-
International Journal of Molecular... May 2023Histones play vital roles in chromatin function and gene transcription; however, they are very harmful in the intercellular space because they stimulate systemic...
Histones play vital roles in chromatin function and gene transcription; however, they are very harmful in the intercellular space because they stimulate systemic inflammatory and toxic responses. Myelin basic protein (MBP) is the major protein of the axon myelin-proteolipid sheath. Antibodies-abzymes with various catalytic activities are specific features of some autoimmune diseases. IgGs against individual histones (H2A, H1, H2B, H3, and H4) and MBP were isolated from the blood of experimental-autoimmune-encephalomyelitis-prone C57BL/6 mice by several affinity chromatographies. These Abs-abzymes corresponded to various stages of EAE development: spontaneous EAE, MOG, and DNA-histones accelerated the onset, acute, and remission stages. IgGs-abzymes against MBP and five individual histones showed unusual polyreactivity in the complex formation and enzymatic cross-reactivity in the specific hydrolysis of the H2A histone. All the IgGs of 3-month-old mice (zero time) against MBP and individual histones demonstrated from 4 to 35 different H2A hydrolysis sites. The spontaneous development of EAE over 60 days led to a significant change in the type and number of H2A histone hydrolysis sites by IgGs against five histones and MBP. Mice treatment with MOG and the DNA-histone complex changed the type and number of H2A hydrolysis sites compared to zero time. The minimum number (4) of different H2A hydrolysis sites was found for IgGs against H2A (zero time), while the maximum (35) for anti-H2B IgGs (60 days after mice treatment with DNA-histone complex). Overall, it was first demonstrated that at different stages of EAE evolution, IgGs-abzymes against individual histones and MBP could significantly differ in the number and type of specific sites of H2A hydrolysis. The possible reasons for the catalytic cross-reactivity and great differences in the number and type of histone H2A cleavage sites were analyzed.
Topics: Animals; Mice; Histones; Hydrolysis; Myelin Basic Protein; Mice, Inbred C57BL; Encephalomyelitis, Autoimmune, Experimental; DNA; Autoantibodies
PubMed: 37239982
DOI: 10.3390/ijms24108636 -
Mucosal Immunology Aug 2023Curative therapies against autoimmune diseases are lacking. Indeed, most of the currently available treatments are only targeting symptoms. We have developed a novel...
Curative therapies against autoimmune diseases are lacking. Indeed, most of the currently available treatments are only targeting symptoms. We have developed a novel strategy for a therapeutic vaccine against autoimmune diseases based on intranasal administration of a fusion protein tolerogen, which consists of a mutant, enzymatically inactive, cholera toxin A1 (CTA1)-subunit genetically fused to disease-relevant high-affinity peptides and a dimer of D-fragments from protein A (DD). The CTA1 R7K mutant - myelin oligodendrocyte glycoprotein (MOG), or proteolipid protein (PLP) - DD (CTA1R7K-MOG/PLP-DD) fusion proteins effectively reduced clinical symptoms in the experimental autoimmune encephalitis model of multiple sclerosis. The treatment induced Tr1 cells, in the draining lymph node, which produced interleukin (IL)-10 and suppressed effector clusters of differentiation 4 T-cell responses. This effect was dependent on IL-27 signaling because treatment was ineffective in bone marrow chimeras lacking IL-27Ra within their hematopoietic compartment. Single-cell RNA sequencing of dendritic cells in draining lymph nodes demonstrated distinct gene transcriptional changes of classic dendritic cells 1, including enhanced lipid metabolic pathways, induced by the tolerogenic fusion protein. Thus, our results with the tolerogenic fusion protein demonstrate the possibility to vaccinate and protect against disease progression by reinstating tolerance in multiple sclerosis and other autoimmune diseases.
Topics: Humans; T-Lymphocytes, Regulatory; Administration, Intranasal; Cholera Toxin; CD4-Positive T-Lymphocytes; Multiple Sclerosis
PubMed: 37192682
DOI: 10.1016/j.mucimm.2023.05.006 -
International Journal of Molecular... May 2023Multiple sclerosis (MS) is the chronic inflammatory demyelinating disease of the CNS. Relapsing-remitting MS (RRMS) is the most common type of MS. However, the...
Involvement of Degenerating 21.5 kDa Isoform of Myelin Basic Protein in the Pathogenesis of the Relapse in Murine Relapsing-Remitting Experimental Autoimmune Encephalomyelitis and MS Autopsied Brain.
