bims-medebr 2024-12-15 papers

bims-medebr

Biomed News

on Metabolism of the developing brain

Issue of 2024–12–15
sixteen papers selected by
Regina F. Fernández, Johns Hopkins University

TMAO is involved in sleep deprivation-induced cognitive dysfunction through regulating astrocytic cholesterol metabolism via SREBP2.
Astrocytic mitochondrial transfer to brain endothelial cells and pericytes in vivo increases with aging.
Providing lysophosphatidylcholine-bound omega-3 fatty acids increased eicosapentaenoic acid, but not docosahexaenoic acid, in the cortex of mice with the apolipoprotein E3 or E4 allele.
Inflammatory Signaling Induces Mitochondrial Dysfunction and Neuronal Death in Traumatic Brain Injury via Downregulation of OXPHOS Genes.
Are stroke-like events in pyruvate dehydrogenase deficiency ischemic, metabolic, or both in nature?
Distinct roles of ascorbic acid in extracellular vesicles and free form: Implications for metabolism and oxidative stress in presymptomatic Huntington's Disease.
Microglial lipid phosphatase SHIP1 limits complement-mediated synaptic pruning in the healthy developing hippocampus.
Differential effects of short-term and long-term ketogenic diet on gene expression in the aging mouse brain.
Carnitine palmitoyltransferase 1 facilitates fatty acid oxidation in a non-cell-autonomous manner.
Ceramide synthase 6 induces mitochondrial dysfunction and apoptosis in hemin-treated neurons by impairing mitophagy through interacting with sequestosome 1.
Tissue specific roles of fatty acid oxidation.
The kinase LRRK2 is required for the physiological function and expression of the glial glutamate transporter EAAT2 (SLC1A2).
Characterization of UGT8 as a monogalactosyl diacylglycerol synthase in mammals.
Mitochondrial Alterations in Alzheimer's Disease: Insight from the 5xFAD Mouse Model.
Nanobodies against the myelin enzyme CNPase as tools for structural and functional studies.
Palmitoylethanolamide causes dose-dependent changes in brain function and the lipidome.

Front Mol Neurosci. 2024 ;17 1499591

TMAO is involved in sleep deprivation-induced cognitive dysfunction through regulating astrocytic cholesterol metabolism via SREBP2.

Shan Zhu, Yue Wang, Yansong Li, Na Li, Yige Zheng, Qiao Li, Hongyan Guo, Jianyu Sun, Qian Zhai, Yaomin Zhu.

  Sleep deprivation (SD) contributes to cognitive impairment. Astrocytic cholesterol biosynthesis is crucial for brain cholesterol homeostasis and cognitive function. However, the underlying mechanism of astrocytic cholesterol metabolism in SD-induced cognitive impairment has not been fully explored. Trimethylamine N-oxide (TMAO), a product of liver flavin-containing monooxygenase-3 (FMO3), has been shown to be increased in the urine of sleep-deprived humans and implicated with peripheral cholesterol metabolism. Nevertheless, how TMAO affects brain cholesterol metabolism remains unclear. In our study, increased FMO3 and brain TMAO levels were observed in the SD mice, and elevated levels of TMAO were confirmed to lead to SD-induced cognitive dysfunction. In addition, we found that the expression of sterol regulatory element-binding protein 2 (SREBP2) is decreased in the brain of SD mice, resulting in the reduction in brain cholesterol content, which in turn causes synaptic damage. Moreover, we demonstrated that TMAO inhibits the expression of SREBP2. In contrast, FMO3 inhibitor 3,3'-diindolylmethane (DIM) alleviates SD-induced cognitive impairment by targeting the liver-brain axis. In conclusion, our study revealed that the TMAO pathway is involved in memory impairment in SD mice through deregulating astrocytic cholesterol metabolism.

Keywords:  FMO3; SREBP2; TMAO; astrocytes; cholesterol metabolism; sleep deprivation

DOI:  https://doi.org/10.3389/fnmol.2024.1499591
J Cereb Blood Flow Metab. 2024 Dec 12. 271678X241306054

Astrocytic mitochondrial transfer to brain endothelial cells and pericytes in vivo increases with aging.

