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Scientific Reports Oct 2023The impact of high-intensity interval training (HIIT) on the central nervous system (CNS) in autoimmune neuroinflammation is not known. The aim of this study was to...
The impact of high-intensity interval training (HIIT) on the central nervous system (CNS) in autoimmune neuroinflammation is not known. The aim of this study was to determine the direct effects of HIIT on the CNS and development of experimental autoimmune encephalomyelitis (EAE). Healthy mice were subjected to HIIT by treadmill running and the proteolipid protein (PLP) transfer EAE model was utilized. To examine neuroprotection, PLP-reactive lymph-node cells (LNCs) were transferred to HIIT and sedentary (SED) mice. To examine immunomodulation, PLP-reactive LNCs from HIIT and SED donor mice were transferred to naïve recipients and analyzed in vitro. HIIT in recipient mice did not affect the development of EAE following exposure to PLP-reactive LNCs. HIIT mice exhibited enhanced migration of systemic autoimmune cells into the CNS and increased demyelination. In contrast, EAE severity in recipient mice injected with PLP-reactive LNCs from HIIT donor mice was significantly diminished. The latter positive effect was associated with decreased migration of autoimmune cells into the CNS and inhibition of very late antigen (VLA)-4 expression in LNCs. Thus, the beneficial effect of HIIT on EAE development is attributed solely to systemic immunomodulatory effects, likely because of systemic inhibition of autoreactive cell migration and reduced VLA-4 integrin expression.
Topics: Mice; Animals; Encephalomyelitis, Autoimmune, Experimental; High-Intensity Interval Training; Central Nervous System; Immunomodulation; Encephalomyelitis; Myelin Proteolipid Protein
PubMed: 37783693
DOI: 10.1038/s41598-023-43534-8 -
BioRxiv : the Preprint Server For... Oct 2023Diverse developmental signals and pro-death stresses converge on regulation of the mitochondrial pathway of apoptosis. BAX, a pro-apoptotic BCL-2 effector, directly...
Diverse developmental signals and pro-death stresses converge on regulation of the mitochondrial pathway of apoptosis. BAX, a pro-apoptotic BCL-2 effector, directly forms proteolipid pores in the outer mitochondrial member to activate the mitochondrial pathway of apoptosis. BAX is a viable pharmacological target for various human diseases, and increasing efforts have been made to study the molecular regulation of BAX and identify small molecules selectively targeting BAX. However, generating large quantities of monomeric and functionally-competent BAX has been challenging due to its aggregation-prone nature. Additionally, there is a lack of detailed and instructional protocols available for investigators who are not already familiar with recombinant BAX production. Here, we present a comprehensive high-yield protocol for expressing, purifying, and storing functional recombinant BAX protein. We utilize an intein-tagged BAX construct and employ a two-step chromatography strategy to capture and purify BAX, and provide example standard assays to observe BAX activation. We also highlight best practices for handling and storing BAX to effectively preserve its quality, shelf-life, and function.
PubMed: 37905126
DOI: 10.1101/2023.10.16.562589 -
Bioelectrochemistry (Amsterdam,... Oct 2024Functional characterization of transporters is impeded by the high cost and technical challenges of current transporter assays. Thus, in this work, we developed a new...
Functional characterization of transporters is impeded by the high cost and technical challenges of current transporter assays. Thus, in this work, we developed a new characterization workflow that combines cell-free protein synthesis (CFPS) and solid supported membrane-based electrophysiology (SSME). For this, membrane protein synthesis was accomplished in a continuous exchange cell-free system (CECF) in the presence of nanodiscs. The resulting transporters expressed in nanodiscs were incorporated into proteoliposomes and assayed in the presence of different substrates using the surface electrogenic event reader. As a proof of concept, we validated this workflow to express and characterize five diverse transporters: the drug/H-coupled antiporters EmrE and SugE, the lactose permease LacY, the Na/H antiporter NhaA from Escherichia coli, and the mitochondrial carrier AAC2 from Saccharomyces cerevisiae. For all transporters kinetic parameters, such as K, I, and pH dependency, were evaluated. This robust and expedite workflow (e.g., can be executed within only five workdays) offers a convenient direct functional assessment of transporter protein activity and has the ability to facilitate applications of transporters in medical and biotechnological research.
Topics: Cell-Free System; Escherichia coli Proteins; Membrane Transport Proteins; Saccharomyces cerevisiae; Escherichia coli; Proteolipids; Sodium-Hydrogen Exchangers; Saccharomyces cerevisiae Proteins; Monosaccharide Transport Proteins; Kinetics; Antiporters; Electrophysiological Phenomena; Symporters
PubMed: 38810322
DOI: 10.1016/j.bioelechem.2024.108732 -
Annals of Clinical and Translational... Sep 2023Hereditary spastic paraplegia (HSP) is a genetically heterogeneous disease caused by over 70 genes, with a significant number of patients still genetically unsolved. In...
