bims-medebr 2024-11-10 papers

bims-medebr

Biomed News

on Metabolism of the developing brain

Issue of 2024‒11‒10
seventeen papers selected by
Regina F. Fernández, Johns Hopkins University

Abnormal Regulation of Mitochondrial Sphingolipids during Aging and Alzheimer's Disease.
Role of cholesterol in modulating brain hyperexcitability.
Impaired brain glucose metabolism as a biomarker for evaluation of dodecyl creatine ester in creatine transporter deficiency: Insights from patient brain-derived organoids and in vivo [18F]FDG PET imaging in a mouse model.
Neuroprotective effect of neuron-specific deletion of the C16 ceramide synthetic enzymes in an animal model of multiple sclerosis.
The Emerging Role of Brain Mitochondria in Fear and Anxiety.
Simultaneous assessment of cerebral glucose and oxygen metabolism and perfusion in rats using interleaved deuterium (2H) and oxygen-17 (17O) MRS.
Differential impact of eicosapentaenoic acid and docosahexaenoic acid in an animal model of Alzheimer's disease.
FREE FATTY ACIDS INHIBIT AN ION-COUPLED MEMBRANE TRANSPORTER BY DISSIPATING THE ION GRADIENT.
Phospholipase A2 group IVD mediates the transacylation of glycerophospholipids and acylglycerols.
Mitochondrial respiratory capacity is not altered in aging rat brains with or without memory impairment.
Regional differences in glucose metabolic decline and tau deposition in the Alzheimer's continuum brain.
Defects in CYB5A and CYB5B impact sterol-C4 oxidation in cholesterol biosynthesis and demonstrate regulatory roles of dimethyl sterols.
Mechanisms of fibrinogen trans-activation of the EGFR/Ca2+ signaling axis to regulate mitochondrial transport and energy transfer and inhibit axonal regeneration following cerebral ischemia.
Defective glycolysis in the cerebrum and cerebellum correlates with the pathology and neurological declines in mice with Angiostrongylus cantonensis infection.
Recent developments in the analytical approaches of acyl-CoAs to assess their role in mitochondrial fatty acid oxidation disorders.
Mass spectrometry-based proteomics to study mutants and interactomes of mitochondrial translocation proteins.
Quantifying acyl-chain diversity in isobaric compound lipids containing monomethyl branched-chain fatty acids.

ASN Neuro. 2024 ;16(1): 2404367

Abnormal Regulation of Mitochondrial Sphingolipids during Aging and Alzheimer's Disease.

Simone M Crivelli, Zainuddin Quadri, Ahmed Elsherbini, Hemendra J Vekaria, Patrick G Sullivan, Wenbo Zhi, Pilar Martinez-Martinez, Stefka D Spassieva, Erhard Bieberich.

  During pathogenesis of Alzheimer's disease (AD), mitochondria suffer alterations that lead to low energy production and reactive oxygen species formation. However, the mechanism of impaired mitochondria homeostasis in AD is not fully understood. We hypothesized that abnormal sphingolipid metabolism in mitochondria could be one of the contributing factors to mitochondrial dysfunction. Synaptic and non-synaptic mitochondria were isolated from 5xFAD and wild type (WT) mice at 3 and 7 months using Ficoll gradient ultracentrifugation, and their function was analyzed using Seahorse assay. Additionally, mitochondria were analyzed using mass spectrometry for proteomics and sphingolipidomics analyses. Sphingolipid levels were also determined in synaptic and non-synaptic mitochondria isolated from AD patients and healthy controls. We found that synaptic mitochondria isolated from 3-months old 5xFAD mice manifest diminished oxygen consumption as compared to WT. Consistently, proteomics analysis showed that proteins related to respiratory electron transport and oxidative phosphorylation were altered in 5xFAD mice. When quantifying the main sphingolipids in mitochondria, we found that Cer 18:0, Cer 22:0, and Cer 24:1 were increased already at 3 months in 5xFAD mice. No increase in ceramides was detected in mitochondria isolated from AD patients. However, increased levels of sphingosine were found in both 5xFAD mice and AD patients when compared to respective controls. We report that the regulation of sphingolipids in mitochondria is abnormal at 3 months of age in 5xFAD mice, as indicated by the accumulation of long-chain ceramides, which increases with age. Sphingosine levels are increased in both the mitochondria of 5xFAD mice and AD patients. Our data suggest that the sphingolipid composition is dysregulated in mitochondria early during AD pathogenesis.

Keywords:  5xFAD; Aging; Alzheimer’s disease; ceramide; mitochondria; sphingosine

DOI:  https://doi.org/10.1080/17590914.2024.2404367
Epilepsia. 2024 Nov 02.

