bims-medebr 2022-11-06 papers

bims-medebr

Biomed News

on Metabolism of the developing brain

Issue of 2022–11–06
twenty-six papers selected by
Regina F. Fernández, Johns Hopkins University

Quantifying brain cholesterol homeostasis.
Glial Glutamine Homeostasis in Health and Disease.
Increasing brain glucose uptake by Gypenoside LXXV ameliorates cognitive deficits in a mouse model of diabetic Alzheimer's disease.
Functional consequences of brain exposure to saturated fatty acids: From energy metabolism and insulin resistance to neuronal damage.
Activation of arcuate nucleus glucagon-like peptide-1 receptor-expressing neurons suppresses food intake.
Evidence of increased sequestration of pro-resolving lipid mediators within brain esterified lipid pools of multiple sclerosis patients.
Role of P2Y receptors in astrocyte physiology and pathophysiology.
The plasma phospholipidome of the bottlenose dolphin (Tursiops truncatus) is modulated by both sex and developmental stage.
Proline Metabolism in Neurological and Psychiatric Disorders.
Dietary Flavonoids and Adult Neurogenesis: Potential Implications for Brain Aging.
Cerebral 18 F-fluorodeoxyglucose metabolism alteration of reward- and motivation-related regions in groups of different BMI classifications.
Impact of postnatal nutrition on neurodevelopmental outcome in rat model of intrauterine growth restriction.
The roles of microglia and astrocytes in myelin phagocytosis in the central nervous system.
Nano-positioning and tubulin conformation contribute to axonal transport regulation of mitochondria along microtubules.
Pyrylium-Based Derivatization for Rapid Labeling and Enhanced Detection of Cholesterol in Mass Spectrometry Imaging.
NRG1/ErbB2 axis regulated mitochondrial function and antioxidant enzymes of neural stem cells in the cochlear nucleus partially through PGC-1α.
All the brain's a stage for serotonin: the forgotten story of serotonin diffusion across cell membranes.
A Heterozygous Mutation in MFF Associated with a Mild Mitochondrial Phenotype.
Nutritional interventions to support acute mTBI recovery.
Oxaloacetate acid ameliorates paraquat-induced acute lung injury by alleviating oxidative stress and mitochondrial dysfunction.
Metabolic and physical function are improved with lifelong 15% calorie restriction in aging male mice.
Preeclampsia and its relationship to pathological brain aging.
HSPB1 overexpression improves hypoxic-ischemic brain damage by attenuating ferroptosis in rats through promoting G6PD expression.
Preterm Brain Injury and Neurodevelopmental Outcomes: A Meta-analysis.
Brain monoamine vesicular transport disease caused by homozygous SLC18A2 variants: A study in 42 affected individuals.
Compound Heterozygote Mutation in the SMPD1 Gene Leading to Nieman-Pick Disease Type A.

Nat Rev Drug Discov. 2022 Nov 02.

Quantifying brain cholesterol homeostasis.

Sarah Crunkhorn.

DOI: https://doi.org/10.1038/d41573-022-00182-3
Neurochem Res. 2022 Nov 02.

Glial Glutamine Homeostasis in Health and Disease.

Jens V Andersen, Arne Schousboe.

  Glutamine is an essential cerebral metabolite. Several critical brain processes are directly linked to glutamine, including ammonia homeostasis, energy metabolism and neurotransmitter recycling. Astrocytes synthesize and release large quantities of glutamine, which is taken up by neurons to replenish the glutamate and GABA neurotransmitter pools. Astrocyte glutamine hereby sustains the glutamate/GABA-glutamine cycle, synaptic transmission and general brain function. Cerebral glutamine homeostasis is linked to the metabolic coupling of neurons and astrocytes, and relies on multiple cellular processes, including TCA cycle function, synaptic transmission and neurotransmitter uptake. Dysregulations of processes related to glutamine homeostasis are associated with several neurological diseases and may mediate excitotoxicity and neurodegeneration. In particular, diminished astrocyte glutamine synthesis is a common neuropathological component, depriving neurons of an essential metabolic substrate and precursor for neurotransmitter synthesis, hereby leading to synaptic dysfunction. While astrocyte glutamine synthesis is quantitatively dominant in the brain, oligodendrocyte-derived glutamine may serve important functions in white matter structures. In this review, the crucial roles of glial glutamine homeostasis in the healthy and diseased brain are discussed. First, we provide an overview of cellular recycling, transport, synthesis and metabolism of glutamine in the brain. These cellular aspects are subsequently discussed in relation to pathological glutamine homeostasis of hepatic encephalopathy, epilepsy, Alzheimer's disease, Huntington's disease and amyotrophic lateral sclerosis. Further studies on the multifaceted roles of cerebral glutamine will not only increase our understanding of the metabolic collaboration between brain cells, but may also aid to reveal much needed therapeutic targets of several neurological pathologies.

Keywords:  Astrocytes; Brain energy and neurotransmitter metabolism; GABA-glutamine cycle; Glutamate-glutamine cycle; Glutamine transporters; Neurodegenerative diseases; Oligodendrocytes

DOI:  https://doi.org/10.1007/s11064-022-03771-1
Phytother Res. 2022 Nov 03.

Increasing brain glucose uptake by Gypenoside LXXV ameliorates cognitive deficits in a mouse model of diabetic Alzheimer's disease.

Xiangbao Meng, Yuan Zhang, Zongyang Li, Guoxu Ma, Xiejun Zhang, Di Zhang, Weiwei Cao, Sicen Wang, Qian Cai, Ping Cui, Guodong Huang.

  We have previously reported that Gypenoside LXXV (GP-75), a novel natural PPARγ agonist isolated from Gynostemma pentaphyllum, ameliorated cognitive deficits in db/db mice. In this study, we further investigated the beneficial effects on cognitive impairment in APP/PS1 mice and a mouse model of diabetic AD (APP/PS1xdb/db mice). Interestingly, intragastric administration of GP-75 (40 mg/kg/day) for 3 months significantly attenuated cognitive deficits in APP/PS1 and APP/PS1xdb/db mice. GP-75 treatment markedly reduced the levels of glucose, HbA1c and insulin in serum and improved glucose tolerance and insulin sensitivity in APP/PS1xdb/db mice. Notably, GP-75 treatment decreased the β-amyloid (Aβ) burden, as measured by 11 C-PIB PET imaging. Importantly, GP-75 treatment increased brain glucose uptake as measured by 18 F-FDG PET imaging. Moreover, GP-75 treatment upregulated PPARγ and increased phosphorylation of Akt (Ser473) and GLUT4 expression levels but decreased phosphorylation of IRS-1 (Ser616) in the hippocampi of both APP/PS1 and APP/PS1xdb/db mice. Furthermore, GP-75-induced increases in GLUT4 membrane translocation in primary hippocampal neurons from APP/PS1xdb/db mice was abolished by cotreatment with the selective PPARγ antagonist GW9662 or the PI3K inhibitor LY294002. In summary, GP-75 ameliorated cognitive deficits in APP/PS1 and APP/PS1xdb/db mice by enhancing glucose uptake via activation of the PPARγ/Akt/GLUT4 signaling pathways.