OBJECTIVES
Hereditary spastic paraplegia (HSP) is a genetically heterogeneous disease caused by over 70 genes, with a significant number of patients still genetically unsolved. In this study, we recruited a suspected HSP family characterized by spasticity, developmental delay, ataxia and hypomyelination, and intended to reveal its molecular etiology by whole exome sequencing (WES) and long-read sequencing (LRS) analyses.
METHODS
WES was performed on 13 individuals of the family to identify the causative mutations, including analyses of SNVs (single-nucleotide variants) and CNVs (copy number variants). Accurate circular consensus (CCS) long-read sequencing (LRS) was used to verify the findings of CNV analysis from WES.
RESULTS
SNVs analysis identified a missense variant c.195G>T (p.E65D) of MORF4L2 at Xq22.2 co-segregating in this family from WES data. Further CNVs analysis revealed a microdeletion, which was adjacent to the MORF4L2 gene, also co-segregating in this family. LRS verified this microdeletion and confirmed the deletion range (chrX: 103,690,507-103,715,018, hg38) with high resolution at nucleotide level accuracy.
INTERPRETATIONS
In this study, we identified an Xq22.2 microdeletion (about 24.5 kb), which contains distal enhancers of the PLP1 gene, as a likely cause of SPG2 in this family. The lack of distal enhancers may result in transcriptional repression of PLP1 in oligodendrocytes, potentially affecting its role in the maintenance of myelin, and causing SPG2 phenotype. This study has highlighted the importance of noncoding genomic alterations in the genetic etiology of SPG2.
Topics: Humans; Spastic Paraplegia, Hereditary; Myelin Proteolipid Protein; Mutation; Mutation, Missense; Phenotype; Transcription Factors
PubMed: 37475517
DOI: 10.1002/acn3.51848 -
International Journal of Molecular... Aug 2023Anti-glycolipid antibodies have been reported to play pathogenic roles in peripheral inflammatory neuropathies, such as Guillain-Barré syndrome. On the other hand, the...
Anti-Glycolipid Antibody Examination in Five EAE Models and Theiler's Virus Model of Multiple Sclerosis: Detection of Anti-GM1, GM3, GM4, and Sulfatide Antibodies in Relapsing-Remitting EAE.
Anti-glycolipid antibodies have been reported to play pathogenic roles in peripheral inflammatory neuropathies, such as Guillain-Barré syndrome. On the other hand, the role in multiple sclerosis (MS), inflammatory demyelinating disease in the central nervous system (CNS), is largely unknown, although the presence of anti-glycolipid antibodies was reported to differ among MS patients with relapsing-remitting (RR), primary progressive (PP), and secondary progressive (SP) disease courses. We investigated whether the induction of anti-glycolipid antibodies could differ among experimental MS models with distinct clinical courses, depending on induction methods. Using three mouse strains, SJL/J, C57BL/6, and A.SW mice, we induced five distinct experimental autoimmune encephalomyelitis (EAE) models with myelin oligodendrocyte glycoprotein (MOG), MOG, or myelin proteolipid protein (PLP), with or without an additional adjuvant curdlan injection. We also induced a viral model of MS, using Theiler's murine encephalomyelitis virus (TMEV). Each MS model had an RR, SP, PP, hyperacute, or chronic clinical course. Using the sera from the MS models, we quantified antibodies against 11 glycolipids: GM1, GM2, GM3, GM4, GD3, galactocerebroside, GD1a, GD1b, GT1b, GQ1b, and sulfatide. Among the MS models, we detected significant increases in four anti-glycolipid antibodies, GM1, GM3, GM4, and sulfatide, in PLP-induced EAE with an RR disease course. We also tested cellular immune responses to the glycolipids and found CD1d-independent lymphoproliferative responses only to sulfatide with decreased interleukin (IL)-10 production. Although these results implied that anti-glycolipid antibodies might play a role in remissions or relapses in RR-EAE, their functional roles need to be determined by mechanistic experiments, such as injections of monoclonal anti-glycolipid antibodies.
Topics: Animals; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Encephalomyelitis, Autoimmune, Experimental; Theilovirus; Sulfoglycosphingolipids; Neoplasm Recurrence, Local; Antibodies; Myelin-Oligodendrocyte Glycoprotein; Glycolipids
PubMed: 37629117
DOI: 10.3390/ijms241612937 -
MBio Aug 2023Magnetosomes of magnetotactic bacteria (MTB) consist of structurally perfect, nano-sized magnetic crystals enclosed within vesicles of a proteo-lipid membrane. In...