Role of cholesterol in modulating brain hyperexcitability.

James W Wheless, Jong M Rho.

  Cholesterol is a critical molecule in the central nervous system, and imbalances in the synthesis and metabolism of brain cholesterol can result in a range of pathologies, including those related to hyperexcitability. The impact of cholesterol on disorders of epilepsy and developmental and epileptic encephalopathies is an area of growing interest. Cholesterol cannot cross the blood-brain barrier, and thus the brain synthesizes and metabolizes its own pool of cholesterol. The primary metabolic enzyme for brain cholesterol is cholesterol 24-hydroxylase (CH24H), which metabolizes cholesterol into 24S-hydroxycholesterol (24HC). Dysregulation of CH24H and 24HC can affect neuronal excitability through a range of mechanisms. 24HC is a positive allosteric modulator of N-methyl-D-aspartate (NMDA) receptors and can increase glutamate release via tumor necrosis factor-α-dependent pathways. Increasing cholesterol metabolism can lead to dysfunction of excitatory amino acid transporter 2 and impair glutamate reuptake. Finally, overstimulation of NMDA receptors can further activate metabolism of cholesterol, leading to a vicious cycle of overactivation. All of these mechanisms increase extracellular glutamate and can lead to hyperexcitability. For these reasons, the cholesterol pathway represents a new potential mechanistic target for antiseizure medications. CH24H inhibition has been shown to decrease seizure behavior and improve survival in multiple animal models of epilepsy and could be a promising new mechanism of action for the treatment of neuronal hyperexcitability and developmental and epileptic encephalopathies.

Keywords:  24S‐hydroxycholesterol; cholesterol 24‐hydroxylase; developmental and epileptic encephalopathies; epilepsy

DOI:  https://doi.org/10.1111/epi.18174
Neurobiol Dis. 2024 Oct 25. pii: S0969-9961(24)00322-X. [Epub ahead of print] 106720

Impaired brain glucose metabolism as a biomarker for evaluation of dodecyl creatine ester in creatine transporter deficiency: Insights from patient brain-derived organoids and in vivo [18F]FDG PET imaging in a mouse model.

Clémence Disdier, Amélie Soyer, Léa Broca-Brisson, Sébastien Goutal, Anne-Cécile Guyot, Nora Ziani, Louise Breuil, Alexandra Winkeler, Gaëlle Hugon, Thomas Joudinaud, Henri Bénech, Jean Armengaud, Matthew R Skelton, Rania Harati, Rifat A Hamoudi, Nicolas Tournier, Aloïse Mabondzo.

  Creatine transporter deficiency (CTD) is an inborn error of creatine (Cr) metabolism in which Cr is not properly distributed to the brain due to a mutation in the Cr transporter (CrT) SLC6A8 gene. CTD is associated with developmental delays and with neurological disability in children. Dodecyl creatine ester (DCE), as a Cr prodrug, is a promising drug to treat CTD after administration by the nasal route, taking advantage of the nose-to-brain pathway. In this study, the potential adaptive response to energy imbalance in glucose metabolism was investigated in CTD using both SLC6A8-deficient mice (CrT KO) and brain organoids derived from CTD patient cells. Longitudinal brain [18F]FDG PET imaging in CrT KO mice compared to wild-type mice demonstrated that CTD was associated with a significant loss and decline in brain glucose metabolism. In CrT KO mice, intranasal supplementation with DCE for a month significantly mitigated the decline in brain glucose metabolism compared to untreated (vehicle) animals. Mechanistic investigations in CrT KO mice and cerebral organoids derived from CTD patient cells suggest that intracellular trafficking of glucose transporter (Glut) may be altered by lack of activation of AMP-activated protein kinase (AMPK). Consistency between observations in the CrT KO mouse model and cerebral organoids derived from CTD patient cells supports the value of this new model for drug discovery and development. In addition, these results suggest that [18F]FDG PET imaging may offer a unique and minimally-invasive biomarker to monitor the impact of investigational treatment on CTD pathophysiology, with translational perspectives.

Keywords:  18F-FDG PET; Cerebral organoids; Creatine transporter deficiency; Dodecyl creatine ester

DOI:  https://doi.org/10.1016/j.nbd.2024.106720
Glia. 2024 Nov 03.

Neuroprotective effect of neuron-specific deletion of the C16 ceramide synthetic enzymes in an animal model of multiple sclerosis.

Mario Amatruda, Damien Marechal, Mar Gacias, Maureen Wentling, Sarah Turpin-Nolan, Johannes Morstein, Mohammed Moniruzzaman, Jens C Brüning, Norman J Haughey, Dirk H Trauner, Patrizia Casaccia.