Gopal V Velmurugan, Hemendra J Vekaria, Samir P Patel, Patrick G Sullivan, W Brad Hubbard.

  Intercellular mitochondrial transfer (IMT) is an intriguing biological phenomenon where mitochondria are transferred between different cells and notably, cell types. IMT is physiological, occurring in normal conditions, but also is utilized to deliver healthy mitochondria to cells in distress. Transferred mitochondria can be integrated to improve cellular metabolism, and mitochondrial function. Research on the mitochondrial transfer axis between astrocytes and brain capillaries in vivo is limited by the cellular heterogeneity of the neurovascular unit. To this end, we developed an inducible mouse model that expresses mitochondrial Dendra2 only in astrocytes and then isolated brain capillaries to remove all intact astrocytes. This method allows the visualization of in vivo astrocyte- endothelial cell (EC) and astrocyte-pericyte IMT. We demonstrate evidence of astrocyte-EC and astrocyte-pericyte mitochondrial transfer within brain capillaries. We also show that healthy aging enhances mitochondrial transfer from astrocytes to brain capillaries, revealing a potential link between brain aging and cellular mitochondrial dynamics. Finally, we observe that astrocyte-derived extracellular vesicles transfer mitochondria to brain microvascular endothelial cells, showing the potential route of in vivo IMT. These results represent a breakthrough in our understanding of IMT in the brain and a new target in brain aging and neurovascular metabolism.

Keywords:  Mitochondrial transfer; aging; astrocyte EV-mito; brain capillaries; capillary isolation

DOI:  https://doi.org/10.1177/0271678X241306054
Prostaglandins Leukot Essent Fatty Acids. 2024 Nov 30. pii: S0952-3278(24)00055-3. [Epub ahead of print]204 102661

Providing lysophosphatidylcholine-bound omega-3 fatty acids increased eicosapentaenoic acid, but not docosahexaenoic acid, in the cortex of mice with the apolipoprotein E3 or E4 allele.

Bijou Andriambelo, Annick Vachon, Marc-André Dansereau, Benoit Laurent, Mélanie Plourde.

   BACKGROUND: Several mechanisms have been proposed for the brain uptake of omega-3 fatty acids (n-3), including passive diffusion of the unesterified form and the use of Mfsd2a transporter for the lysophosphatidylcholine (LPC) form. We hypothesize that the accumulation of LPC n-3 in the brain is lower in mice carrying the apolipoprotein E ε4 allele (APOE4), a major genetic risk factor for developing sporadic Alzheimer's disease in humans.
OBJECTIVE: Determine whether two or four months of supplementation with LPC n-3 increases the levels of docosahexaenoic acids (DHA) and eicosapentaenoic acids (EPA) in the frontal cortex of APOE3 and APOE4 mice.
METHODS: APOE3 and APOE4 mice were administered LPC n-3 (9.6 mg DHA + 18.3 mg EPA) or sunflower oil (control) by oral gavage for two or four months (n = 5-8 per genotype, per treatment, and per treatment duration). At the end of the treatment period, frontal cortices were collected, and their FA profiles analyzed by gas chromatography with flame ionization detection.
RESULTS: After two months of gavage with LPC n-3, APOE3 mice showed increased levels of EPA in their cortex, but not DHA. In APOE4 mice, neither EPA nor DHA levels were significantly affected. After four months of LPC n-3, both APOE3 and APOE4 mice exhibited higher EPA levels, while changes in DHA levels were not statistically significant.
CONCLUSION: LPC n-3 supplementation increased EPA, but not DHA, levels in the frontal cortex of mice in a duration- and APOE genotype-dependent manner. Further research is needed to explore the implications for brain health.

Keywords:  APOE4; Brain; Cortex; Docosahexaenoic acids; Eicosapentaenoic acids; Lysophosphatidylcholine; Omega-3 fatty acids

DOI:  https://doi.org/10.1016/j.plefa.2024.102661
Biochem Genet. 2024 Dec 10.

Inflammatory Signaling Induces Mitochondrial Dysfunction and Neuronal Death in Traumatic Brain Injury via Downregulation of OXPHOS Genes.