Magnetosomes of magnetotactic bacteria (MTB) consist of structurally perfect, nano-sized magnetic crystals enclosed within vesicles of a proteo-lipid membrane. In species of biosynthesis of their cubo-octahedral-shaped magnetosomes was recently demonstrated to be a complex process, governed by about 30 specific genes that are comprised within compact magnetosome gene clusters (MGCs). Similar, yet distinct gene clusters were also identified in diverse MTB that biomineralize magnetosome crystals with different, genetically encoded morphologies. However, since most representatives of these groups are inaccessible by genetic and biochemical approaches, their analysis will require the functional expression of magnetosome genes in foreign hosts. Here, we studied whether conserved essential magnetosome genes from closely and remotely related MTB can be functionally expressed by rescue of their respective mutants in the tractable model of the . Upon chromosomal integration, single orthologues from other magnetotactic restored magnetosome biosynthesis to different degrees, while orthologues from distantly related and were found to be expressed but failed to re-induce magnetosome biosynthesis, possibly due to poor interaction with their cognate partners within multiprotein magnetosome organelle of the host. Indeed, co-expression of the known interactors MamB and MamM from the alphaproteobacterium increased functional complementation. Furthermore, a compact and portable version of the entire MGCs of was assembled by transformation-associated recombination cloning, and it restored the ability to biomineralize magnetite both in deletion mutants of the native donor and , while co-expression of gene clusters from both and resulted in overproduction of magnetosomes. IMPORTANCE We provide proof of principle that is a suitable surrogate host for the functional expression of foreign magnetosome genes and extended the transformation-associated recombination cloning platform for the assembly of entire large magnetosome gene cluster, which could then be transplanted to different magnetotactic bacteria. The reconstruction, transfer, and analysis of gene sets or entire magnetosome clusters will be also promising for engineering the biomineralization of magnetite crystals with different morphologies that would be valuable for biotechnical applications.
PubMed: 37318230
DOI: 10.1128/mbio.03282-22 -
Frontiers in Cell and Developmental... 2023Diverse developmental signals and pro-death stresses converge on the regulation of the mitochondrial pathway of apoptosis. BAX, a proapoptotic BCL-2 effector, directly...
Diverse developmental signals and pro-death stresses converge on the regulation of the mitochondrial pathway of apoptosis. BAX, a proapoptotic BCL-2 effector, directly forms proteolipid pores in the outer mitochondrial membrane to activate the mitochondrial pathway of apoptosis. BAX is a viable pharmacological target for various human diseases, and increasing efforts have been made to study the molecular regulation of BAX while identifying small molecules selectively targeting BAX. However, generating large quantities of monomeric and functionally competent BAX has been challenging due to its aggregation-prone nature. Additionally, there is a lack of detailed and instructional protocols available for investigators who are not already familiar with recombinant BAX production. Here, we present a comprehensive protocol for expressing, purifying, and storing functional monomeric recombinant BAX protein. We use an intein-chitin binding domain-tagged BAX-expressing construct and employ a two-step chromatography strategy to capture and purify BAX. We also provide examples of standard assays to observe BAX activation, and highlight the best practices for handling and storing BAX to effectively preserve its quality, shelf life, and function.
PubMed: 38143925
DOI: 10.3389/fcell.2023.1322816 -
PeerJ 2023Multiple sclerosis (MS) is a chronic inflammatory neurologic disease characterized by the demyelinating injury of the central nervous system (CNS). It was reported that...
Multiple sclerosis (MS) is a chronic inflammatory neurologic disease characterized by the demyelinating injury of the central nervous system (CNS). It was reported that the mutant peptide came from myelin proteolipid protein (PLP) and myelin basic protein (MBP) might play a critical role in immunotherapy function of MS. However, endogenous peptides in demyelinating brain tissue of MS and their role in the pathologic process of MS have not been revealed. Here, we performed peptidomic analysis of freshly isolated callosum (CC) from the brains of CPZ-treated mice and normal diet controls of male C57BL/6 mice by LC-MS/MS. Identified a total of 217 peptides were expressed at different levels in MS mice model compared with controls. By performed GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis, we found that the precursor protein of these differently expressed peptides (DEPs) were associated with myelin sheath and oxidative phosphorylation. Our study is the first brain peptidomic of MS mice model, revealing the distinct features of DEPs in demyelination brain tissue. These DPEs may provide further insight into the pathogenesis and complexity of MS, which would facilitate the discovery of the potential novel and effective strategy for the treatment of MS.