Keywords:  Alzheimer's disease; diabetes; glucose transporter 4; peroxisome proliferator-activated receptor gamma

DOI:  https://doi.org/10.1002/ptr.7639
Endocrinol Diabetes Metab. 2022 Nov 02. e386

Functional consequences of brain exposure to saturated fatty acids: From energy metabolism and insulin resistance to neuronal damage.

Karina Sánchez-Alegría, Clorinda Arias.

   INTRODUCTION: Saturated fatty acids (FAs) are the main component of high-fat diets (HFDs), and high consumption has been associated with the development of insulin resistance, endoplasmic reticulum stress and mitochondrial dysfunction in neuronal cells. In particular, the reduction in neuronal insulin signaling seems to underlie the development of cognitive impairments and has been considered a risk factor for Alzheimer's disease (AD).
METHODS: This review summarized and critically analyzed the research that has impacted the field of saturated FA metabolism in neurons.
RESULTS: We reviewed the mechanisms for free FA transport from the systemic circulation to the brain and how they impact neuronal metabolism. Finally, we focused on the molecular and the physiopathological consequences of brain exposure to the most abundant FA in the HFD, palmitic acid (PA).
CONCLUSION: Understanding the mechanisms that lead to metabolic alterations in neurons induced by saturated FAs could help to develop several strategies for the prevention and treatment of cognitive impairment associated with insulin resistance, metabolic syndrome, or type II diabetes.

Keywords:  energy metabolism; insulin resistance; mitochondrial dysfunction; neurodegeneration; palmitic acid; saturated fatty acids

DOI:  https://doi.org/10.1002/edm2.386
Cell Biosci. 2022 Oct 29. 12(1): 178

Activation of arcuate nucleus glucagon-like peptide-1 receptor-expressing neurons suppresses food intake.

Ishnoor Singh, Le Wang, Baijuan Xia, Ji Liu, Azeddine Tahiri, Abdelfattah El Ouaamari, Michael B Wheeler, Zhiping P Pang.

   BACKGROUND: Central nervous system (CNS) control of metabolism plays a pivotal role in maintaining energy balance. In the brain, Glucagon-like peptide 1 (GLP-1), encoded by the proglucagon 'Gcg' gene, produced in a distinct population of neurons in the nucleus tractus solitarius (NTS), has been shown to regulate feeding behavior leading to the suppression of appetite. However, neuronal networks that mediate endogenous GLP-1 action in the CNS on feeding and energy balance are not well understood.
RESULTS: We analyzed the distribution of GLP-1R-expressing neurons and axonal projections of NTS GLP-1-producing neurons in the mouse brain. GLP-1R neurons were found to be broadly distributed in the brain and specific forebrain regions, particularly the hypothalamus, including the arcuate nucleus of the hypothalamus (ARC), a brain region known to regulate energy homeostasis and feeding behavior, that receives dense NTSGcg neuronal projections. The impact of GLP-1 signaling in the ARC GLP-1R-expressing neurons and the impact of activation of ARC GLP-1R on food intake was examined. Application of GLP-1R specific agonist Exendin-4 (Exn-4) enhanced a proportion of the ARC GLP-1R-expressing neurons and pro-opiomelanocortin (POMC) neuronal action potential firing rates. Chemogenetic activation of the ARC GLP-1R neurons by using Cre-dependent hM3Dq AAV in the GLP-1R-ires-Cre mice, established that acute activation of the ARC GLP-1R neurons significantly suppressed food intake but did not have a strong impact on glucose homeostasis.
CONCLUSIONS: These results highlight the importance of central GLP-1 signaling in the ARC that express GLP-1R that upon activation, regulate feeding behavior.

Keywords:  Chemogenetics; Exendin-4; Feeding; Glucagon-like peptide-1; Glucose tolerance; Hypothalamus; Pro-opiomelanocortin

DOI:  https://doi.org/10.1186/s13578-022-00914-3
Mult Scler Relat Disord. 2022 Oct 09. pii: S2211-0348(22)00740-4. [Epub ahead of print]68 104236

Evidence of increased sequestration of pro-resolving lipid mediators within brain esterified lipid pools of multiple sclerosis patients.

Qing Shen, Yurika Otoki, Raymond A Sobel, Rashed M Nagra, Ameer Y Taha.

   BACKGROUND: Unresolved inflammation in multiple sclerosis (MS) is associated with progressive demyelination and symptom worsening. In the brain, both inflammation and resolution pathways are mediated by free lipid mediators (i.e., oxylipins) that can be derived from the enzymatic hydrolysis of esterified oxylipins . It is not known whether disturbances in the turnover of free lipid mediators from esterified pools exist in postmortem brain of MS patients. We hypothesized that resolution pathways are impaired in MS patients because of disturbances in the turnover of free pro-resolving lipid mediators from esterified lipids. The objective was to characterize free and esterified oxylipins in postmortem prefrontal cortex of MS and unaffected control participants.
METHODS: Oxylipins in free, neutral lipid and phospholipid pools were extracted from prefrontal cortex of 10 MS participants and 5 unaffected controls, separated by solid phase extraction columns, and quantified by ultra-high-pressure liquid chromatography-tandem mass spectrometry. Significant differences between the control and MS groups were determined by an unpaired t-test with Benjamini and Hochberg False Discovery Rate correction (10%) applied to oxylipins within each lipid pool.
RESULTS: The concentration of 7 esterified pro-resolving fatty acid epoxides within neutral lipids were significantly higher by 126%-285% in postmortem prefrontal cortex of MS compared to control participants. The concentration of esterified linoleic acid-derived 9(10)-epoxy-octadecenoic acid, a pro-inflammatory epoxide, was higher by 206% in MS compared to controls. No significant changes were observed in free or phospholipid-bound oxylipins.
CONCLUSION: In MS, several pro-resolving lipid mediators are trapped within prefrontal cortex neutral lipids, potentially limiting their supply and availability in the free bioactive form. This may explain why inflammation resolution is impaired in MS patients.

