Comparative metabolic profiling of posterior parietal cortex, amygdala, and hippocampus in conditioned fear memory

Fear conditioning and retrieval are suitable models to investigate the biological basis of various mental disorders. Hippocampus and amygdala neurons consolidate conditioned stimulus (CS)-dependent fear memory. Posterior parietal cortex is considered important for the CS-dependent conditioning and retrieval of fear memory. Metabolomic screening among functionally related brain areas provides molecular signatures and biomarkers to improve the treatment of psychopathologies. Herein, we analyzed and compared changes of metabolites in the hippocampus, amygdala, and posterior parietal cortex under the fear retrieval condition. Metabolite profiles of posterior parietal cortex and amygdala were similarly changed after fear memory retrieval. While the retrieval of fear memory perturbed various metabolic pathways, most metabolic pathways that overlapped among the three brain regions had high ranks in the enrichment analysis of posterior parietal cortex. In posterior parietal cortex, the most perturbed pathways were pantothenate and CoA biosynthesis, purine metabolism, glutathione metabolism, and NAD+ dependent signaling. Metabolites of posterior parietal cortex including 4′-phosphopantetheine, xanthine, glutathione, ADP-ribose, ADP-ribose 2′-phosphate, and cyclic ADP-ribose were significantly regulated in these metabolic pathways. These results point to the importance of metabolites of posterior parietal cortex in conditioned fear memory retrieval and may provide potential biomarker candidates for traumatic memory-related mental disorders. © 2021, The Author(s).1