  Ceramide C16 is a sphingolipid detected at high levels in several neurodegenerative disorders, including multiple sclerosis (MS). It can be generated de novo or from the hydrolysis of other sphingolipids, such as sphingomyelin or through the recycling of sphingosine, in what is known as the salvage pathway. While the myelin damage occurring in MS suggests the importance of the hydrolytic and salvage pathways, the growing interest on the importance of diet in demyelinating disorders, prompted us to investigate the involvement of de novo ceramide C16 synthesis on disease severity. A diet rich in saturated fats such as palmitic acid, as found in many highly processed foods, provides substrates for the ceramide C16 synthetic enzymes ceramide synthase 6 (CERS6) and 5 (CERS5), which are expressed in the central nervous system. Using the experimental autoimmune encephalomyelitis (EAE) model of inflammatory demyelination, we show here that mice with CamK2a+ neuronal specific deletion of both CerS6 and CerS5 show a milder course of EAE than wild type mice, even when fed a diet enriched in palmitic acid. At a cellular level, neurons lacking both CerS6 and CerS5 are protected from the mitochondrial dysfunction arising from exposure to oxidative stress and palmitic acid in the medium. These data underscore the importance of a healthy diet avoiding processed foods for demyelinating disorders and identifies endogenous neuronal synthesis of ceramide C16 as an important determinant of disease severity.

Keywords:  diet; experimental autoimmune encephalomyelitis; mitochondria; neurodegeneration; palm‐oil

DOI:  https://doi.org/10.1002/glia.24631
Curr Top Behav Neurosci. 2024 Nov 07.

The Emerging Role of Brain Mitochondria in Fear and Anxiety.

Alessandro Chioino, Carmen Sandi.

  The functional complexity of brain circuits underlies the broad spectrum of behaviors, cognitive functions, and their associated disorders. Mitochondria, traditionally known for their role in cellular energy metabolism, are increasingly recognized as central to brain function and behavior. This review examines how mitochondria are pivotal in linking cellular energy processes with the functioning of neural circuits that govern fear and anxiety. Following an introductory section in which we summarize current knowledge about fear and anxiety neural circuits, we provide a brief summary of mitochondria fundamental roles (e.g., from energy production and calcium buffering to their involvement in reactive oxygen species (ROS) generation, mitochondrial dynamics, and signaling), particularly emphasizing their contribution to synaptic plasticity, neurodevelopment, and stress response mechanisms. The review's core focuses on the current state of knowledge regarding how mitochondrial function and dysfunction impact the neural substrates of fear and anxiety. Furthermore, we explore the implications of mitochondrial alterations in the context of posttraumatic stress disorder (PTSD) and anxiety disorders, underscoring the potential of mitochondrial pathways as new therapeutic targets. Integrating insights from genetic, biochemical, neurobiological, behavioral, and clinical studies, we propose a model in which mitochondrial function is critical for regulating the neural circuits that underpin fear and anxiety behaviors, highlighting how mitochondrial dysfunction can lead to their pathological manifestations. This integration emphasizes the potential for developing novel treatments targeting the biological roots of fear, anxiety, and related disorders. By merging mitochondrial biology with behavioral and circuit neuroscience, we enrich our neurobiological understanding of fear and anxiety, uncovering promising avenues for therapeutic intervention.

Keywords:  Anxiety; Fear; Mitochondria; Neural circuits; Nutritional interventions

DOI:  https://doi.org/10.1007/7854_2024_537
NMR Biomed. 2024 Nov 06. e5284

Simultaneous assessment of cerebral glucose and oxygen metabolism and perfusion in rats using interleaved deuterium (2H) and oxygen-17 (17O) MRS.

Guangle Zhang, Parker Jenkins, Wei Zhu, Wei Chen, Xiao-Hong Zhu.

  Cerebral glucose and oxygen metabolism and blood perfusion play key roles in neuroenergetics and oxidative phosphorylation to produce adenosine triphosphate (ATP) energy molecules in supporting cellular activity and brain function. Their impairments have been linked to numerous brain disorders. This study aimed to develop an in vivo magnetic resonance spectroscopy (MRS) method capable of simultaneously assessing and quantifying the major cerebral metabolic rates of glucose (CMRGlc) and oxygen (CMRO2) consumption, lactate formation (CMRLac), and tricarboxylic acid (TCA) cycle (VTCA); cerebral blood flow (CBF); and oxygen extraction fraction (OEF) via a single dynamic MRS measurement using an interleaved deuterium (2H) and oxygen-17 (17O) MRS approach. We introduced a single-loop multifrequency radio-frequency (RF) surface coil that can be used to acquire proton (1H) magnetic resonance imaging (MRI) or interleaved low-γ X-nuclei 2H and 17O MRS. By combining this RF coil with a modified MRS pulse sequence, 17O-isotope-labeled oxygen gas inhalation, and intravenous 2H-isotope-labeled glucose administration, we demonstrate for the first time the feasibility of simultaneously and quantitatively measuring six important physiological parameters, CMRGlc, CMRO2, CMRLac, VTCA, CBF, and OEF, in rat brains at 16.4 T. The interleaved 2H-17O MRS technique should be readily adapted to image and study cerebral energy metabolism and perfusion in healthy and diseased brains.