Keywords:  Inflammation; Lipid mediators; Multiple sclerosis; Neutral lipids; Oxylipins; Post mortem

DOI:  https://doi.org/10.1016/j.msard.2022.104236
Neuropharmacology. 2022 Oct 31. pii: S0028-3908(22)00370-7. [Epub ahead of print] 109311

Role of P2Y receptors in astrocyte physiology and pathophysiology.

Christian Lohr.

  Astrocytes are active constituents of the brain that manage ion homeostasis and metabolic support of neurons and directly tune synaptic transmission and plasticity. Astrocytes express all known P2Y receptors. These regulate a multitude of physiological functions such as cell proliferation, Ca2+ signalling, gliotransmitter release and neurovascular coupling. In addition, P2Y receptors are fundamental in the transition of astrocytes into reactive astrocytes, as occuring in many brain disorders such as neurodegenerative diseases, neuroinflammation and epilepsy. This review summarizes the current literature addressing the function of P2Y receptors in astrocytes in the healthy brain as well as in brain diseases.

Keywords:  Astrocyte; Ca(2+) signalling; Gliotransmission; Neuropathology; P2Y(1); Purinergic signalling

DOI:  https://doi.org/10.1016/j.neuropharm.2022.109311
Mol Omics. 2022 Oct 31.

The plasma phospholipidome of the bottlenose dolphin (Tursiops truncatus) is modulated by both sex and developmental stage.

João P Monteiro, Helena B Ferreira, Tânia Melo, Carla Flanagan, Nuno Urbani, João Neves, Pedro Domingues, M Rosário Domingues.

Lipidomics represent a valid complementary tool to the biochemical analysis of plasma in humans. However, in cetaceans, these tools have been unexplored. Here, we evaluated how the plasma lipid composition of Tursiops truncatus is modulated by developmental stage and sex, aiming at a potential use of lipidomics in integrated strategies to monitor cetacean health. We characterized the fatty acid profile and detected a total of 26 fatty acids in T. truncatus plasma. The most abundant fatty acids were palmitic acid (C16:0), stearic acid (C18:0) and oleic acid (C18:1n-9). Interestingly, there are consistent differences between the fatty acid profile of mature female and mature male specimens. Phospholipidome analysis identified 320 different lipid species belonging to phosphatidylcholine (PC, 105 lipid species), lysophosphatidylcholine (42), phosphatidylethanolamine (PE, 67), lysophosphatidylethanolamine (18), phosphatidylglycerol (14), lysophosphatidylglycerol (8), phosphatidylinositol (14), lysophosphatidylinositol (2), phosphatidylserine (3), sphingomyelin (45) and ceramides (2) classes. The statistical analysis of the phospholipidome showed that its composition allows discriminating mature animals between sexes and mature males from immature males. Notably, discrimination between sexes is mainly determined by the contents of PE plasmalogens and lysophospholipids (LPC and LPE), while the differences between mature and immature male animals were mainly determined by the levels of PC lipids. This is the first time that a correlation between developmental stage and sex and the lipid composition of the plasma has been established in cetaceans. Being able to discern between age and sex-related changes is an encouraging step towards using these tools to also detect differences related to disease/dysfunction processes.

DOI: https://doi.org/10.1039/d2mo00202g
Mol Cells. 2022 Nov 02.

Proline Metabolism in Neurological and Psychiatric Disorders.

Yuxiao Yao, Weiping Han.

  Proline plays a multifaceted role in protein synthesis, redox balance, cell fate regulation, brain development, and other cellular and physiological processes. Here, we focus our review on proline metabolism in neurons, highlighting the role of dysregulated proline metabolism in neuronal dysfunction and consequently neurological and psychiatric disorders. We will discuss the association between genetic and protein function of enzymes in the proline pathway and the development of neurological and psychiatric disorders. We will conclude with a potential mechanism of proline metabolism in neuronal function and mental health.

Keywords:  cell metabolism; neurological disease; neuron; proline; psychiatric disease

DOI:  https://doi.org/10.14348/molcells.2022.0115
Curr Neuropharmacol. 2022 Oct 31.

Dietary Flavonoids and Adult Neurogenesis: Potential Implications for Brain Aging.

Sergio Davinelli, Alessandro Medoro, Sawan Ali, Daniela Passarella, Mariano Intrieri, Giovanni Scapagnini.

  Adult neurogenesis deficiency has been proposed to be a common hallmark in different age- related neurodegenerative diseases. The administration of flavonoids is currently reported as a potentially beneficial strategy for preventing brain aging alterations, including adult neurogenesis decline. Flavonoids are a class of plant-derived dietary polyphenols that have drawn attention for their neuroprotective and pro-cognitive effects. Although they undergo extensive metabolism and localize in the brain at low concentrations, flavonoids are now believed to improve cerebral vasculature and interact with signal transduction cascades involved in the regulation of adult neurogenesis. Furthermore, many dietary flavonoids have been shown to reduce oxidative stress and neuroinflammation, improving the neuronal microenvironment where adult neurogenesis occurs. The overall goal of this review is to summarize the evidence supporting the role of flavonoids in modulating adult neurogenesis as well as to highlight how these dietary agents may be promising candidates in restoring healthy brain function during physiological and pathological aging.

Keywords:  Adult Neurogenesis; Aging; Brain; Diet; Flavonoids; Neurodegeneration

DOI:  https://doi.org/10.2174/1570159X21666221031103909
Obesity (Silver Spring). 2022 Nov;30(11): 2213-2221

Cerebral 18 F-fluorodeoxyglucose metabolism alteration of reward- and motivation-related regions in groups of different BMI classifications.

Yu-Jie Duan, Mou-Xiong Zheng, Jia-Jia Wu, Jie Ma, Xiang-Xin Xing, Zhen-Zhen Ma, Si-Si Li, Yu-Lin Li, Xin Xue, Xu-Yun Hua, Jian-Guang Xu.

