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Biomedicines May 2023Cathpesin B is a multi-functional protease that plays numerous roles in physiology and pathophysiology. We hypothesized that actin cytoskeleton proteins that are...
Decreased MARCKS Protein Expression in Kidney Cortex Membrane Fractions of Cathepsin B Knockout Mice Is Associated with Reduced Lysophosphatidylcholine and Protein Kinase C Activity.
Cathpesin B is a multi-functional protease that plays numerous roles in physiology and pathophysiology. We hypothesized that actin cytoskeleton proteins that are substrates of cathepsin B, various lipids, and kinases that are regulated by lipids would be down-regulated in the kidney of cathepsin B knockout mice. Here, we show by Western blot and densitometric analysis that the expression and proteolysis of the actin cytoskeleton proteins myristoylated alanine-rich C-kinase substrate (MARCKS) and spectrin are significantly reduced in kidney cortex membrane fractions of cathepsin B knockout mice compared to C57B6 wild-type control mice. Lipidomic results show that specific lipids are increased while other lipids, including lysophosphatidylcholine (LPC) species LPC (16:0), LPC (18:0), LPC (18:1), and LPC (18:2), are significantly decreased in membrane fractions of the kidney cortex from Cathepsin B null mice. Protein Kinase C (PKC) activity is significantly lower in the kidney cortex of cathepsin B knockout mice compared to wild-type mice, while calcium/calmodulin-dependent protein kinase II (CaMKII) activity and phospholipase D (PLD) activity are comparable between the two groups. Together, these results provide the first evidence of altered actin cytoskeleton organization, membrane lipid composition, and PKC activity in the kidneys of mice lacking cathepsin B.
PubMed: 37239160
DOI: 10.3390/biomedicines11051489 -
Biomedicines May 2023Glaucoma is one of the most common sight-threatening eye disorders and one of the main causes of irreversible blindness worldwide. The current therapies focusing on...
Neuroprotective Effect of a Nutritional Supplement Containing Spearmint Extract, Forskolin, Homotaurine and Group B Vitamins in a Mouse Model of Transient Ocular Hypertension.
Glaucoma is one of the most common sight-threatening eye disorders and one of the main causes of irreversible blindness worldwide. The current therapies focusing on reducing intraocular pressure (IOP) are often insufficient to prevent the progression of the disease, so the therapeutic management of glaucoma remains a challenge. The aim of this study was to evaluate the neuroprotective, IOP-lowering independent effects of a nutritional supplement containing forskolin, homotaurine, spearmint extract and vitamins of the B group in a model of acute glaucoma developed in mice. Glaucoma was induced in adult wild-type C57BL/6J mice by transient elevation of IOP. The dietary supplement, branded as Gangliomix (125 mg/kg/day), was administered by oral gavage for 17 days and ocular hypertension was induced on the 10th day of treatment. A histological analysis of the retinas was performed and RGC survival was evaluated with fluorogold labeling and Brn3a immunostaining on wholemount and retinal sections. Expression of alpha-spectrin, caspase-3, PARP-1 and GFAP was studied with western blotting or immunofluorescence. A significant increase in RGC survival was reported in the retina of mice treated with the dietary supplement as compared to vehicle-treated animals. The observed neuroprotection was associated with a calpain activity decrease, reduction in caspase-3 and PARP-1 activation, and prevention of GFAP upregulation. These effects were independent from the hypotensive effects of the supplement. Altogether, our data suggest that the dietary supplementation with forskolin, homotaurine, spearmint extract and vitamins of the B group supports RGC survival and may offer beneficial effects in glaucoma patients in combination with the currently used IOP-lowering therapy.
PubMed: 37239149
DOI: 10.3390/biomedicines11051478 -
Diagnostics (Basel, Switzerland) May 2023Acute traumatic spinal cord injury (SCI) is recognized as a global problem that can lead to a range of acute and secondary complications impacting morbidity and...
Association between Cerebrospinal Fluid and Serum Biomarker Levels and Diagnosis, Injury Severity, and Short-Term Outcomes in Patients with Acute Traumatic Spinal Cord Injury.
Acute traumatic spinal cord injury (SCI) is recognized as a global problem that can lead to a range of acute and secondary complications impacting morbidity and mortality. There is still a lack of reliable diagnostic and prognostic biomarkers in patients with SCI that could help guide clinical care and identify novel therapeutic targets for future drug discovery. The aim of this prospective controlled study was to determine the cerebral spinal fluid (CSF) and serum profiles of 10 biomarkers as indicators of SCI diagnosis, severity, and prognosis to aid in assessing appropriate treatment modalities. CSF and serum samples of 15 SCI and ten healthy participants were included in the study. The neurological assessments were scored on admission and at discharge from the hospital using the American Spinal Injury Association Impairment Score (AIS) grades. The CSF and serum concentrations of SBDP150, S100B, GFAP, NF-L, UCHL-1, Tau, and IL-6 were significantly higher in SCI patients when compared with the control group. The CSF GBDP 38/44K, UCHL-L1, S100B, GFAP, and Tau levels were significantly higher in the AIS A patients. This study demonstrated a strong correlation between biomarker levels in the diagnosis and injury severity of SCI but no association with short-term outcomes. Future prospective controlled studies need to be done to support the results of this study.