Multiple sclerosis (MS) is the chronic inflammatory demyelinating disease of the CNS. Relapsing-remitting MS (RRMS) is the most common type of MS. However, the mechanisms of relapse and remission in MS have not been fully understood. While SJL mice immunized with proteolipid protein (PLP) develop relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE), we have recently observed that some of these mice were resistant to the active induction of relapsing EAE after initial clinical and histological symptoms of EAE with a severity similar to the relapsing EAE mice. To clarify the mechanism of relapsing, we examined myelin morphology during PLP-induced RR-EAE in the SJL mice. While RR-EAE mice showed an increased EAE severity (relapse) with CNS inflammation, demyelination with abnormal myelin morphology in the spinal cord, the resistant mice exhibited a milder EAE phenotype with diminished relapse. Compared with the RR-EAE mice, the resistant mice showed less CNS inflammation, demyelination, and abnormalities of the myelin structure. In addition, scanning electron microscopic (SEM) analysis with the osmium-maceration method displayed ultrastructural abnormalities of the myelin structure in the white matter of the RR-EAE spinal cord, but not in that of the resistant mice. While the intensity of myelin staining was reduced in the relapsing EAE spinal cord, immunohistochemistry and immunoblot analysis revealed that the 21.5 kDa isoform of degenerating myelin basic protein (MBP) was specifically induced in the relapsing EAE spinal cord. Taken together, the neuroinflammation-induced degenerating 21 kDa isoform of MBP sheds light on the development of abnormal myelin on the relapse of MS pathogenesis.
Topics: Mice; Animals; Encephalomyelitis, Autoimmune, Experimental; Myelin Basic Protein; Myelin Proteolipid Protein; Neoplasm Recurrence, Local; Spinal Cord; Multiple Sclerosis; Mice, Inbred Strains; Chronic Disease; Inflammation; Brain; Protein Isoforms
PubMed: 37175866
DOI: 10.3390/ijms24098160 -
Scientific Reports May 2023The genes enconding proteins containing plasma membrane proteolipid 3 (PMP3) domain are responsive to abiotic stresses, but their functions in maize drought tolerance...
The genes enconding proteins containing plasma membrane proteolipid 3 (PMP3) domain are responsive to abiotic stresses, but their functions in maize drought tolerance remain largely unknown. In this study, the transgenic maize lines overexpressing maize ZmPMP3g gene were featured by enhanced drought tolerance; increases in total root length, activities of superoxide dismutase and catalase, and leaf water content; and decreases in leaf water potential, levels of Oand HO, and malondialdehyde content under drought. Under treatments with foliar spraying with abscisic acid (ABA), drought tolerance of both transgenic line Y7-1 overexpressing ZmPMP3g and wild type Ye478 was enhanced, of which Y7-1 showed an increased endogenous ABA and decreased endogenous gibberellin (GA) 1 (significantly) and GA3 (very slightly but not significantly) and Ye478 had a relatively lower ABA and no changes in GA1 and GA3. ZmPMP3g overexpression in Y7-1 affected the expression of multiple key transcription factor genes in ABA-dependent and -independent drought signaling pathways. These results indicate that ZmPMP3g overexpression plays a role in maize drought tolerance by harmonizing ABA-GA1-GA3 homeostasis/balance, improving root growth, enhancing antioxidant capacity, maintaining membrane lipid integrity, and regulating intracellular osmotic pressure. A working model on ABA-GA-ZmPMP3g was proposed and discussed.
Topics: Zea mays; Drought Resistance; Hydrogen Peroxide; Plant Proteins; Plants, Genetically Modified; Abscisic Acid; Stress, Physiological; Droughts; Water; Gene Expression Regulation, Plant
PubMed: 37147346
DOI: 10.1038/s41598-023-32989-4 -
Research Square Apr 2023Relapses in patients with relapsing-remitting multiple sclerosis (RRMS) are typically treated with high-dose corticosteroids including methylprednisolone. However,...
OBJECTIVE
Relapses in patients with relapsing-remitting multiple sclerosis (RRMS) are typically treated with high-dose corticosteroids including methylprednisolone. However, high-dose corticosteroids are associated with significant adverse effects, can increase the risk for other morbidities, and often do not impact disease course. Multiple mechanisms are proposed to contribute to acute relapses in RRMS patients, including neuroinflammation, fibrin formation and compromised blood vessel barrier function. The protein C activator, E-WE thrombin is a recombinant therapeutic in clinical development for its antithrombotic and cytoprotective properties, including protection of endothelial cell barrier function. In mice, treatment with E-WE thrombin reduced neuroinflammation and extracellular fibrin formation in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). We therefore tested the hypothesis that E-WE thrombin could reduce disease severity in a relapsing-remitting model of EAE.
METHODS
Female SJL mice were inoculated with proteolipid protein (PLP) peptide and treated with E-WE thrombin (25 μg/kg; iv) or vehicle at onset of detectable disease. In other experiments, E-WE thrombin was compared to methylprednisolone (100 mg/kg; iv) or the combination of both.
