatty acid products through liquid chromatography-mass spectrometry in the peri-infarct area on day 3 after MCAO, respectively. BBB permeability was determined by extravasation of Evans blue, IgG and dextran, and levels of tight junction proteins in the peri-infarct area on day 7 after MCAO, respectively. Infarct size and neurological defects were assessed on day 7 after MCAO. Brain atrophy on day 30 and long-term sensorimotor abilities after MCAO were analyzed as well. The inhibitor of FASN, C75 and the virus-delivered FASN shRNA were used to evaluate the role of FASN-driven lipogenesis in GTA-improved BBB repair. Finally, the therapeutic potential of recombinant IL-33 on BBB repair and neurological recovery was evaluated.
RESULTS: We found that treatment with GTA increased the lipogenesis as evidenced by lipid droplets level and lauric acid content, but not the FASN protein level. Treatment with GTA increased the IL-33 level in the peri-infarct area and decreased the BBB permeability after MCAO. However, infarct size and neurological defect score were unchanged on day 7 after MCAO, while the long-term recovery of sensorimotor function and brain atrophy were improved by GTA. Inhibition of lipogenesis using C75 or FASN shRNA reversed the beneficial effect of GTA. Finally, exogenous IL-33 improved BBB repair and long-term functional recovery after stroke.
CONCLUSION: Collectively, we concluded that treatment with GTA improved the BBB repair and functional recovery after ischemic stroke, probably by the enhancement of lipogenesis and IL-33 expression.

DOI: https://doi.org/10.1186/s12974-023-02942-3
Bio Protoc. 2023 Nov 05. 13(21): e4873

Identification of Acetylation Sites of Fatty Acid Synthase (FASN) by Mass Spectrometry and FASN Activity Assay.

Ting Miao, Hua Bai.

  Lysine acetylation is a conserved post-translational modification and a key regulatory mechanism for various cellular processes, including metabolic control, epigenetic regulation, and cellular signaling transduction. Recent advances in mass spectrometry (MS) enable the extensive identification of acetylated lysine residues of histone and non-histone proteins. However, protein enrichment before MS analysis may be necessary to improve the detection of low-abundant proteins or proteins that exhibit low acetylation levels. Fatty acid synthase (FASN), an essential enzyme catalyzing the de novo synthesis of fatty acids, has been found to be acetylated in various species, from fruit flies to humans. Here, we describe a step-by-step process of antibody-based protein enrichment and sample preparation for acetylation identification of endogenous FASN protein by MS-based proteomics analysis. Meanwhile, we provide a protocol for nicotinamide adenine dinucleotide phosphate (NADPH) absorbance assay for FASN activity measurement, which is one of the primary functional readouts of de novo lipogenesis. Key features • A comprehensive protocol for protein immunoprecipitation and sample preparation for acetylation site identification by mass spectrometry. • Step-by-step procedures for measurement of FASN activity of fruit fly larvae using an absorbance assay.

Keywords:  Acetyl-CoA; Auto-acetylation; De novo lipogenesis; FASN activity; Mass spectrometry; Post-translational modification

DOI:  https://doi.org/10.21769/BioProtoc.4873
Adv Sci (Weinh). 2023 Nov 15. e2303489

Branched-Chain Amino Acid Accumulation Fuels the Senescence-Associated Secretory Phenotype.

Yaosi Liang, Christopher Pan, Tao Yin, Lu Wang, Xia Gao, Ergang Wang, Holly Quang, De Huang, Lianmei Tan, Kun Xiang, Yu Wang, Peter B Alexander, Qi-Jing Li, Tso-Pang Yao, Zhao Zhang, Xiao-Fan Wang.

  The essential branched-chain amino acids (BCAAs) leucine, isoleucine, and valine play critical roles in protein synthesis and energy metabolism. Despite their widespread use as nutritional supplements, BCAAs' full effects on mammalian physiology remain uncertain due to the complexities of BCAA metabolic regulation. Here a novel mechanism linking intrinsic alterations in BCAA metabolism is identified to cellular senescence and the senescence-associated secretory phenotype (SASP), both of which contribute to organismal aging and inflammation-related diseases. Altered BCAA metabolism driving the SASP is mediated by robust activation of the BCAA transporters Solute Carrier Family 6 Members 14 and 15 as well as downregulation of the catabolic enzyme BCAA transaminase 1 during onset of cellular senescence, leading to highly elevated intracellular BCAA levels in senescent cells. This, in turn, activates the mammalian target of rapamycin complex 1 (mTORC1) to establish the full SASP program. Transgenic Drosophila models further indicate that orthologous BCAA regulators are involved in the induction of cellular senescence and age-related phenotypes in flies, suggesting evolutionary conservation of this metabolic pathway during aging. Finally, experimentally blocking BCAA accumulation attenuates the inflammatory response in a mouse senescence model, highlighting the therapeutic potential of modulating BCAA metabolism for the treatment of age-related and inflammatory diseases.

Keywords:  BCAA; SASP; age-related inflammation; mTORC1; senescence

DOI:  https://doi.org/10.1002/advs.202303489
Hum Mol Genet. 2023 Nov 17. pii: ddad196. [Epub ahead of print]

Mitochondrial aminoacyl-tRNA synthetases trigger unique compensatory mechanisms in neurons.

Oliver Podmanicky, Fei Gao, Benjamin Munro, Matthew J Jennings, Veronika Boczonadi, Denisa Hathazi, Juliane S Mueller, Rita Horvath.

  Mitochondrial aminoacyl-tRNA synthetase (mt-ARS) mutations cause severe, progressive, and often lethal diseases with highly heterogeneous and tissue-specific clinical manifestations. This study investigates the molecular mechanisms triggered by three different mt-ARS defects caused by biallelic mutations in AARS2, EARS2, and RARS2, using an in vitro model of human neuronal cells. We report distinct molecular mechanisms of mitochondrial dysfunction among the mt-ARS defects studied. Our findings highlight the ability of proliferating neuronal progenitor cells (iNPCs) to compensate for mitochondrial translation defects and maintain balanced levels of oxidative phosphorylation (OXPHOS) components, which becomes more challenging in mature neurons. Mutant iNPCs exhibit unique compensatory mechanisms, involving specific branches of the integrated stress response, which may be gene-specific or related to the severity of the mitochondrial translation defect. RNA sequencing revealed distinct transcriptomic profiles showing dysregulation of neuronal differentiation and protein translation. This study provides valuable insights into the tissue-specific compensatory mechanisms potentially underlying the phenotypes of patients with mt-ARS defects. Our novel in vitro model may more accurately represent the neurological presentation of patients and offer an improved platform for future investigations and therapeutic development.

Keywords:  aminoacyl-tRNA synthetase; mitochondrial biology; neurological disease; protein synthesis

DOI:  https://doi.org/10.1093/hmg/ddad196