Hui Dong, Hui Zhang, Lei Cai, Quanyi Ye, Heping Wang, Bo Liu, Wenhu Zhang, Junxin Li.

  Traumatic brain injury (TBI) is a major cause of neurological dysfunction and disability. This study aimed to investigate the transcriptomic changes and the functional consequences in TBI, focusing on the interplay between inflammation and mitochondrial impairment. Brain tissue samples from TBI patients and healthy controls were subjected to RNA-sequencing analysis. Mouse hippocampal HT-22 cells were treated with inflammatory cytokine and the PGC-1α activator ZLN005. Mitochondrial function, oxidative stress, and apoptosis were assessed using Seahorse respirometry, electron microscopy, flow cytometry, and molecular assays. A TBI mouse model was established to evaluate the therapeutic effects of ZLN005. Transcriptome profiling revealed downregulation of mitochondrial oxidative phosphorylation (OXPHOS) genes, particularly those encoded by the mitochondrial genome, along with enrichment of neurodegenerative pathways in TBI patients. Concomitantly, pro-inflammatory signaling pathways showed upregulation. In vitro studies demonstrated that inflammatory cytokine TNF-α treatment impaired mitochondrial respiration, induced oxidative stress and apoptosis in HT-22 cells, which could be rescued by ZLN005-mediated PGC-1α activation and restoration of OXPHOS gene expression. Administration of ZLN005 in the TBI mouse model alleviated neuronal cell death, preserved mitochondrial integrity, normalized OXPHOS gene levels in brain tissues, and improved cognitive function. This study uncovers a mechanistic link between inflammation-induced downregulation of mitochondrial OXPHOS genes and neuronal damage in TBI. Targeting this pathway by activating PGC-1α represents a potential therapeutic strategy for TBI.

Keywords:  Inflammation; Mitochondria; Neuronal death; OXPHOS; Traumatic brain injury

DOI:  https://doi.org/10.1007/s10528-024-10980-6
Neurol Sci. 2024 Dec 10.

Are stroke-like events in pyruvate dehydrogenase deficiency ischemic, metabolic, or both in nature?

Mehri Sounira, Josef Finsterer.

  The interesting article by Fecarotta et al. reports a 6-year-old female with pyruvate dehydrogenase (PDHC) deficiency due to the variant c.869 A > C in PDHA1, which manifested phenotypically itself with microcephaly, developmental delay, lactic acidosis, global cerebral atrophy, and subependymal gliosis lateral to the left ventricle. At the age of 6, the patient suffered acute-onset right hemiparesis, which was attributed to a stroke-like lesion (SLL) in the left cerebral peduncle. However, there are some arguments against a SLL in the index patient. First, SLLs have not yet been reported in PDHC, Second, the lesion shown in Fig. 1 does not meet the criteria for SLL. Third, the authors themselves speculate the hyperintense DWI lesion in the right globus pallidus may represent hyperperfusion. Before a peduncular DWI hyperintensity can be interpreted as SLL, ischemic stroke must be thoroughly ruled out.

Keywords:  Lactic acidosis; Mitochondrial; Multimodal MRI; Pyruvate-dehydrogenase deficiency; Stroke-like episode

DOI:  https://doi.org/10.1007/s10072-024-07927-5
Free Radic Biol Med. 2024 Dec 09. pii: S0891-5849(24)01099-2. [Epub ahead of print]

Distinct roles of ascorbic acid in extracellular vesicles and free form: Implications for metabolism and oxidative stress in presymptomatic Huntington's Disease.

Felipe A Beltrán, Leandro Torres, Paulina Troncoso-Escudero, Juan Villalobos-González, Gonzalo Mayorga-Weber, Marcelo Lara, Adriana Covarrubias-Pinto, Sharin Valdivia, Isidora Vicencio, Eduardo Papic, Carolina Paredes-Martínez, Mara E Silva-Januàrio, Alejandro Rojas, Luis L P daSilva, Felipe Court, Abraham Rosas-Arellano, Federico Bátiz, Patricio Rojas, Francisco J Rivera, Maite A Castro.

  Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the first exon of the huntingtin gene. The huntingtin protein (Htt) is ubiquitously expressed and localized in several organelles, including endosomes, where it plays an essential role in intracellular trafficking. Presymptomatic HD is associated with a failure in energy metabolism and oxidative stress. Ascorbic acid is a potent antioxidant that plays a key role in modulating neuronal metabolism and is highly concentrated in the brain. During synaptic activity, neurons take up ascorbic acid released by glial cells; however, this process is disrupted in HD. In this study, we aim to elucidate the molecular and cellular mechanisms underlying this dysfunction. Using an electrophysiological approach in presymptomatic YAC128 HD slices, we observed decreased ascorbic acid flux from astrocytes to neurons, which altered neuronal metabolic substrate preferences. Ascorbic acid efflux and recycling were also decreased in cultured astrocytes from YAC128 HD mice. We confirmed our findings using GFAP-HD160Q, an HD mice model expressing mutant N-terminal Htt mainly in astrocytes. For the first time, we demonstrated that ascorbic acid is released from astrocytes via extracellular vesicles (EVs). Decreased number of particles and exosomal markers were observed in EV fractions from cultured YAC128 HD astrocytes and Htt-KD cells. We observed reduced number of multivesicular bodies (MVBs) in YAC128 HD striatum via electron microscopy, suggesting mutant Htt alters MVB biogenesis. EVs containing ascorbic acid effectively reduced reactive oxygen species, whereas "free" ascorbic acid played a role in modulating neuronal metabolic substrate preferences. These findings suggest that the early redox imbalance observed in HD arises from a reduced release of ascorbic acid-containing EVs by astrocytes. Meanwhile, a decrease in "free" ascorbic acid likely contributes to presymptomatic metabolic impairment.

Keywords:  ascorbic acid; exosomes; glucose; lactate; neurodegeneration

DOI:  https://doi.org/10.1016/j.freeradbiomed.2024.12.001
Immunity. 2024 Dec 06. pii: S1074-7613(24)00513-2. [Epub ahead of print]

Microglial lipid phosphatase SHIP1 limits complement-mediated synaptic pruning in the healthy developing hippocampus.

Alessandro Matera, Anne-Claire Compagnion, Chiara Pedicone, M Kotah Janssen, Andranik Ivanov, Katia Monsorno, Gwenaël Labouèbe, Loredana Leggio, Marta Pereira, Dieter Beule, Virginie Mansuy-Aubert, Tim L Williams, Nunzio Iraci, Amanda Sierra, Samuele G Marro, Alison M Goate, Bart J L Eggen, William G Kerr, Rosa C Paolicelli.

  The gene inositol polyphosphate-5-phosphatase D (INPP5D), which encodes the lipid phosphatase SH2-containing inositol polyphosphate 5-phosphatase 1 (SHIP1), is associated with the risk of Alzheimer's disease (AD). How it influences microglial function and brain physiology is unclear. Here, we showed that SHIP1 was enriched in early stages of healthy brain development. By combining in vivo loss-of-function approaches and proteomics, we discovered that mice conditionally lacking microglial SHIP1 displayed increased complement and synapse loss in the early postnatal brain. SHIP1-deficient microglia showed altered transcriptional signatures and abnormal synaptic pruning that was dependent on the complement system. Mice exhibited cognitive defects in adulthood only when microglial SHIP1 was depleted early postnatally but not at later stages. Induced pluripotent stem cell (iPSC)-derived microglia lacking SHIP1 also showed increased engulfment of synaptic structures. These findings suggest that SHIP1 is essential for proper microglia-mediated synapse remodeling in the healthy developing brain. Disrupting this process has lasting behavioral effects and may be linked to vulnerability to neurodegeneration.

Keywords:  AD risk genes; Alzheimer’s disease; INPP5D; SHIP1; cognitive dysfunction; complement; microglia; synaptic pruning

DOI:  https://doi.org/10.1016/j.immuni.2024.11.003
J Nutr Health Aging. 2024 Dec 10. pii: S1279-7707(24)00515-3. [Epub ahead of print]29(2): 100427

Differential effects of short-term and long-term ketogenic diet on gene expression in the aging mouse brain.

Matthew S Stratton, José Alberto López-Domínguez, Alessandro Canella, Jon J Ramsey, Gino A Cortopassi.