Keywords:  X‐nuclear MRS and imaging; brain energy metabolism and perfusion; interleaved 2H–17O MRS technique; multifrequency RF surface coil; ultrahigh field

DOI:  https://doi.org/10.1002/nbm.5284
J Lipid Res. 2024 Oct 25. pii: S0022-2275(24)00187-1. [Epub ahead of print] 100682

Differential impact of eicosapentaenoic acid and docosahexaenoic acid in an animal model of Alzheimer's disease.

Méryl-Farelle Oye Mintsa Mi-Mba, Meryem Lebbadi, Waël Alata, Carl Julien, Vincent Emond, Cyntia Tremblay, Samuel Fortin, Colin J Barrow, Jean-François Bilodeau, Frédéric Calon.

  Dietary supplementation with n-3 polyunsaturated fatty acids (n-3 PUFA) improves cognitive performance in several animal models of Alzheimer's disease (AD), an effect often associated with reduced amyloid-beta (Aβ) and/or tau pathologies. However, it remains unclear to what extent eicosapentaenoic (EPA) provides additional benefits compared to docosahexaenoic acid (DHA). Here, male and female 3xTg-AD mice were fed for 3 months (13 to 16 months of age) the following diets: (1) control (no DHA/EPA), (2) DHA (1.1g/kg) and low EPA (0.4g/kg), or (3) DHA (0.9g/kg) with high EPA (9.2g/kg). The DHA and DHA+EPA diets respectively increased DHA by 19% and 8% in the frontal cortex of 3xTg-AD mice, compared to controls. Levels of EPA, which were below the detection limit after the control diet, reached 0.14% and 0.29% of total brain fatty acids after the DHA and DHA+EPA diet, respectively. DHA and DHA+EPA diets lowered brain arachidonic acid (ARA) levels and the n-6:n-3 docosapentaenoic acid (DPA) ratio. Brain uptake of free 14C-DHA measured through intracarotid brain perfusion, but not of 14C-EPA, was lower in 3xTg-AD compared to NonTg mice. DHA and DHA+EPA diets in 3xTg-AD mice reduced cortical soluble phosphorylated tau (pS202) (-34% high-DHA, -34% DHA+EPA, p<0.05) while increasing p21 activated kinase (+58% and +83%, p<0.001; respectively). High EPA intake lowered insoluble phosphorylated tau (-31% versus DHA, p<0.05). No diet effect on Aβ levels was observed. In conclusion, dietary intake of DHA and EPA leads to differential changes in brain PUFA while altering cerebral biomarkers consistent with beneficial effects against AD-like neuropathology.

Keywords:  3xTg-AD mice; Alzheimer’s disease; docosahexaenoic acid; eicosapentaenoic acid; n-3 polyunsaturated fatty acids

DOI:  https://doi.org/10.1016/j.jlr.2024.100682
J Biol Chem. 2024 Nov 02. pii: S0021-9258(24)02457-8. [Epub ahead of print] 107955

FREE FATTY ACIDS INHIBIT AN ION-COUPLED MEMBRANE TRANSPORTER BY DISSIPATING THE ION GRADIENT.

Xiaoyu Wang, Radda Rusinova, G Glenn Gregorio, Olga Boudker.

  Glutamate is the main excitatory transmitter in the mammalian central nervous system; glutamate transporters keep the synaptic glutamate concentrations at bay for normal brain function. Arachidonic acid (AA), docosahexaenoic acid (DHA), and other unsaturated fatty acids modulate glutamate transporters in cell- and tissue slices-based studies. Here, we investigated their effect and mechanism using a purified archaeal glutamate transporter homolog reconstituted into the lipid membranes. AA, DHA, and related fatty acids irreversibly inhibited the sodium-dependent concentrative substrate uptake into lipid vesicles within the physiologically relevant concentration range. In contrast, AA did not inhibit amino acid exchange across the membrane. The length and unsaturation of the aliphatic tail affect inhibition, and the free carboxylic headgroup is necessary. The inhibition potency did not correlate with the fatty acid effects on the bilayer deformation energies. AA does not affect the conformational dynamics of the protein, suggesting it does not inhibit structural transitions necessary for transport. Single-transporter and membrane voltage assays showed that AA and related fatty acids mediate cation leak, dissipating the driving sodium gradient. Thus, such fatty acids can act as cation ionophores, suggesting a general modulatory mechanism of membrane channels and ion-coupled transporters.