OBJECTIVE: This study explored the relationship between BMI and regional cerebral glucose metabolism and explicitly detected regions with significant differences in cerebral metabolism using positron emission tomography (PET)/magnetic resonance imaging in the resting state.
METHODS: Corresponding PET images acquired from 220 participants were sorted into four groups according to Asian BMI standards: underweight, normal weight, overweight, and obesity. Pearson correlation coefficient analysis was performed to assess the association between BMI and standard uptake value. The regional cerebral glucose metabolism was measured in the fasted state. The PET images were analyzed using statistical parameter maps. One-way ANOVA was used to explore differences in the standard uptake value as an indicator of regional cerebral glucose metabolism.
RESULTS: This study found that lower cerebral glucose metabolism in reward- and motivation-related regions was accompanied by more severe obesity and that regional cerebral glucose metabolism activities were negatively correlated with BMI. In addition, more severe obesity was accompanied by a larger range of areas with significant differences independent of current dietary status.
CONCLUSIONS: These findings suggest that the reward and motivation circuits may be a factor regulating energy balance and influencing the degree of obesity.

DOI: https://doi.org/10.1002/oby.23553
Eur Rev Med Pharmacol Sci. 2022 Oct;pii: 30020. [Epub ahead of print]26(20): 7498-7505

Impact of postnatal nutrition on neurodevelopmental outcome in rat model of intrauterine growth restriction.

B Baysal, S Miçili, D Engür, P Akokay, A R Karabulut, P Keskinoğlu, O Yilmaz, A Kumral.

OBJECTIVE: There are limited data on nutritional management of infants with intrauterine growth restriction (IUGR). Postnatal protein supplementation for promoting growth is a common clinical practice in neonatology. The present study aims to investigate the consequences of protein supplementation on long-term growth, brain and body weight, brain histology and behavioral outcome in a rat model of IUGR.
MATERIALS AND METHODS: Twenty-four IUGR-formed rat puppies and 12 healthy puppies were included in the study. IUGR model was established by low (10%) protein diet throughout pregnancy together with intraperitoneal injection of lipopolysaccharide (LPS). Pups were started to be fed with either standard protein (SP), or high protein (HP) diet until postnatal day (PN) 35. Puppies in the control group were given SP diet for 35 days. Six pups from each group were sacrificed at PN7, remaining six were evaluated by Morris water maze test between PN 30 to 35 days and then sacrificed at PN35. Histologic evaluation of brain tissue was performed at PN7 and PN35.
RESULTS: IUGR group displayed lower body and brain weights at PN7 when compared with control. At PN35, SP group achieved similar brain/body weight ratios with control, whereas HP group displayed lowest brain/body weight ratio. The number of TUNEL positive cells was significantly higher and myelin basic protein and oligodendrocyte marker O4 immunoreactivity were significantly lower in HP group when compared with SP at PN35. Neuronal density in prefrontal cortex and hippocampus at PN7 were similar among SP and HP groups, but significantly lower in HP group when compared with SP at PN35. SP group displayed better results in the Morris water maze test when compared with HP group.
CONCLUSIONS: Although postnatal HP support is associated with increase in body weight at PN35, it did not result in better brain/body weight ratios in the rat model of IUGR. In IUGR rats, HP diet was associated with increased apoptosis in brain tissue with lower neuronal density and decreased myelination when compared to SP. Furthermore, better neurodevelopmental scores were achieved by SP diet rather than HP support in IUGR.

DOI: https://doi.org/10.26355/eurrev_202210_30020
J Cereb Blood Flow Metab. 2022 Nov 02. 271678X221137762

The roles of microglia and astrocytes in myelin phagocytosis in the central nervous system.

Tongtong Xu, Chang Liu, Shiyu Deng, Lin Gan, Zhijun Zhang, Guo-Yuan Yang, Hengli Tian, Yaohui Tang.

  Myelination is an important process in the central nervous system (CNS). Oligodendrocytes (OLs) extend multiple layers to densely sheath on axons, composing the myelin to achieve efficient electrical signal conduction. The myelination during developmental stage maintains a balanced state. However, numerous CNS diseases including neurodegenerative and cerebrovascular diseases cause demyelination and disrupt the homeostasis, resulting in inflammation and white matter deficits. Effective clearance of myelin debris is needed in the region of demyelination, which is a key step for remyelination and tissue regeneration. Microglia and astrocytes are the major resident phagocytic cells in the brain, which may play different or collaborative roles in myelination. Microglia and astrocytes participate in developmental myelination through engulfing excessive unneeded myelin. They are also involved in the clearance of degenerated myelin debris for accelerating remyelination, or engulfing healthy myelin sheath for inhibiting remyelination. This review focuses on the roles of microglia and astrocytes in phagocytosing myelin in the developmental brain and diseased brain. In addition, the interaction between microglia and astrocytes to mediate myelin engulfment is also summarized.

Keywords:  Astrocyte; brain; microglia; myelin engulfment; myelination

DOI:  https://doi.org/10.1177/0271678X221137762
Proc Natl Acad Sci U S A. 2022 Nov 08. 119(45): e2203499119

Nano-positioning and tubulin conformation contribute to axonal transport regulation of mitochondria along microtubules.

Valérie Van Steenbergen, Flavie Lavoie-Cardinal, Youcef Kazwiny, Marianna Decet, Tobie Martens, Patrik Verstreken, Werend Boesmans, Paul De Koninck, Pieter Vanden Berghe.

  Correct spatiotemporal distribution of organelles and vesicles is crucial for healthy cell functioning and is regulated by intracellular transport mechanisms. Controlled transport of bulky mitochondria is especially important in polarized cells such as neurons that rely on these organelles to locally produce energy and buffer calcium. Mitochondrial transport requires and depends on microtubules that fill much of the available axonal space. How mitochondrial transport is affected by their position within the microtubule bundles is not known. Here, we found that anterograde transport, driven by kinesin motors, is susceptible to the molecular conformation of tubulin in neurons both in vitro and in vivo. Anterograde velocities negatively correlate with the density of elongated tubulin dimers like guanosine triphosphate (GTP)-tubulin. The impact of the tubulin conformation depends primarily on where a mitochondrion is positioned, either within or at the rim of microtubule bundle. Increasing elongated tubulin levels lowers the number of motile anterograde mitochondria within the microtubule bundle and increases anterograde transport speed at the microtubule bundle rim. We demonstrate that the increased kinesin velocity and density on microtubules consisting of elongated dimers add to the increased mitochondrial dynamics. Our work indicates that the molecular conformation of tubulin contributes to the regulation of mitochondrial motility and as such to the local distribution of mitochondria along axons.