   BACKGROUND: Aging is associated with multiple neurodegenerative conditions that severely limit quality of life and can shorten lifespan. Studies in rodents indicate that in addition to extending lifespan, the ketogenic diet (KD) improves cognitive function in aged animals, yet long term adherence to KD in Humans is poor.
OBJECTIVES: To broadly investigate what mechanisms might be activated in the brain in response to ketogenic diet.
METHODS: We conducted transcriptome wide analysis on whole brain samples from 13-month-old mice, 13-month-old mice fed a ketogenic diet for 1 month, 26-month-old mice, and 26-month-old mice fed a ketogenic diet for 14 months.
RESULTS: As expected, analysis of differently expressed genes between the old (26 month) vs younger mice (13 month) showed clear activation of inflammation and complement system pathways with aging. Analysis between the 26-month-old animals fed ketogenic diet for 14 months with 26-month-old animals fed control diet indicate that long-term KD resulted in activation of LRP, TCF7L2 (WNT pathway), and IGF1 signaling. There was also a significant increase in the expression of SOX2-dependent oligodendrocyte/myelination markers, though TCF7L2 and SOX2 dependent gene sets were largely overlapping. Remarkably, the effect of 1 month of ketogenic diet was minimal and there was no congruence between gene expression effects of short-term KD vs long-term KD.
CONCLUSIONS: This work informs target identification efforts for aging and neurodegenerative disorder therapeutics discovery while also establishing differential effects of short-term vs long-term KD on gene expression in the brain.

Keywords:  Brain aging; Ketogenic diet; Target discovery; Transcriptomics

DOI:  https://doi.org/10.1016/j.jnha.2024.100427
Cell Rep. 2024 Dec 12. pii: S2211-1247(24)01357-3. [Epub ahead of print]43(12): 115006

Carnitine palmitoyltransferase 1 facilitates fatty acid oxidation in a non-cell-autonomous manner.

Joseph Choi, Danielle M Smith, Susanna Scafidi, Ryan C Riddle, Michael J Wolfgang.

  Mitochondrial fatty acid oxidation is facilitated by the combined activities of carnitine palmitoyltransferase 1 (Cpt1) and Cpt2, which generate and utilize acylcarnitines, respectively. We compare the response of mice with liver-specific deficiencies in the liver-enriched Cpt1a or the ubiquitously expressed Cpt2 and discover that they display unique metabolic, physiological, and molecular phenotypes. The loss of Cpt1a or Cpt2 results in the induction of the muscle-enriched isoenzyme Cpt1b in hepatocytes in a Pparα-dependent manner. However, hepatic Cpt1b does not contribute substantively to hepatic fatty acid oxidation when Cpt1a is absent. Liver-specific double knockout of Cpt1a and Cpt1b or Cpt2 eliminates the mitochondrial oxidation of non-esterified fatty acids. However, Cpt1a/Cpt1b double knockout mice retain fatty acid oxidation by utilizing extracellular long-chain acylcarnitines that are dependent on Cpt2. These data demonstrate the non-cell-autonomous intercellular metabolism of fatty acids in hepatocytes.

Keywords:  CP: Metabolism; Cpt1; Cpt2; acylcarnitine; biochemistry; fasting; liver; metabolism

DOI:  https://doi.org/10.1016/j.celrep.2024.115006
Free Radic Biol Med. 2024 Dec 04. pii: S0891-5849(24)01122-5. [Epub ahead of print]227 282-295

Ceramide synthase 6 induces mitochondrial dysfunction and apoptosis in hemin-treated neurons by impairing mitophagy through interacting with sequestosome 1.

Aoqian Xu, Yikui Liu, Baofeng Wang, Qixiang Zhang, Yuxiao Ma, Yuxiao Xue, Zhuohang Wang, Qingfang Sun, Yuhao Sun, Liuguan Bian.