Topics: Male; Animals; Mice; Mice, Inbred C57BL; Chromatography, Liquid; Tandem Mass Spectrometry; Central Nervous System; Multiple Sclerosis; Disease Models, Animal; Peptides
PubMed: 37637167
DOI: 10.7717/peerj.15846 -
Neurotrauma Reports 2023Myelin loss and oligodendrocyte death are well documented in patients with traumatic brain injury (TBI), as well as in experimental animal models after...
Myelin loss and oligodendrocyte death are well documented in patients with traumatic brain injury (TBI), as well as in experimental animal models after moderate-to-severe TBI. In comparison, mild TBI (mTBI) does not necessarily result in myelin loss or oligodendrocyte death, but causes structural alterations in the myelin. To gain more insight into the impact of mTBI on oligodendrocyte lineage in the adult brain, we subjected mice to mild lateral fluid percussion injury (mFPI) and characterized the early impact (1 and 3 days post-injury) on oligodendrocytes in the corpus callosum using multiple oligodendrocyte lineage markers (platelet-derived growth factor receptor [PDGFR]-α, glutathione -transferase [GST]-π, CC1, breast carcinoma-amplified sequence 1 [BCAS1], myelin basic protein [MBP], myelin-associated glycoprotein [MAG], proteolipid protein [PLP], and FluoroMyelin™). Two regions of the corpus callosum in relation to the impact site were analyzed: areas near (focal) and anterior (distal) to the impact site. mFPI did not result in oligodendrocyte death in either the focal or distal corpus callosum, nor impact on oligodendrocyte precursors (PDGFR-α) and GST-π oligodendrocyte numbers. In the focal but not distal corpus callosum, mFPI caused a decrease in CC1 as well as BCAS1 actively myelinating oligodendrocytes and reduced FluoroMyelin intensity without altering myelin protein expression (MBP, PLP, and MAG). Disruption in node-paranode organization and loss of Nav1.6 nodes were observed in both the focal and distal regions, even in areas without obvious axonal damage. Altogether, our study shows regional differences in mature and myelinating oligodendrocyte in response to mFPI. Further, mFPI elicits a widespread impact on node-paranode organization that affects regions both close to and remotely located from the site of injury.
PubMed: 37435356
DOI: 10.1089/neur.2023.0037 -
Biomedicine & Pharmacotherapy =... Mar 2024Melatonin acute treatment limits obesity of young Zücker diabetic fatty (ZDF) rats by non-shivering thermogenesis (NST). We recently showed melatonin chronically...
Chronic melatonin treatment improves obesity by inducing uncoupling of skeletal muscle SERCA-SLN mediated by CaMKII/AMPK/PGC1α pathway and mitochondrial biogenesis in female and male Zücker diabetic fatty rats.
Melatonin acute treatment limits obesity of young Zücker diabetic fatty (ZDF) rats by non-shivering thermogenesis (NST). We recently showed melatonin chronically increases the oxidative status of vastus lateralis (VL) in both obese and lean adult male animals. The identification of VL skeletal muscle-based NST by uncoupling of sarcoendoplasmic reticulum Ca-ATPase (SERCA)- sarcolipin (SLN) prompted us to investigate whether melatonin is a SERCA-SLN calcium futile cycle uncoupling and mitochondrial biogenesis enhancer. Obese ZDF rats and lean littermates (ZL) of both sexes were subdivided into two subgroups: control (C) and 12 weeks orally melatonin treated (M) (10 mg/kg/day). Compared to the control groups, melatonin decreased the body weight gain and visceral fat in ZDF rats of both sexes. Melatonin treatment in both sex obese rats restored the VL muscle skin temperature and sensitized the thermogenic effect of acute cold exposure. Moreover, melatonin not only raised SLN protein levels in the VL of obese and lean rats of both sexes; also, the SERCA activity. Melatonin treatment increased the SERCA2 expression in obese and lean rats (both sexes), with no effects on SERCA1 expression. Melatonin increased the expression of thermogenic genes and proteins (PGC1-α, PPARγ, and NRF1). Furthermore, melatonin treatment enhanced the expression ratio of P-CaMKII/CaMKII and P-AMPK/AMPK. In addition, it rose mitochondrial biogenesis. These results provided the initial evidence that chronic oral melatonin treatment triggers the CaMKII/AMPK/PGC1α axis by upregulating SERCA2-SLN-mediated NST in ZDF diabetic rats of both sexes. This may further contribute to the body weight control and metabolic benefits of melatonin.
Topics: Female; Male; Animals; Rats; AMP-Activated Protein Kinases; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Melatonin; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Rats, Zucker; Diabetes Mellitus, Experimental; Organelle Biogenesis; Muscle, Skeletal; Obesity; Muscle Proteins; Proteolipids
PubMed: 38387135
DOI: 10.1016/j.biopha.2024.116314