Keywords:  arachidonic acid (AA) (ARA); docosahexaenoic acid (DHA); fatty acid; free fatty acid; glutamate; glutamate transporter; membrane bilayer; membrane permeation; membrane transport; membrane transporter; polyunsaturated fatty acid (PUFA)

DOI:  https://doi.org/10.1016/j.jbc.2024.107955
J Lipid Res. 2024 Oct 25. pii: S0022-2275(24)00190-1. [Epub ahead of print] 100685

Phospholipase A2 group IVD mediates the transacylation of glycerophospholipids and acylglycerols.

Johannes Breithofer, Dominik Bulfon, Nermeen Fawzy, Martin Tischitz, Clara Zitta, Lennart Hartig, Gernot F Grabner, Anita Pirchheim, Hubert Hackl, Ulrike Taschler, Achim Lass, Carmen Tam-Amersdorfer, Herbert Strobl, Dagmar Kratky, Robert Zimmermann.

In mammalian cells, glycerolipids are mainly synthesized using acyl-CoA-dependent mechanisms. The acyl-CoA-independent transfer of fatty acids between lipids, designated as transacylation reaction, represents an additional mechanism for lipid remodeling and synthesis pathways. Here, we demonstrate that human and mouse phospholipase A2 group IVD (PLA2G4D) catalyzes transacylase reactions using both phospholipids and acylglycerols as substrates. In the presence of mono- and diacylglycerol (MAG and DAG), purified PLA2G4D generates DAG and triacylglycerol (TAG), respectively. The enzyme also transfers fatty acids between phospholipids and from phospholipids to acylglycerols. Overexpression of PLA2G4D in COS7 cells enhances the incorporation of polyunsaturated fatty acids into TAG stores and induces the accumulation of lysophospholipids. In the presence of exogenously added MAG, the enzyme strongly increases cellular DAG formation, while MAG levels are decreased. PLA2G4D is not or poorly detectable in commonly used cell lines. It is expressed in keratinocytes, where it is strongly upregulated by proinflammatory cytokines. Pla2g4d-deficient mouse keratinocytes exhibit complex lipidomic changes in response to cytokine treatment, indicating that PLA2G4D is involved in the remodeling of the lipidome under inflammatory conditions. Transcriptomic analysis revealed that PLA2G4D modulates fundamental biological processes including cell proliferation, differentiation, and signaling. Together, our observations demonstrate that PLA2G4D has broad substrate specificity for fatty acid donor and acceptor lipids, allowing the acyl-CoA-independent synthesis of both phospholipids and acylglycerols. Loss-of-function studies indicate that PLA2G4D affects metabolic and signaling pathways in keratinocytes, which is associated with complex lipidomic and transcriptomic alterations.

DOI: https://doi.org/10.1016/j.jlr.2024.100685
MicroPubl Biol. 2024 ;2024

Mitochondrial respiratory capacity is not altered in aging rat brains with or without memory impairment.

Pamela J Yao, Jeffrey M Long, Peter R Rapp, Dimitrios Kapogiannis.

Mitochondria are essential for supporting the high metabolic demands that are required for brain function. Impairments in mitochondria have been linked to age-related decline in brain functions. Here, we investigate whether the mitochondrial respiratory capacity of brain cells is changed in cognitive aging. We used a rat model of normal cognitive aging and analyzed mitochondrial oxidative phosphorylation in frozen brain samples. Mitochondrial oxygen consumption rate analysis of the frontal cortex did not show any differences between young rats and aged rats with either intact memory or impaired spatial memory. Mitochondrial ATP synthase activity and quantity also did not differ between young and aged rats. These results suggest that the total level of mitochondrial respiratory capacity is preserved in the frontal cortex of aged rats and may not explain aging-associated cognitive impairment.