  Intracerebral hemorrhage (ICH) is a severe subtype of stroke linked to high morbidity and mortality rates. However, the underlying mechanisms of neuronal injury post-ICH remain poorly understood. In this study, we investigated sphingolipid metabolism alterations in neurons using lipidomics and explored the regulatory mechanisms involved. Western blot and live-cell imaging were applied to detect mitochondrial quality and mitophagy level. We found a significant upregulation of ceramide synthase 6 (CERS6)-related C16 ceramide biosynthesis after hemin treatment. Knockdown of CERS6 notably ameliorated mitochondrial dysfunction and reduced neuronal apoptosis. Additionally, impaired neuronal mitophagy was observed after hemin treatment, which was restored by CERS6 knockdown. Mechanistically, CERS6 impaired mitophagy by interacting with sequestosome 1, leading to mitochondrial dysfunction and neuronal apoptosis. Our study explored the relationship between ceramide metabolism and mitophagy in neurons, revealing the pro-apoptotic role of CERS6 while providing a potential therapeutic target for patients with ICH.

Keywords:  Apoptosis; Ceramide synthase 6; Intracerebral hemorrhage; Lipidomic; Mitochondria; Mitophagy; Neuron; Sphingolipid

DOI:  https://doi.org/10.1016/j.freeradbiomed.2024.12.018
Adv Biol Regul. 2024 Dec 05. pii: S2212-4926(24)00058-7. [Epub ahead of print] 101070

Tissue specific roles of fatty acid oxidation.

Danielle M Smith, Joseph Choi, Michael J Wolfgang.

Mitochondrial long chain fatty acid β-oxidation is a critical central carbon catabolic process. The importance of fatty acid oxidation is made evident by the life-threatening disease associated with diverse inborn errors in the pathway. While inborn errors show multisystemic requirements for fatty acid oxidation, it is not clear from the clinical presentation of these enzyme deficiencies what the tissue specific roles of the pathway are compared to secondary systemic effects. To understand the cell or tissue specific contributions of fatty acid oxidation to systemic physiology, conditional knockouts in mice have been employed to determine the requirements of fatty acid oxidation in disparate cell types. This has produced a host of surprising results that sometimes run counter to the canonical view of this metabolic pathway. The rigor of conditional knockouts has also provided clarity over previous research utilizing cell lines in vitro or small molecule inhibitors with dubious specificity. Here we will summarize current research using mouse models of Carnitine Palmitoyltransferases to determine the tissue specific roles and requirements of long chain mitochondrial fatty acid β-oxidation.

DOI: https://doi.org/10.1016/j.jbior.2024.101070
J Neurochem. 2025 Jan;169(1): e16265

The kinase LRRK2 is required for the physiological function and expression of the glial glutamate transporter EAAT2 (SLC1A2).

Angela Di Iacovo, Chiara D'Agostino, Manan Bhatt, Tiziana Romanazzi, Stefano Giovannardi, Raffaella Cinquetti, Cristina Roseti, Elena Bossi.

  Neurotransmitter transporters (NTTs) control synaptic responses by modulating the concentration of neurotransmitters at the synaptic cleft. Glutamate is the most abundant excitatory neurotransmitter in the brain and needs to be finely tuned in time and space to maintain a healthy brain and precise neurotransmission. The glutamate transporter EAAT2 (SLC1A2) is primarily responsible for glutamate clearance. EAAT2 impairment has been associated with Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), and Parkinson's disease (PD). Mutations in leucine-rich repeat kinase 2 (LRRK2) contribute to both monogenic and sporadic forms of PD, of which the common substitution Gly2019Ser is associated with a significant deficit in EAAT2 expression. The role of pathological mutants of the LRRK2 is intensively studied and reviewed. Here we have focused the attention on the physiological role of LRRK2 on EAAT2, comparing the activity of NTTs with or without the LRRK2 kinase. By heterologous expression in Xenopus laevis oocytes and two-electrode voltage clamp, the current amplitudes of the selected NTTs and kinetic parameters have been collected in the presence and absence of LRRK2. The results show that EAAT2 expression and function are impaired in the absence of the kinase and also under its pharmacological inhibition via MLi-2 treatment. LRRK2 stabilizes EAAT2 expression increasing the amount of transporter at the plasma membrane. Interestingly, the LRRK2 action is EAAT2-specific, as we observed no significant changes in the transport current amplitude and kinetic parameters obtained for the other excitatory and inhibitory NTTs studied. This study, for the first time, demonstrates the physiological importance of LRRK2 in EAAT2 function, highlighting the specificity of LRRK2-mediated modulation of EAAT2 and suggesting a potential role for the kinase as a checkpoint for preserving neurons from excitotoxicity. In brain conditions associated with impaired glutamate clearance, targeting LRRK2 for EAAT2 regulation may offer novel therapeutic opportunities.