액체 크로마토그래피 사중극자 비행시간형 질량분석기에 의한 조건화된 공포 기억에서 뇌 하위 영역의 대사 프로파일링

Conditioned fear memory, Metabolomics, Posterior parietal cortex, Amygdala, HippocampusFear conditioning and retrieval are suitable models to investigate the biological basis of various mental disorders. Among the subregions of the brain, hippocampus and amygdala are known to consolidate conditioned stimulus (CS)-dependent fear memory, and posterior parietal cortex is considered important for the CS-dependent conditioning and retrieval of fear memory. To provide molecular signatures and biomarkers for the improved treatment of psychopathologies, functionally related brain subregions are analyzed by UPLC-Q-TOF-MS/MS, which is an instrument commonly used in untargeted metabolomics. Herein, I analyzed and compared changed metabolites of posterior parietal cortex, amygdala, and hippocampus in response to conditioned fear memory. Metabolic profiles of posterior parietal cortex and amygdala were similarly changed after fear memory retrieval compared to that of hippocampus. Overlapping metabolic pathways were identified in three brain subregions through metabolite set enrichment analysis, and most of them were in high ranks in posterior parietal cortex. In pathway analysis of posterior parietal cortex, the most perturbed pathways were pantothenate and CoA biosynthesis, purine metabolism, glutathione metabolism, and NAD+ dependent signaling. Metabolites of posterior parietal cortex including 4′-phosphopantetheine, xanthine, glutathione, ADP-ribose, ADP-ribose phosphate, and cyclic ADP-ribose were significantly changed in these metabolic pathways. These findings point to the importance of metabolites in posterior parietal cortex when conditioned fear memory is given and suggest candidates for potential biomarkers in fear memory-related mental disorders.|공포 조건화(Fear conditioning) 및 인출(retrieval)은 다양한 정신 장애의 생물학적 기반을 조사하는 데 적합한 모델이다. 뇌의 하위 영역 중, 해마와 편도체는 중성적인 조건 자극 의존적 공포 기억을 고착하는 역할로 알려져 있고, 후두정엽피질은 중성적인 조건 의존적 공포 기억의 고착과 인출에 중요한 역할을 할 것으로 고려된다. 본 논문에서는 정신 장애의 치료 발달을 위한 분자 서명 및 생체 표지자를 제공하기 위해, 공포 기억과 관련된 세 뇌 하위 영역을 비표적화 대사체 분석에 일반적으로 사용되는 액체 크로마토그래피 사중극자 비행시간형 질량분석기로 분석했다. 그리고 조건화된 공포 기억에 의해 후두정엽피질, 편도체, 해마에서 변화된 대사산물을 관찰하고 비교했다. 공포 기억 인출 후, 후두정엽피질과 편도체의 대사산물 프로파일은 해마와 비교할 때보다 유사하게 변경됐다. 또한 대사 산물 세트 농축 분석을 수행하여 확인된 대사 경로 중에서, 후두정엽피질과 편도체 사이에서 겹치는 대사 경로가 가장 많았으며 세 뇌 하위 영역 사이에서 겹치는 대부분의 대사 경로는 후두정엽피질에서 통계적으로 높은 순위를 보였다. 후두정엽피질의 경로 분석에서 가장 교란된 경로는 판토텐산 및 조효소 A 생합성, 퓨린 대사, 글루타티온 대사 및 NAD+ 의존적 신호전달 경로였다. 4'-포스포판테테인(4′-phosphopantetheine), 잔틴(xanthine), 글루타티온(glutathione), 아데노신 이인산 리보스(ADP-ribose), 아데노신 이인산 리보스 인산염(ADP-ribose phosphate), 순환 아데노신 이인산 리보스(cyclic ADP-ribose)를 포함한 후두정엽피질의 대사 산물은 이러한 대사 경로에서 유의하게 조절됐다. 이 결과는 조건화된 공포 기억에서 후두정엽피질의 대사 산물의 중요성을 시사하고 공포 기억 관련 정신 장애에 대한 잠재적인 생체 표지자 후보를 제안한다.YⅠ. Introduction 1 1. Fear memory and Pavlovian fear conditioning paradigms 1 1.1 Neural circuits of processing conditioned fear memory 1 1.2 Potential roles of PPC in fear memory formation and processing 2 2. Metabolomics 5 2.1 Applications of metabolomics to fear memory-related diseases 5 2.2 Strategies of metabolomics 6 3. Aims of the study 10 Ⅱ. Materials and methods 11 1. Animals 11 2. Fear conditioning procedure 11 3. Tissue collection 12 4. Sample preparation 12 5. UPLC-MS analysis for data acquisition 13 6. Data filtering and visualization 13 7. UPLC-MS/MS analysis for identification of features 14 8. Pathway analysis 15 9. Real-time quantitative PCR 15 10. Statistical analysis 16 Ⅲ. Results 18 1. Contextual and cued fear conditioning and retrieval test for mice 18 2. Metabolic profiling of AMG in conditioned fear memory 22 3. Metabolic profiling of HPC in conditioned fear memory 26 4. Metabolic profiling of PPC in conditioned fear memory 30 5. Intersection analysis of metabolic profiling in three brain subregions 34 6. Metabolite set enrichment analysis of AMG in conditioned fear memory 39 7. Metabolite set enrichment analysis of HPC in conditioned fear memory 41 8. Metabolite set enrichment analysis of PPC in conditioned fear memory 43 9. Intersection analysis of metabolic pathways from three brain subregions 45 10. Pathway analysis of PPC metabolites altered in conditioned fear memory 53 11. Representative metabolite and metabolic pathways of PPC in conditioned fear memory 56 Ⅳ. Discussion 60 Ⅴ. Conclusion 64 Ⅵ. References 65 Abstract in Korean 71DoctordCollectio

Hypothalamic AMP-activated Protein Kinase as a Regulator of Food Intake and Energy Balance

The maintenance of appetite at proper levels, depending on the energy status, is important; otherwise abnormal appetite may cause a series of disorders, such as anorexia, hyperphagia, obesity, and its complications (diabetes mellitus, hypertension, cardiovascular disease, and fatty liver disease). Hypothalamic AMPactivated protein kinase (AMPK) integrates diverse hormonal and nutritional signals to regulate food intake and energy metabolism. Recent evidence suggests that different hormones, nutrients and synthetic chemicals can modulate AMPK activity in the hypothalamus, thereby regulating food intake and body weight, through neuropeptide expressions. In order to elucidate the mechanisms that control hypothalamic AMPK activity, a variety of studies have focused on finding upstream and downstream modulators of hypothalamic AMPK for the regulation of food intake and energy balance. This review highlights the current evidence for understanding how hypothalamic AMPK regulates food intake and energy balance, and will help in the development of effective interventions for the treatment of food intake-related disorders. In the future, it is hoped that new pharmaceutical developments targeting hypothalamic AMPK, in combination with careful clinical trials, will lead to improved and effective therapeutic strategies for complications caused by abnormal appetite and energy balance. © 2016 Bentham Science Publishers.1