DOI: https://doi.org/10.17912/micropub.biology.001359
J Alzheimers Dis. 2024 Nov;102(1): 228-236

Regional differences in glucose metabolic decline and tau deposition in the Alzheimer's continuum brain.

for BATON Study Group

  BACKGROUND: Alzheimer's disease (AD) is characterized by the accumulation of amyloid-β and tau proteins, leading to neurofibrillary tangles. A biomarker-based diagnostic method called the ATN system categorizes AD pathology into amyloid-β (A), tau (T), and neurodegeneration (N). The relationship between regional tau deposition and reduced glucose metabolism in the preclinical AD stage is not well understood.OBJECTIVE: We presented voxel-by-voxel metabolic/tau deposition ratio (MTR) images to investigate the effects of tau deposition on metabolism in AD brains on a stage-by-stage basis.
METHODS: We selected 174 subjects who underwent 3D-MRI, FDG-PET, amyloid PET, and tau PET scans. MTR images were created by normalizing FDG-PET toMK6240 PET images. Voxel-wise comparisons among 63 cognitively normal amyloid-negative (CNA) subjects, 49 subjects with AD dementia (ADD), 23 subjects with mild cognitive impairment due to AD (MCA), and 39 preclinical AD (PRC) subjects were conducted.
RESULTS: There was reduced glucose metabolism in ADD and MCA groups compared to CNA, predominantly in parietotemporal areas. Tau deposition was observed in wider areas in ADD and restricted to the medial temporal lobes in MCA. MTR exhibited significant reductions in broader regions in ADD and MCA, indicating simultaneous glucose metabolism decrease and tau deposition. At the MCA and PRC stages, glucose metabolism impairment and tau deposition were shown in separate regions by FDG PET and tau PET, respectively, while MTR images showed impairment in both regions.
CONCLUSIONS: Our findings suggest that MTR imaging provides insights into AD pathophysiology by simultaneously assessing glucose metabolism and tau deposition. In the early stage of the AD continuum (MCA and PRC), metabolic decline and tau deposition occur independently in different brain regions.

Keywords:  Alzheimer's disease; PET; amyloid; glucose metabolism; tau

DOI:  https://doi.org/10.1177/13872877241284314
Cell Rep. 2024 Oct 26. pii: S2211-1247(24)01263-4. [Epub ahead of print]43(11): 114912

Defects in CYB5A and CYB5B impact sterol-C4 oxidation in cholesterol biosynthesis and demonstrate regulatory roles of dimethyl sterols.

Mei-Yan Ma, Gang Deng, Wen-Zhuo Zhu, Ming Sun, Lu-Yi Jiang, Wei-Hui Li, Yuan-Bin Liu, Lin Guo, Bao-Liang Song, Xiaolu Zhao.

  Cytochrome b5 (CYB5) is a hemoprotein crucial for electron transfer to oxygenases. Although microsomal CYB5A is required for sterol C4-demethylation in vitro, cholesterol biosynthesis remains intact in Cyb5a knockout mice. Here, we show that knockout of mitochondrial CYB5B, rather than CYB5A, blocks cholesterol biosynthesis at the sterol-C4 oxidation step in HeLa cells, causing an accumulation of testis meiosis-activating sterol (T-MAS) and dihydro-T-MAS. Surprisingly, liver-specific Cyb5b knockout (L-Cyb5b-/-) mice exhibit normal cholesterol metabolism. Further knockdown of Cyb5a in L-Cyb5b-/- (L-Cyb5b-/-/short hairpin [sh]Cyb5a) mice leads to a marked accumulation of T-MAS and dihydro-T-MAS, indicating that either CYB5A or CYB5B is required for sterol C4-demethylation. The L-Cyb5b-/-/shCyb5a mice are largely normal, with lower sterol regulatory element-binding protein (SREBP)-target gene expression during refeeding and higher liver triglyceride levels while fasting, as T-MAS and dihydro-T-MAS inhibit the SREBP pathway and activate the PPARγ pathway. In summary, CYB5A and CYB5B compensate for sterol C4-demethylation, and T-MAS and dihydro-T-MAS can modulate the SREBP and PPARγ pathways.

Keywords:  CP: Metabolism; CYB5A/CYB5B; SREBP and PPARγ pathway; T-MAS/dihydro-T-MAS; cholesterol biosynthesis; sterol C4-demethylation

DOI:  https://doi.org/10.1016/j.celrep.2024.114912
J Neuropathol Exp Neurol. 2024 Nov 04. pii: nlae114. [Epub ahead of print]

Mechanisms of fibrinogen trans-activation of the EGFR/Ca2+ signaling axis to regulate mitochondrial transport and energy transfer and inhibit axonal regeneration following cerebral ischemia.

Shengqiang Zhou, Bo Li, Dahua Wu, Yanjun Chen, Wen Zeng, Jia Huang, Lingjuan Tan, Guo Mao, Fang Liu.