Keywords:  EAAT2 (SLC1A2); LRRK2; SLC1; SLC6; dopamine transporter; excitatory/inhibitory balance

DOI:  https://doi.org/10.1111/jnc.16265
J Biochem. 2024 Dec 10. pii: mvae084. [Epub ahead of print]

Characterization of UGT8 as a monogalactosyl diacylglycerol synthase in mammals.

Yohsuke Ohba, Mizuki Motohashi, Makoto Arita.

  Monogalactosyl diacylglycerol (MGDG) is a major membrane lipid component in plants and is crucial for proper thylakoid functioning. However, MGDG in mammals has not received much attention, partly because of its relative scarcity in mammalian tissues. In addition, the biosynthetic pathway of MGDG in mammals has not been thoroughly analyzed, although some reports have suggested that UGT8, a ceramide galactosyltransferase, has the potential to catalyze MGDG biosynthesis. Here, we successfully captured the endogenous levels of MGDG in HeLa cells using LC-MS/MS-based lipidomics. Cellular MGDG was completely depleted in CRISPR/Cas9-mediated UGT8 knockout HeLa cells. Transient overexpression of UGT8 enhanced MGDG production in HeLa cells, and the corresponding cell lysates displayed MGDG biosynthetic activity in vitro. Site-directed mutagenesis revealed that His358 within the UGT signature sequence was important for its activity. UGT8 was localized in the endoplasmic reticulum and activation of the unfolded protein response by membrane lipid saturation was impaired in UGT8 knockout cells. These results demonstrate that UGT8 is an MGDG synthase in mammals and that UGT8 regulates membrane lipid saturation signals in cells.

Keywords:  endoplasmic reticulum stress (ER stress); glycolipid; mass spectrometry (MS); membrane lipid; unfolded protein response (UPR)

DOI:  https://doi.org/10.1093/jb/mvae084
Mol Neurobiol. 2024 Dec 11.

Mitochondrial Alterations in Alzheimer's Disease: Insight from the 5xFAD Mouse Model.

Elif Nedret Keskinoz, Musa Celik, Ezgi Sila Toklucu, Kerem Birisik, Alev Erisir, Devrim Oz-Arslan.

  Mitochondrial dysfunction is increasingly recognized as a key factor in Alzheimer's disease (AD) pathogenesis, but the precise relationship between mitochondrial dynamics and proteinopathies in AD remains unclear. This study investigates the role of mitochondrial dynamics and function in the hippocampal tissue and peripheral blood mononuclear cells (PBMCs) of 5xFAD transgenic mice, as a model of AD. The levels of mitochondrial fusion proteins OPA1 and MFN2 and fission proteins DRP1 and phospho-DRP1 (S616) at 3, 6, and 9 months of age were assessed. Western blot analysis revealed significantly lower levels of OPA1 and MFN2 in the hippocampus of 6- and 9-month-old transgenic (TG) 5xFAD mice compared to controls (CTR), while DRP1 and pDRP1 levels were increased in 9-month-old TG mice. Additionally, MFN2 were decreased in the PBMCs of 9-month-old TG mice, indicating systemic mitochondrial alterations. Ultrastructural analysis of hippocampal tissues showed substantial alterations in mitochondrial morphology, including abnormalities in size and shape, a preponderance of teardrop-shaped mitochondria, and alterations in the somatic mitochondria-ER complex. Notably, mitochondria-associated ER contact sites were more distant in TG mice, suggesting functional impairments. Flow cytometric measurements demonstrated decreased mitochondrial membrane potential and mass, along with increased superoxide production, in the PBMCs of TG mice, particularly at 9 months, highlighting compromised mitochondrial function. Levels of key mitochondrial proteins including VDAC, TOM2O, and mitophagy-related protein PINK1 levels altered in both central and peripheral tissue of TG mice. These findings suggest that mitochondrial dysfunction and altered dynamics are early events in AD development in 5xFAD mice, manifesting in both central and peripheral tissues, and support the notion that mitochondrial abnormalities are an integral component of AD pathology. These insights might lead to the development of targeted therapies that modulate mitochondrial dynamics and function to mitigate AD progression.