Keywords:  STED; microtubules; mitochondria; neuronal axon; transport

DOI:  https://doi.org/10.1073/pnas.2203499119
J Am Soc Mass Spectrom. 2022 Nov 04.

Pyrylium-Based Derivatization for Rapid Labeling and Enhanced Detection of Cholesterol in Mass Spectrometry Imaging.

Shuai Huang, Xinxin Liu, Dan Liu, Xiaozhe Zhang, Lihua Zhang, Weidong Le, Yukui Zhang.

Cholesterol in the central nervous system has been increasingly found to be closely related to neurodegenerative diseases. Defects in cholesterol metabolism can cause structural and functional disorders of the central nervous system. The detection of abnormal cholesterol is of great significance for the cognition of physiological and pathological states of organisms, and the spatial distribution of cholesterol can also provide more clues for our understanding of the complex mechanism of disease. Here, we developed a novel pyrylium-based derivatization reagent combined with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to visualize cholesterol in biological tissues. A new class of charged hydroxyl derivatization reagents was designed and synthesized, and finally 1-(carboxymethyl)-2,4,6-trimethylpyridinium (CTMP) was screened for tissue derivatization of cholesterol. Different from the shortcomings of traditional hydroxyl labeling methods such as harsh reaction conditions and long reaction time, in our study, we combined the advantages of CTMP itself and the EDCl/HOBt reaction system to achieve instant labeling of cholesterol on tissues through two-step activation. In addition, we also reported changes in cholesterol content in different stages and different brain regions during disease development in SOD1 mutant mouse model. The cholesterol derivatization method we developed provides an efficient way to explore the distribution and spatial metabolic network of cholesterol in biological tissues.

DOI: https://doi.org/10.1021/jasms.2c00271
Neurosci Lett. 2022 Oct 31. pii: S0304-3940(22)00503-1. [Epub ahead of print] 136942

NRG1/ErbB2 axis regulated mitochondrial function and antioxidant enzymes of neural stem cells in the cochlear nucleus partially through PGC-1α.

Jian Wang, Li Wei, Keyong Tian, Min Xu, Xiaodong Chen, Fuquan Chen, Dingjun Zha, Tao Xue.

  Neuregulin-1 (NRG1)/erythroblastic leukaemia viral oncogene homologues 2 (ErbB2) pathway had been implicated in promoting differentiation and suppressing apoptosis of neuronal stem cells (NSCs) isolated from cochlear nucleus. In the current study, we aimed at determining the effects of NRG1/ErbB2 on mitochondrial (mt) function of NSCs. As expected, NRG1 increased the expression of mitofusin (Mfn) 1 and Mfn2 and decreased the expression of mitochondrial fission protein 1 (Fis1) and dynamin-related protein 1 (Drp1). However, after ErbB2 knockout, Mfn1 and Mfn2 expression decreased while Fis1 and Drp1 increased. Moreover, the increased mtDNA copy number and intracellular ATP level, elevated ATPase activities as well as decreased lactate production induced by NRG1 were partially reversed by ErbB2 knockout. Additionally, NRG1 treatment increased the activities of catalase, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and upregulated the protein expression of catalase, manganese superoxide dismutase (MnSOD), peroxisome proliferator-activated receptor-γ coactlvator-1α (PGC-1α), nuclear respiratory factor 1 (NRF1) and transcription factor A, mitochondrial (TFAM), which were also reversed by ErbB2 knockout. Furthermore, PGC-1α overexpression partially reversed the above effects of ErbB2 knockout. In conclusion, these findings suggest that the promotion of mitochondrial function of NRG1/ErbB2 axis is at least in part mediated by PGC-1α in NSCs from cochlear nucleus.

Keywords:  NRG1/ErbB2 axis; PGC-1α; cochlear nucleus; mitochondrial biogenesis; mitochondrial function; neural stem cells

DOI:  https://doi.org/10.1016/j.neulet.2022.136942
Proc Biol Sci. 2022 11 09. 289(1986): 20221565

All the brain's a stage for serotonin: the forgotten story of serotonin diffusion across cell membranes.

Paul W Andrews, Catherine Bosyj, Luke Brenton, Laura Green, Paul J Gasser, Christopher A Lowry, Virginia M Pickel.

  In the conventional model of serotonin neurotransmission, serotonin released by neurons in the midbrain raphe nuclei exerts its actions on forebrain neurons by interacting with a large family of post-synaptic receptors. The actions of serotonin are terminated by active transport of serotonin back into the releasing neuron, which is mediated by the serotonin reuptake transporter (SERT). Because SERT is expressed pre-synaptically and is widely thought to be the only serotonin transporter in the forebrain, the conventional model does not include serotonin transport into post-synaptic neurons. However, a large body of evidence accumulating since the 1970s has shown that serotonin, despite having a positive charge, can cross cell membranes through a diffusion-like process. Multiple low-affinity, high-capacity, sodium-independent transporters, widely expressed in the brain, allow the carrier-mediated diffusion of serotonin into forebrain neurons. The amount of serotonin crossing cell membranes through this mechanism under physiological conditions is considerable. Most prominent textbooks fail to include this alternative method of serotonin uptake in the brain, and even most neuroscientists are unaware of it. This failure has limited our understanding of a key regulator of serotonergic neurotransmission, impeded research on the potential intracellular actions of serotonin in post-synaptic neurons and glial cells, and may have impeded our understanding of the mechanism by which antidepressant medications reduce depressive symptoms.

Keywords:  carrier-mediated diffusion; mitochondria; organic cation transporters; serotonin

DOI:  https://doi.org/10.1098/rspb.2022.1565
J Neuromuscul Dis. 2022 Oct 28.

A Heterozygous Mutation in MFF Associated with a Mild Mitochondrial Phenotype.

Daisuke Murata, Christopher Grunseich, Miho Iijima, David Chan, Andrea Corse, Ahmet Hoke, Alice Schindler, Hiromi Sesaki, Ricardo H Roda.