  Ischemic stroke results in inhibition of axonal regeneration but the roles of fibrinogen (Fg) in neuronal signaling and energy crises in experimental stroke are under-investigated. We explored the mechanism of Fg modulation of axonal regeneration and neuronal energy crisis after cerebral ischemia using a permanent middle cerebral artery occlusion (MCAO) rat model and primary cortical neurons under low glucose-low oxygen. Behavioral tests assessed neurological deficits; immunofluorescence, immunohistochemistry, and Western-blot analyzed Fg and protein levels. Fluo-3/AM fluorescence measured free Ca2+ and ATP levels were gauged via specific assays and F560nm/F510nm ratio calculations. Mito-Tracker Green labeled mitochondria and immunoprecipitation studied protein interactions. Our comprehensive study revealed that Fg inhibited axonal regeneration post-MCAO as indicated by reduced GAP43 expression along with elevated free Ca2+, both suggesting an energy crisis. Fg impeded mitochondrial function and mediated impairment through the EGFR/Ca2+ axis by trans-activating EGFR via integrin αvβ3 interaction. These results indicate that the binding of Fg with integrin αvβ3 leads to the trans-activation of the EGFR/Ca2+ signaling axis thereby disrupting mitochondrial energy transport and axonal regeneration and exacerbating the detrimental effects of ischemic neuronal injury.

Keywords:  EGFR/Ca2+ signaling; axonal regeneration; fibrinogen; middle cerebral artery occlusion; neuronal energy crisis

DOI:  https://doi.org/10.1093/jnen/nlae114
Parasitol Int. 2023 Oct 27. pii: S1383-5769(23)00099-5. [Epub ahead of print] 102821

Defective glycolysis in the cerebrum and cerebellum correlates with the pathology and neurological declines in mice with Angiostrongylus cantonensis infection.

Shih-Yi Peng, Ho Yin Pekkle Lam, Yu-Ting Huang.

  A significant complication of angiostrongyliasis remains eosinophilic meningoencephalitis, leading to patients' neurological deterioration, cerebral palsy, and respiratory changes, resulting in death. Clinically, A. cantonensis-infected patients sometimes showed decreased CSF glucose levels. Animal models infected with A. cantonensis have also reported a reduced serum glucose profile. While the brain uses glucose as the primary fuel source, glycolysis is essential for various neural activities in the brain. The defection of the glycolytic pathway has also been found to closely correlate to neurodegenerative diseases such as Alzheimer's disease. However, the role of glycolysis in the pathology and neurological declines associated with A. cantonensis infection remains unknown. Our current study has shown that A. cantonensis infection increases glucose content in the brain and suppresses the expression of the glycolytic enzymes in the brain. Glycolytic products such as pyruvate and ATP were also decreased in their level in the brain. This suppression of brain glycolysis was found to be correlated to the host's histopathology and neurological symptoms. Further analysis using mice infected with a different number of third-stage larvae (L3) A. cantonensis revealed that the defection of glycolysis was indeed caused by the presence of fifth-stage larvae (L5) of A. cantonensis in the brain of experimental mice. However, it may not be directly related to the damage that L5 caused to the brain. Our study delineates some aspects of the pathophysiology of angiostrongyliasis and may provide potential therapeutic targets for the future.

Keywords:  Angiostrongyliasis; Angiostrongylus cantonensis; Glycolysis; Meningoencephalitis

DOI:  https://doi.org/10.1016/j.parint.2023.102821
Mol Genet Metab. 2023 Oct 20. pii: S1096-7192(23)00341-4. [Epub ahead of print] 107711

Recent developments in the analytical approaches of acyl-CoAs to assess their role in mitochondrial fatty acid oxidation disorders.

Madhulika Singh, Hyung L Elfrink, Amy C Harms, Thomas Hankemeier.

  Fatty acid oxidation disorders (FAOD) are inborn errors of metabolism that occur due to deficiency of specific enzyme activities and transporter proteins involved in the mitochondrial metabolism of fatty acids, causing a deficiency in ATP production. The identification of suitable biomarkers plays a crucial role in predicting the future risk of disease and monitoring responses to therapies. Acyl-CoAs are directly involved in the steps of fatty acid oxidation and are the primary biomarkers associated with FAOD. However, acyl-CoAs are not used as diagnostic biomarkers in hospitals and clinics as they are present intracellularly with low endogenous levels. Additionally, the analytical method development of acyl-CoAs is quite challenging due to diverse physicochemical properties and instability. Hence, secondary biomarkers such as acylcarnitines are used for the identification of FAOD. In this review, the focus is on the analytical techniques that have evolved over the years for the identification and quantitation of acyl-CoAs. Among these techniques, liquid chromatography-mass spectrometry clearly has an advantage in terms of sensitivity and selectivity. Stable isotope labeling by essential nutrients in cell culture (SILEC) enables the generation of labeled internal standards. Each acyl-CoA species has a distinct pattern of instability and degradation, and the use of appropriately matched internal standards can compensate for such issues. Although significant progress has been made in measuring acyl-CoAs, more efforts are needed for bringing these technical advancements to hospitals and clinics. This review also highlights the difficulties involved in the routine use of acyl-CoAs as a diagnostic biomarker and some of the measures that can be adopted by clinics and hospitals for overcoming these limitations.