D-chiro-inositol glycan reduces food intake by regulating hypothalamic neuropeptide expression via AKT-FoxO1 pathway

The regulation of food intake is important for body energy homeostasis. Hypothalamic insulin signaling decreases food intake by upregulating the expression of anorexigenic neuropeptides and downregulating the expression of orexigenic neuropeptides. INS-2, a Mn2+ chelate of 4-O-(2-amino-2-deoxy-β-d-galactopyranosyl)-3-O-methyl-d-chiro-inositol, acts as an insulin mimetic and sensitizer. We found that intracerebroventricular injection of INS-2 decreased body weight and food intake in mice. In hypothalamic neuronal cell lines, INS-2 downregulated the expression of neuropeptide Y (NPY), an orexigenic neuropeptide, but upregulated the expression of proopiomelanocortin (POMC), an anorexigenic neuropeptide, via modulation of the AKT-forkhead box-containing protein-O1 (FoxO1) pathway. Pretreatment of these cells with INS-2 enhanced the action of insulin on downstream signaling, leading to a further decrease in NPY expression and increase in POMC expression. These data indicate that INS-2 reduces food intake by regulating the expression of the hypothalamic neuropeptide genes through the AKT-FoxO1 pathway downstream of insulin. © 2016 Elsevier Inc. All rights reserved.

Tanycytic TSPO Inhibition Induces Lipophagy to Regulate Lipid Metabolism and Improve Energy Balance

Hypothalamic glial cells named tanycytes, which line the 3rd ventricle (3V), are components of the hypothalamic network that regulates a diverse array of metabolic functions for energy homeostasis. Herein, we report that TSPO (translocator protein), an outer mitochondrial protein, is highly enriched in tanycytes and regulates homeostatic responses to nutrient excess as a potential target for an effective intervention in obesity. Administration of a TSPO ligand, PK11195, into the 3V, and tanycyte-specific deletion of Tspo reduced food intake and elevated energy expenditure, leading to negative energy balance in a high-fat diet challenge. Ablation of tanycytic Tspo elicited AMPK-dependent lipophagy, breaking down lipid droplets into free fatty acids, thereby elevating ATP in a lipid stimulus. Our findings suggest that tanycytic TSPO affects systemic energy balance through macroautophagy/autophagy-regulated lipid metabolism, and highlight the physiological significance of TSPO in hypothalamic lipid sensing and bioenergetics in response to overnutrition. Abbreviations: 3V: 3rd ventricle; ACAC: acetyl-Coenzyme A carboxylase; AGRP: agouti related neuropeptide; AIF1/IBA1: allograft inflammatory factor 1; AMPK: AMP-activated protein kinase; ARC: arcuate nucleus; Atg: autophagy related; Bafilo: bafilomycin A1; CAMKK2: calcium/calmodulin-dependent protein kinase kinase 2, beta; CCCP: carbonyl cyanide m-chlorophenylhydrazone; CNS: central nervous system; COX4I1: cytochrome c oxidase subunit 4I1; FFA: free fatty acid; GFAP: glial fibrillary acidic protein; HFD: high-fat diet; ICV: intracerebroventricular; LAMP2: lysosomal-associated membrane protein 2; LD: lipid droplet; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MBH: mediobasal hypothalamus; ME: median eminence; MEF: mouse embryonic fibroblast; NCD: normal chow diet; NEFM/NFM: neurofilament medium; NPY: neuropeptide Y; OL: oleic acid; POMC: pro-opiomelanocortin-alpha; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; Rax: retina and anterior neural fold homeobox; RBFOX3/NeuN: RNA binding protein, fox-1 homolog (C. elegans) 3; RER: respiratory exchange ratio; siRNA: small interfering RNA; SQSTM1: sequestosome 1; TG: triglyceride; TSPO: translocator protein; ULK1: unc-51 like kinase 1; VCO2: carbon dioxide production; VMH: ventromedial hypothalamus; VO2: oxygen consumption. © 2019, © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.TRU