   BACKGROUND: The number of mutations in nuclear encoded genes causing mitochondrial disease is ever increasing. Identification of these mutations is particularly important in the diagnosis of neuromuscular disorders as their presentation may mimic other acquired disorders.We present a novel heterozygous variant in mitochondrial fission factor (MFF) which mimics myasthenia gravis.
OBJECTIVE: To determine if the MFF c.937G>A, p.E313K variant causes a mild mitochondrial phenotype.
METHODS: We used whole exome sequencing (WES) to identify a novel heterozygous variant in MFF in a patient with ptosis, fatigue and muscle weakness. Using patient derived fibroblasts, we performed assays to evaluate mitochondrial and peroxisome dynamics.
RESULTS: We show that fibroblasts derived from this patient are defective in mitochondrial fission, despite normal recruitment of Drp1 to the mitochondria.
CONCLUSIONS: The MFF c.937G>A, p.E313K variant leads to a mild mitochondrial phenotype and is associated with defective mitochondrial fission in patient-derived fibroblasts.

Keywords:  MFF; Mitochondrial myopathies; mitochondrial dynamics; mitochondrial fission factor

DOI:  https://doi.org/10.3233/JND-221532
Front Nutr. 2022 ;9 977728

Nutritional interventions to support acute mTBI recovery.

Emma Finnegan, Ed Daly, Alan J Pearce, Lisa Ryan.

  When mild traumatic brain injury (mTBI) occurs following an impact on the head or body, the brain is disrupted leading to a series of metabolic events that may alter the brain's ability to function and repair itself. These changes may place increased nutritional demands on the body. Little is known on whether nutritional interventions are safe for patients to implement post mTBI and whether they may improve recovery outcomes. To address this knowledge gap, we conducted a systematic review to determine what nutritional interventions have been prescribed to humans diagnosed with mTBI during its acute period (<14 days) to support, facilitate, and result in measured recovery outcomes.
Methods: Databases CINAHL, PubMed, SPORTDiscus, Web of Science, and the Cochrane Library were searched from inception until January 6, 2021; 4,848 studies were identified. After removing duplicates and applying the inclusion and exclusion criteria, this systematic review included 11 full papers.
Results: Patients that consumed enough food to meet calorie and macronutrient (protein) needs specific to their injury severity and sex within 96 h post mTBI had a reduced length of stay in hospital. In addition, patients receiving nutrients and non-nutrient support within 24-96 h post mTBI had positive recovery outcomes. These interventions included omega-3 fatty acids (DHA and EPA), vitamin D, mineral magnesium oxide, amino acid derivative N-acetyl cysteine, hyperosmolar sodium lactate, and nootropic cerebrolysin demonstrated positive recovery outcomes, such as symptom resolution, improved cognitive function, and replenished nutrient deficiencies (vitamin D) for patients post mTBI.
Conclusion: Our findings suggest that nutrition plays a positive role during acute mTBI recovery. Following mTBI, patient needs are unique, and this review presents the potential for certain nutritional therapies to support the brain in recovery, specifically omega-3 fatty acids. However, due to the heterogenicity nature of the studies available at present, it is not possible to make definitive recommendations.
Systematic review registration: The systematic review conducted following the PRISMA guidelines protocol was registered (CRD42021226819), on Prospero.

Keywords:  brain injury - traumatic; concussion; mild traumatic brain injury (mTBI); nutrition; omega 3 (n-3) polyunsaturated fatty acids; supplementation; vitamin

DOI:  https://doi.org/10.3389/fnut.2022.977728
Front Pharmacol. 2022 ;13 1029775

Oxaloacetate acid ameliorates paraquat-induced acute lung injury by alleviating oxidative stress and mitochondrial dysfunction.

Wenwen Li, Mengxuan Li, Kaiyuan Chen, Yahui Tang, Ran Yin, Linhua Lan, Guangliang Hong.

  Acute lung injury (ALI) is the primary cause of death among patients with acute paraquat (PQ) poisoning, whereby peroxidative damage is an important mechanism underlying PQ-induced lung injury. There is a lack of effective interventional drugs for patients with PQ poisoning. Oxaloacetic acid (OAA) participates in multiple in vivo metabolic processes, whereby it facilitates the clearance of reactive oxygen species (ROS) and improves mitochondrial function. The study aimed to assess the protective effects of OAA on PQ-induced ALI and elucidate the underlying molecular mechanism. Our data demonstrated that OAA treatment significantly alleviated PQ-induced ALI and improved the survival rate of PQ-poisoned mice, and also alleviated PQ-induced cellular oxidative stress and mitochondrial dysfunction. OAA-mediated alleviation of PQ-induced mitochondrial dysfunction depends on the following mechanisms which may explain the above findings: 1) OAA effectively cleared intracellular ROS, inhibited ROS accumulation, and mitochondrial depolarization; 2) OAA inhibited the downregulation of L-OPA1 and MFN2 caused by PQ and promoted a dynamic balance of mitochondrial fusion and fission, and 3) the expression of PGC-1α, TFAM, COX2, and COX4I1, increased significantly following OAA intervention which improved mitochondrial respiratory functions and promoted its biogenesis and energy metabolism in damaged cells. In conclusion, OAA effectively cleared ROS and improved mitochondrial dysfunction, thereby significantly improving ALI caused by PQ poisoning and the animal survival rate. Therefore, OAA may be a potential drug for the treatment of PQ poisoning.

Keywords:  acute lung injury; mitochondrial biogenesis; mitochondrial dysfunction; oxaloacetate acid; oxidative stress; paraquat

DOI:  https://doi.org/10.3389/fphar.2022.1029775
Biogerontology. 2022 Oct 31.

Metabolic and physical function are improved with lifelong 15% calorie restriction in aging male mice.

Emily C Peters, Luke Safayan, Tyler J Marx, Emily Ngu, Anastasiia Vasileva, India Zappia, William H Powell, Frank A Duca, Jennifer H Stern.

  Chronic calorie restriction (CR) results in lengthened lifespan and reduced disease risk. Many previous studies have implemented 30-40% calorie restriction to investigate these benefits. The goal of our study was to investigate the effects of calorie restriction, beginning at 4 months of age, on metabolic and physical changes induced by aging. Male C57BL/6NCrl calorie restricted and ad libitum fed control mice were obtained from the National Institute on Aging (NIA) and studied at 10, 18, 26, and 28 months of age to better understand the metabolic changes that occur in response to CR in middle age and advanced age. Food intake was measured in ad libitum fed controls to assess the true degree of CR (15%) in these mice. We found that 15% CR decreased body mass and liver triglyceride content, improved oral glucose clearance, and increased all limb grip strength in 10- and 18-month-old mice. Glucose clearance in ad libitum fed 26- and 28-month-old mice is enhanced relative to younger mice but was not further improved by CR. CR decreased basal insulin concentrations in all age groups and improved insulin sensitivity and rotarod time to fall in 28-month-old mice. The results of our study demonstrate that even a modest reduction (15%) in caloric intake may improve metabolic and physical health. Thus, moderate calorie restriction may be a dietary intervention to promote healthy aging with improved likelihood for adherence in human populations.