RESULTS
Compared to vehicle, administration of E-WE thrombin significantly improved disease severity of the initial attack and relapse and delayed onset of relapse as effectively as methylprednisolone. Both methylprednisolone and E-WE thrombin reduced demyelination and immune cell recruitment, and the combination of both treatments had an additive effect.
CONCLUSION
The data presented herein demonstrate that E-WE thrombin is protective in mice with relapsing-remitting EAE, a widely used model of MS. Our data indicate that E-WE thrombin is as effective as high-dose methylprednisolone in improving disease score and may exert additional benefit when administered in combination. Taken together, these data suggest that E-WE thrombin may be an effective alternative to high-dose methylprednisolone for managing acute MS attacks.
PubMed: 37131631
DOI: 10.21203/rs.3.rs-2802415/v1 -
Acta Biomaterialia Jul 2023Implantation of electrodes in the brain can be used to record from or stimulate neural tissues to treat neurological disease and injury. However, the tissue response to...
Implantation of electrodes in the brain can be used to record from or stimulate neural tissues to treat neurological disease and injury. However, the tissue response to implanted devices can limit their functional longevity. Recent RNA-seq datasets identify hundreds of genes associated with gliosis, neuronal function, myelination, and cellular metabolism that are spatiotemporally expressed in neural tissues following the insertion of microelectrodes. To validate mRNA as a predictor of protein expression, this study evaluates a sub-set of RNA-seq identified proteins (RSIP) at 24-hours, 1-week, and 6-weeks post-implantation using quantitative immunofluorescence methods. This study found that expression of RSIPs associated with glial activation (Glial fibrillary acidic protein (GFAP), Polypyrimidine tract binding protein-1 (Ptbp1)), neuronal structure (Neurofilament heavy chain (Nefh), Proteolipid protein-1 (Plp1), Myelin Basic Protein (MBP)), and iron metabolism (Transferrin (TF), Ferritin heavy chain-1 (Fth1)) reinforce transcriptional data. This study also provides additional context to the cellular distribution of RSIPs using a MATLAB-based approach to quantify immunofluorescence intensity within specific cell types. Ptbp1, TF, and Fth1 were found to be spatiotemporally distributed within neurons, astrocytes, microglia, and oligodendrocytes at the device interface relative to distal and contralateral tissues. The altered distribution of RSIPs relative to distal tissue is largely localized within 100µm of the device injury, which approaches the functional recording range of implanted electrodes. This study provides evidence that RNA-sequencing can be used to predict protein-level changes in cortical tissues and that RSIPs can be further investigated to identify new biomarkers of the tissue response that influence signal quality. STATEMENT OF SIGNIFICANCE: Microelectrode arrays implanted into the brain are useful tools that can be used to study neuroscience and to treat pathological conditions in a clinical setting. The tissue response to these devices, however, can severely limit their functional longevity. Transcriptomics has deepened the understandings of the tissue response by revealing numerous genes which are differentially expressed following device insertion. This manuscript provides validation for the use of transcriptomics to characterize the tissue response by evaluating a subset of known differentially expressed genes at the protein level around implanted electrodes over time. In additional to validating mRNA-to-protein relationships at the device interface, this study has identified emerging trends in the spatiotemporal distribution of proteins involved with glial activation, neuronal remodeling, and essential iron binding proteins around implanted silicon devices. This study additionally provides a new MATLAB based methodology to quantify protein distribution within discrete cell types at the device interface which may be used as biomarkers for further study or therapeutic intervention in the future.
Topics: Rats; Animals; Rats, Sprague-Dawley; RNA-Seq; Neurons; Astrocytes; Electrodes, Implanted; Microelectrodes
PubMed: 37116634
DOI: 10.1016/j.actbio.2023.04.028 -
Brain Communications 2023Hypertension in midlife contributes to cognitive decline and is a modifiable risk factor for dementia. The relationship between late-life hypertension and dementia is...