Keywords:  Acyl-CoA; Biomarkers; Fatty acid oxidation disorders; LC-MS; Newborn screening; SILEC

DOI:  https://doi.org/10.1016/j.ymgme.2023.107711
Methods Enzymol. 2024 ;pii: S0076-6879(24)00404-X. [Epub ahead of print]707 101-152

Mass spectrometry-based proteomics to study mutants and interactomes of mitochondrial translocation proteins.

Silke Oeljeklaus, Lakshita Sharma, Julian Bender, Bettina Warscheid.

  The multiple functions of mitochondria are governed by their proteome comprising 1000-1500 proteins depending on the organism. However, only few proteins are synthesized inside mitochondria, whereas most are "born" outside mitochondria. To reach their destined location, these mitochondrial proteins follow specific import routes established by a mitochondrial translocase network. A detailed understanding of the role and interplay of the different translocases is imperative to understand mitochondrial biology and how mitochondria are integrated into the cellular network. Mass spectrometry (MS) proved to be effective to study the translocase network regarding composition, functions, interplay, and cellular responses evoked by dysfunction. In this chapter, we provide protocols tailored to MS-enabled functional analysis of mutants and interactomes of mitochondrial translocation proteins. In the first part, we exemplify the MS-based proteomics analysis of translocation mutants for delineating the human mitochondrial importome following depletion of the central translocation protein TOMM40. The protocol comprises metabolic stable isotope labeling, TOMM40 knockdown, preparation of mitochondrial fractions, and sample preparation for liquid chromatography (LC)-MS. For deep MS analysis, prefractionation of peptide mixtures by high pH reversed-phase LC is described. In the second part, we outline an affinity purification MS approach to reveal the association of an orphaned protein with the translocase TIM23. The protocol covers FLAG-tag affinity purification of protein complexes from mitochondrial fractions and downstream sample preparation for interactome analysis. In the last unifying part, we describe methods for LC-MS, data processing, statistical analysis and visualization of quantitative MS data, and provide a Python code for effective, customizable analysis.

Keywords:  Affinity-purification mass spectrometry; Data analysis; Importome; Interactome; Mass spectrometry; Mitochondria; Protein quantification; Proteomics; SILAC; Translocation mutants

DOI:  https://doi.org/10.1016/bs.mie.2024.07.059
J Lipid Res. 2024 Oct 25. pii: S0022-2275(24)00182-2. [Epub ahead of print] 100677

Quantifying acyl-chain diversity in isobaric compound lipids containing monomethyl branched-chain fatty acids.

C R Green, M J Kolar, G H McGregor, A T Nelson, M Wallace, C M Metallo.

  Compound lipids comprise a diverse group of metabolites present in living systems, and metabolic- and environmentally-driven structural distinctions across this family is increasingly linked to biological function. However, methods for deconvoluting these often isobaric lipid species are lacking or require specialized instrumentation. Notably, acyl-chain diversity within cells may be influenced by nutritional states, metabolic dysregulation, or genetic alterations. Therefore, a reliable, validated method of quantifying structurally similar even-, odd-, and branched-chain acyl groups within intact compound lipids will be invaluable for gaining molecular insights into their biological functions. Here we demonstrate the chromatographic resolution of isobaric lipids containing distinct combinations of straight-chain and branched-chain acyl groups via ultra-high-pressure liquid chromatography (UHPLC)-mass spectrometry (MS) using a C30 liquid chromatography column. Using metabolically-engineered adipocytes lacking branched-keto acid dehydrogenase A (Bckdha), we validate this approach through a combination of fatty acid supplementation and metabolic tracing using monomethyl branched-chain fatty acids and valine. We observe resolution of numerous isobaric triacylglycerols and other compound lipids, demonstrating the resolving utility of this method. This approach adds to the toolbox for laboratories to quantify and characterize acyl chain diversity across the lipidome.

Keywords:  BCKDH; Branched-chain fatty acids; C30 chromatography; stable isotope tracing

DOI:  https://doi.org/10.1016/j.jlr.2024.100677