Keywords:  Calorie restriction; Glucose homeostasis; Healthspan; Insulin sensitivity; Liver fat

DOI:  https://doi.org/10.1007/s10522-022-09996-5
Front Physiol. 2022 ;13 979547

Preeclampsia and its relationship to pathological brain aging.

Abigail A Testo, Carole McBride, Ira M Bernstein, Julie A Dumas.

  The development of preeclampsia during pregnancy may have long-term effects on brain aging in women. Associations between preeclampsia and vascular dementia have been established, however the connection between preeclampsia and Alzheimer's disease has not been as thoroughly explored. Both preeclampsia and Alzheimer's disease have been associated with misfolded amyloid beta proteins and inflammation; due to these similarities, in this minireview, we examined the potential links between a history of preeclampsia and the development of dementia. We also discussed how hypertensive disorders of pregnancy may relate to both normal brain aging and dementia to highlight the need for additional research regarding the long-term cognitive effects of preeclampsia on the brain.

Keywords:  Alzheimer’s disease; brain aging (normal); dementia; preeclampsia; vascular dementia

DOI:  https://doi.org/10.3389/fphys.2022.979547
J Neurophysiol. 2022 Nov 02.

HSPB1 overexpression improves hypoxic-ischemic brain damage by attenuating ferroptosis in rats through promoting G6PD expression.

Yi Dai, Lan Hu.

   BACKGROUND: Heat-shock protein B (HSPB1) has a neuroprotective effect on brain injury and is a negative regulator of ferroptosis. Therefore, we infer that HSPB1 plays a protective role in hypoxic-ischemic (HI) brain damage by inhibiting ferroptosis.
METHODS: A neonatal rat model of hypoxic-ischemic (HI) brain damage was established. HSPB1 overexpression plasmid and the negative control were injected into the lateral ventricle of rats 48 h before HI brain damage surgery. HSPB1 and glucose-6-phosphate dehydrogenase (G6PD) levels, infarction rate, iron accumulation, apoptosis, and ferroptosis-related markers were estimated with the assistance of qRT-PCR, TTC staining, Prussian blue staining, iron assay kit, TUNEL staining, and western blot. In vitro, after transfection, HSPB1 and G6PD levels, oxygen-glucose deprivation (OGD)-mediated hippocampal neuron cell viability, apoptosis, iron content, and ferroptosis-related markers were assessed using qRT-PCR, MTT, flow cytometry, iron assay kit, and western blot.
RESULTS: HSPB1 and G6PD were overexpressed in the hippocampus tissues of HI rats. High expression of HSPB1 in HI rats lessened infarction rate and ferritin level, hindered iron accumulation and apoptosis, and promoted GPX4, SLC7A11 and TFR1 levels. In OGD-mediated hippocampal neuron cells, HSPB1 up-regulation intensified the viability and repressed apoptosis and ferroptosis, while G6PD silencing reversed the effects of HSPB1 up-regulation.
CONCLUSION: We documented that HSPB1 overexpression unleashes neuroprotective effects via modulating G6PD expression, which offers a novel target for the prevention and treatment of HI brain damage.

Keywords:  ferroptosis; glucose-6-phosphate dehydrogenase; heat-shock protein B; hypoxic-ischemic brain damage

DOI:  https://doi.org/10.1152/jn.00306.2022
Pediatrics. 2022 Nov 04. pii: e2022057442. [Epub ahead of print]

Preterm Brain Injury and Neurodevelopmental Outcomes: A Meta-analysis.

Philippa Rees, Caitriona Callan, Karan R Chadda, Meriel Vaal, James Diviney, Shahad Sabti, Fergus Harnden, Julian Gardiner, Cheryl Battersby, Chris Gale, Alastair Sutcliffe.

CONTEXT: Preterm brain injuries are common; neurodevelopmental outcomes following contemporary neonatal care are continually evolving.
OBJECTIVE: To systematically review and meta-analyze neurodevelopmental outcomes among preterm infants after intraventricular hemorrhage (IVH) and white matter injury (WMI).
DATA SOURCES: Published and gray literature were searched across 10 databases between 2000 and 2021.
STUDY SELECTION: Observational studies reporting 3-year neurodevelopmental outcomes for preterm infants with IVH or WMI compared with preterm infants without injury.
DATA EXTRACTION: Study characteristics, population characteristics, and outcome data were extracted.
RESULTS: Thirty eight studies were included. There was an increased adjusted risk of moderate-severe neurodevelopmental impairment after IVH grade 1 to 2 (adjusted odds ratio 1.35 [95% confidence interval 1.05-1.75]) and IVH grade 3 to 4 (adjusted odds ratio 4.26 [3.25-5.59]). Children with IVH grade 1 to 2 had higher risks of cerebral palsy (odds ratio [OR] 1.76 [1.39-2.24]), cognitive (OR 1.79 [1.09-2.95]), hearing (OR 1.83 [1.03-3.24]), and visual impairment (OR 1.77 [1.08-2.9]). Children with IVH grade 3 to 4 had markedly higher risks of cerebral palsy (OR 4.98 [4.13-6.00]), motor (OR 2.7 [1.52-4.8]), cognitive (OR 2.3 [1.67-3.15]), hearing (OR 2.44 [1.42-4.2]), and visual impairment (OR 5.42 [2.77-10.58]). Children with WMI had much higher risks of cerebral palsy (OR 14.91 [7.3-30.46]), motor (OR 5.3 [3-9.36]), and cognitive impairment (OR 3.48 [2.18-5.53]).
LIMITATIONS: Heterogeneity of outcome data.
CONCLUSIONS: Mild IVH, severe IVH, and WMI are associated with adverse neurodevelopmental outcomes. Utilization of core outcome sets and availability of open-access study data would improve our understanding of the nuances of these outcomes.

DOI: https://doi.org/10.1542/peds.2022-057442
Genet Med. 2022 Oct 31. pii: S1098-3600(22)00948-0. [Epub ahead of print]

Brain monoamine vesicular transport disease caused by homozygous SLC18A2 variants: A study in 42 affected individuals.