Keywords:  Electron microscopy; Flow cytometry; MERCs; Mitochondrial dynamics; Western blot

DOI:  https://doi.org/10.1007/s12035-024-04632-4
J Neurochem. 2025 Jan;169(1): e16274

Nanobodies against the myelin enzyme CNPase as tools for structural and functional studies.

Sigurbjörn Markusson, Arne Raasakka, Marcel Schröder, Shama Sograte-Idrissi, Amir Mohammad Rahimi, Ommolbanin Asadpour, Henrike Körner, Dmitri Lodygin, Maria A Eichel-Vogel, Risha Chowdhury, Aleksi Sutinen, Gopinath Muruganandam, Manasi Iyer, Madeline H Cooper, Maya K Weigel, Nicholas Ambiel, Hauke B Werner, J Bradley Zuchero, Felipe Opazo, Petri Kursula.

  2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNPase) is an abundant constituent of central nervous system non-compact myelin, and its loss in mice and humans causes neurodegeneration. Additionally, CNPase is frequently used as a marker antigen for myelinating cells. The catalytic activity of CNPase, the 3'-hydrolysis of 2',3'-cyclic nucleotides, is well characterised in vitro, but the in vivo function of CNPase remains unclear. CNPase interacts with the actin cytoskeleton to counteract the developmental closure of cytoplasmic channels that travel through compact myelin; its enzymatic activity may be involved in adenosine metabolism and RNA degradation. We developed a set of high-affinity nanobodies recognising the phosphodiesterase domain of CNPase, and the crystal structures of each complex show that the five nanobodies have distinct epitopes. One of the nanobodies bound deep into the CNPase active site and acted as an inhibitor. Moreover, the nanobodies were characterised in imaging applications and as intrabodies, expressed in mammalian cells, such as primary oligodendrocytes. Fluorescently labelled nanobodies functioned in imaging of teased nerve fibres and whole brain tissue sections, as well as super-resolution microscopy. These anti-CNPase nanobodies provide new tools for structural and functional studies on myelin formation, dynamics, and disease, including high-resolution imaging of nerve tissue.

Keywords:  CNPase; function; imaging; myelin; nanobody; structure

DOI:  https://doi.org/10.1111/jnc.16274
Front Neurosci. 2024 ;18 1506352

Palmitoylethanolamide causes dose-dependent changes in brain function and the lipidome.

Shreyas Balaji, Taylor J Woodward, Emily Richter, Arnold Chang, Richard Otiz, Praveen P Kulkarni, Kaashyap Balaji, Heather B Bradshaw, Craig F Ferris.

  The present studies were undertaken to understand the effects of the commonly used nutraceutical PEA on brain function and lipid chemistry. These studies using MRI and broad-scale lipidomics are without precedent in animal or human research. During the MRI scanning session awake rats were given one of three doses of PEA (3, 10, or 30 mg/kg) or vehicle and imaged for changes in BOLD signal and functional connectivity. There was an inverse dose-response for negative BOLD suggesting a decrease in brain activity affecting the prefrontal ctx, sensorimotor cortices, basal ganglia and thalamus. However, there was a dose-dependent increase in functional connectivity in these same brain areas. Plasma and CNS levels of PEA and over 80 endogenous lipids (endolipids) were determined post treatment. While levels of PEA in the CNS were significantly higher after 30 mg/kg treatment, levels of the endocannabinoid, Anandamide, and at least 20 additional endolipids, were significantly lower across the CNS. Of the 78 endolipids that were detected in all CNS regions evaluated, 51 of them were modulated in at least one of the regions. Taken together, the functional connectivity and lipidomics changes provide evidence that PEA treatment drives substantial changes in CNS activity.

Keywords:  BOLD imaging; cerebellum; endocannabinoids; functional connectivity; lipidomics; sensorimotor cortex

DOI:  https://doi.org/10.3389/fnins.2024.1506352