Hypertension in midlife contributes to cognitive decline and is a modifiable risk factor for dementia. The relationship between late-life hypertension and dementia is less clear. We have investigated the relationship of blood pressure and hypertensive status during late life (after 65 years) to post-mortem markers of Alzheimer's disease (amyloid-β and tau loads); arteriolosclerosis and cerebral amyloid angiopathy; and to biochemical measures of ante-mortem cerebral oxygenation (the myelin-associated glycoprotein:proteolipid protein-1 ratio, which is reduced in chronically hypoperfused brain tissue, and the level of vascular endothelial growth factor-A, which is upregulated by tissue hypoxia); blood-brain barrier damage (indicated by an increase in parenchymal fibrinogen); and pericyte content (platelet-derived growth factor receptor β, which declines with pericyte loss), in Alzheimer's disease ( = 75), vascular ( = 20) and mixed dementia ( = 31) cohorts. Systolic and diastolic blood pressure measurements were obtained retrospectively from clinical records. Non-amyloid small vessel disease and cerebral amyloid angiopathy were scored semiquantitatively. Amyloid-β and tau loads were assessed by field fraction measurement in immunolabelled sections of frontal and parietal lobes. Homogenates of frozen tissue from the contralateral frontal and parietal lobes (cortex and white matter) were used to measure markers of vascular function by enzyme-linked immunosorbent assay. Diastolic (but not systolic) blood pressure was associated with the preservation of cerebral oxygenation, correlating positively with the ratio of myelin-associated glycoprotein to proteolipid protein-1 and negatively with vascular endothelial growth factor-A in both the frontal and parietal cortices. Diastolic blood pressure correlated negatively with parenchymal amyloid-β in the parietal cortex. In dementia cases, elevated late-life diastolic blood pressure was associated with more severe arteriolosclerosis and cerebral amyloid angiopathy, and diastolic blood pressure correlated positively with parenchymal fibrinogen, indicating blood-brain barrier breakdown in both regions of the cortex. Systolic blood pressure was related to lower platelet-derived growth factor receptor β in controls in the frontal cortex and in dementia cases in the superficial white matter. We found no association between blood pressure and tau. Our findings demonstrate a complex relationship between late-life blood pressure, disease pathology and vascular function in dementia. We suggest that hypertension helps to reduce cerebral ischaemia (and may slow amyloid-β accumulation) in the face of increasing cerebral vascular resistance, but exacerbates vascular pathology.
PubMed: 37113314
DOI: 10.1093/braincomms/fcad112 -
Nature Methods Jun 2023Hierarchical organization of integral membrane proteins (IMP) and lipids at the membrane is essential for regulating myriad downstream signaling. A quantitative...
Hierarchical organization of integral membrane proteins (IMP) and lipids at the membrane is essential for regulating myriad downstream signaling. A quantitative understanding of these processes requires both detections of oligomeric organization of IMPs and lipids directly from intact membranes and determination of key membrane components and properties that regulate them. Addressing this, we have developed a platform that enables native mass spectrometry (nMS) analysis of IMP-lipid complexes directly from intact and customizable lipid membranes. Both the lipid composition and membrane properties (such as curvature, tension, and fluidity) of these bilayers can be precisely customized to a target membrane. Subsequent direct nMS analysis of these intact proteolipid vesicles can yield the oligomeric states of the embedded IMPs, identify bound lipids, and determine the membrane properties that can regulate the observed IMP-lipid organization. Applying this method, we show how lipid binding regulates neurotransmitter release and how membrane composition regulates the functional oligomeric state of a transporter.
Topics: Mass Spectrometry; Biological Transport; Lipids; Membrane Proteins; Lipid Bilayers
PubMed: 37106230
DOI: 10.1038/s41592-023-01864-5 -
Journal of Cancer Research and Clinical... Sep 2023Treatment of advanced colorectal cancer (CRC) depends on the correct selection of personalized strategies. HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is...
PURPOSE
Treatment of advanced colorectal cancer (CRC) depends on the correct selection of personalized strategies. HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a natural proteolipid milk compound that might serve as a novel cancer prevention and therapy candidate. Our purpose was to investigate HAMLET effect on viability, death pathway and mitochondrial bioenergetics of CRC cells with different KRAS/BRAF mutational status in vitro.
METHODS
We treated three cell lines (Caco-2, LoVo, WiDr) with HAMLET to evaluate cell metabolic activity and viability, flow cytometry of apoptotic and necrotic cells, pro- and anti-apoptotic genes, and protein expressions. Mitochondrial respiration (oxygen consumption) rate was recorded by high-resolution respirometry system Oxygraph-2 k.
RESULTS
The HAMLET complex was cytotoxic to all investigated CRC cell lines and this effect is irreversible. Flow cytometry revealed that HAMLET induces necrotic cell death with a slight increase in an apoptotic cell population. WiDr cell metabolism, clonogenicity, necrosis/apoptosis level, and mitochondrial respiration were affected significantly less than other cells.
CONCLUSION
HAMLET exhibits irreversible cytotoxicity on human CRC cells in a dose-dependent manner, leading to necrotic cell death and inhibiting the extrinsic apoptosis pathway. BRAF-mutant cell line is more resistant than other type lines. HAMLET decreased mitochondrial respiration and ATP synthesis in CaCo-2 and LoVo cell lines but did not affect WiDr cells' respiration. Pretreatment of cancer cells with HAMLET has no impact on mitochondrial outer and inner membrane permeability.
Topics: Humans; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); Caco-2 Cells; Cell Death; Apoptosis; Colorectal Neoplasms; Mutation; Respiration
PubMed: 37099199
DOI: 10.1007/s00432-023-04777-0