Ken Saida, Reza Maroofian, Toru Sengoku, Tadahiro Mitani, Alistair T Pagnamenta, Dana Marafi, Maha S Zaki, Thomas J O'Brien, Ehsan Ghayoor Karimiani, Rauan Kaiyrzhanov, Marina Takizawa, Sachiko Ohori, Huey Yin Leong, Gulsen Akay, Hamid Galehdari, Mina Zamani, Ratna Romy, Christopher J Carroll, Mehran Beiraghi Toosi, Farah Ashrafzadeh, Shima Imannezhad, Hadis Malek, Najmeh Ahangari, Hoda Tomoum, Vykuntaraju K Gowda, Varunvenkat M Srinivasan, David Murphy, Natalia Dominik, Hasnaa M Elbendary, Karima Rafat, Sanem Yilmaz, Seda Kanmaz, Mine Serin, Deepa Krishnakumar, Alice Gardham, Anna Maw, Tekki Sreenivasa Rao, Sarah Alsubhi, Myriam Srour, Daniela Buhas, Tamison Jewett, Rachel E Goldberg, Hanan Shamseldin, Eirik Frengen, Doriana Misceo, Petter Strømme, José Ricardo Magliocco Ceroni, Chong Ae Kim, Gozde Yesil, Esma Sengenc, Serhat Guler, Mariam Hull, Mered Parnes, Dilek Aktas, Banu Anlar, Yavuz Bayram, Davut Pehlivan, Jennifer E Posey, Shahryar Alavi, Seyed Ali Madani Manshadi, Hamad Alzaidan, Mohammad Al-Owain, Lama Alabdi, Ferdous Abdulwahab, Futoshi Sekiguchi, Kohei Hamanaka, Atsushi Fujita, Yuri Uchiyama, Takeshi Mizuguchi, Satoko Miyatake, Noriko Miyake, Reem M Elshafie, Kamran Salayev, Ulviyya Guliyeva, Fowzan S Alkuraya, Joseph G Gleeson, Kristin G Monaghan, Katherine G Langley, Hui Yang, Mahsa Motavaf, Saeid Safari, Mozhgan Alipour, Kazuhiro Ogata, André E X Brown, James R Lupski, Henry Houlden, Naomichi Matsumoto.

   PURPOSE: Brain monoamine vesicular transport disease is an infantile-onset movement disorder that mimics cerebral palsy. In 2013, the homozygous SLC18A2 variant, p.Pro387Leu, was first reported as a cause of this rare disorder, and dopamine agonists were efficient for treating affected individuals from a single large family. To date, only 6 variants have been reported. In this study, we evaluated genotype-phenotype correlations in individuals with biallelic SLC18A2 variants.
METHODS: A total of 42 affected individuals with homozygous SLC18A2 variant alleles were identified. We evaluated genotype-phenotype correlations and the missense variants in the affected individuals based on the structural modeling of rat VMAT2 encoded by Slc18a2, with cytoplasm- and lumen-facing conformations. A Caenorhabditis elegans model was created for functional studies.
RESULTS: A total of 19 homozygous SLC18A2 variants, including 3 recurrent variants, were identified using exome sequencing. The affected individuals typically showed global developmental delay, hypotonia, dystonia, oculogyric crisis, and autonomic nervous system involvement (temperature dysregulation/sweating, hypersalivation, and gastrointestinal dysmotility). Among the 58 affected individuals described to date, 16 (28%) died before the age of 13 years. Of the 17 patients with p.Pro237His, 9 died, whereas all 14 patients with p.Pro387Leu survived. Although a dopamine agonist mildly improved the disease symptoms in 18 of 21 patients (86%), some affected individuals with p.Ile43Phe and p.Pro387Leu showed milder phenotypes and presented prolonged survival even without treatment. The C. elegans model showed behavioral abnormalities.
CONCLUSION: These data expand the phenotypic and genotypic spectra of SLC18A2-related disorders.

Keywords:  Brain monoamine vesicular transport disease; Dopamine agonist; Dystonia; SLC18A2; VMAT2

DOI:  https://doi.org/10.1016/j.gim.2022.09.010
Am J Case Rep. 2022 Nov 05. 23 e937220

Compound Heterozygote Mutation in the SMPD1 Gene Leading to Nieman-Pick Disease Type A.

Alja Kavčič, Matjaž Homan, Milanka Živanović, Maruša Debeljak, Tita Butenko, Ana Drole Torkar, Mojca Žerjav Tanšek, Sara Bertok, Tadej Battelino, Urh Groselj.

BACKGROUND Niemann-Pick disease (NPD) type A is an autosomal recessive lipid storage disorder caused by acid sphingomyelinase deficiency due to a mutation in the SMPD1 gene. Type A is the most severe phenotype of NPD, with early onset in infancy and unfavorable outcome in early childhood. CASE REPORT An 11-month-old boy with hepatosplenomegaly, elevated liver transaminases, and faltering growth was admitted to our hospital for further assessment of potential liver disease. He had severe generalized muscular hypotonia, muscular hypotrophy, reduced muscular strenght, joint laxity, weak deep tendon reflexes, and severe motor developmental delay. Leukodystrophy was seen on the brain MRI, and brainstem auditory evoked potentials were characteristic for auditory neuropathy. A chest X-ray showed signs of interstitial lung disease, which was not further evaluated due to absence of respiratory distress. Liver biopsy histopathologic findings were indicative for lipid storage disease. Genetic analysis showed that the patient is a compound heterozygote in the SMPD1 gene - (NM_000543.5): c.573delT p.(Ser192Alafs*65), which was inherited from the mother and c.1267C>T p.(His423Tyr) was inherited from the father. Both variants were previously individually reported in NPD type A and B. The clinical phenotype in our patient was characteristic of NPD type A, with an early onset and a rapidly progresive neurodegeneration. The patient was included in multidisciplinary follow-up, providing him symptomatic treatment and support. CONCLUSIONS We present a case of NPD type A caused by a rare compound heterozygote mutation in the SMPD1 gene. Most clinical findings and the disease course were typical for NPD type A, except for bilateral auditory neuropathy, which seems to be an uncommon finding in this phenotype and could be underestimated due to infrequent testing for auditory dysfunction.

DOI: https://doi.org/10.12659/AJCR.937220