PubMed: 37238298
DOI: 10.3390/diagnostics13101814 -
Developmental Neuroscience 2024The developing brain is uniquely susceptible to oxidative stress, and endogenous antioxidant mechanisms are not sufficient to prevent injury from a hypoxic-ischemic...
The developing brain is uniquely susceptible to oxidative stress, and endogenous antioxidant mechanisms are not sufficient to prevent injury from a hypoxic-ischemic challenge. Glutathione peroxidase (GPX1) activity reduces hypoxic-ischemic injury. Therapeutic hypothermia (HT) also reduces hypoxic-ischemic injury, in the rodent and the human brain, but the benefit is limited. Here, we combined GPX1 overexpression with HT in a P9 mouse model of hypoxia-ischemia (HI) to test the effectiveness of both treatments together. Histological analysis showed that wild-type (WT) mice with HT were less injured than WT with normothermia. In the GPX1-tg mice, however, despite a lower median score in the HT-treated mice, there was no significant difference between HT and normothermia. GPX1 protein expression was higher in the cortex of all transgenic groups at 30 min and 24 h, as well as in WT 30 min after HI, with and without HT. GPX1 was higher in the hippocampus of all transgenic groups and WT with HI and normothermia, at 24 h, but not at 30 min. Spectrin 150 was higher in all groups with HI, while spectrin 120 was higher in HI groups only at 24 h. There was reduced ERK1/2 activation in both WT and GPX1-tg HI at 30 min. Thus, with a relatively moderate insult, we see a benefit with cooling in the WT but not the GPX1-tg mouse brain. The fact that we see no benefit with increased GPx1 here in the P9 model (unlike in the P7 model) may indicate that oxidative stress in these older mice is elevated to an extent that increased GPx1 is insufficient for reducing injury. The lack of benefit of overexpressing GPX1 in conjunction with HT after HI indicates that pathways triggered by GPX1 overexpression may interfere with the neuroprotective mechanisms provided by HT.
Topics: Animals; Mice; Humans; Animals, Newborn; Hypothermia; Spectrin; Hypoxia-Ischemia, Brain; Hypoxia; Glutathione Peroxidase; Antioxidants; Ischemia; Hypothermia, Induced
PubMed: 37231852
DOI: 10.1159/000531204 -
Frontiers in Cell and Developmental... 2023In this review, we discuss FHOD formins with a focus on recent studies that reveal a new role for them as critical links for nuclear mechanotransduction. The FHOD family... (Review)
Review
In this review, we discuss FHOD formins with a focus on recent studies that reveal a new role for them as critical links for nuclear mechanotransduction. The FHOD family in vertebrates comprises two structurally related proteins, FHOD1 and . Their similar biochemical properties suggest overlapping and redundant functions. is widely expressed, less so, with highest expression in skeletal () and cardiac () muscle where specific splice isoforms are expressed. Unlike other formins, FHODs have strong F-actin bundling activity and relatively weak actin polymerization activity. These activities are regulated by phosphorylation by ROCK and Src kinases; bundling is additionally regulated by ERK1/2 kinases. FHODs are unique among formins in their association with the nuclear envelope through direct, high affinity binding to the outer nuclear membrane proteins nesprin-1G and nesprin-2G. Recent crystallographic structures reveal an interaction between a conserved motif in one of the spectrin repeats (SRs) of nesprin-1G/2G and a site adjacent to the regulatory domain in the amino terminus of FHODs. Nesprins are components of the LINC (linker of nucleoskeleton and cytoskeleton) complex that spans both nuclear membranes and mediates bidirectional transmission of mechanical forces between the nucleus and the cytoskeleton. FHODs interact near the actin-binding calponin homology (CH) domains of nesprin-1G/2G enabling a branched connection to actin filaments that presumably strengthens the interaction. At the cellular level, the tethering of FHODs to the outer nuclear membrane mechanically couples perinuclear actin arrays to the nucleus to move and position it in fibroblasts, cardiomyocytes, and potentially other cells. FHODs also function in adhesion maturation during cell migration and in the generation of sarcomeres, activities distant from the nucleus but that are still influenced by it. Human genetic studies have identified multiple variants linked to dilated and hypertrophic cardiomyopathies, with many mutations mapping to "hot spots" in domains. We discuss how FHOD1/3's role in reinforcing the LINC complex and connecting to perinuclear actin contributes to functions of mechanically active tissues such as striated muscle.
PubMed: 37215084
DOI: 10.3389/fcell.2023.1160219 -
Experimental and Therapeutic Medicine Jun 2023Ovarian cancer is as a major contributor to gynaecologic death globally. The present study aimed to investigate the regulatory role of spectrin β non-erythrocytic 2...
Ovarian cancer is as a major contributor to gynaecologic death globally. The present study aimed to investigate the regulatory role of spectrin β non-erythrocytic 2 gene (SPTBN2) in endometroid ovarian cancer and its mechanism of action. According to the Gene Expression Profiling Interactive Analysis (GEPIA) database, SPTBN2 expression is elevated in ovarian cancer tissues and higher SPTBN2 expression indicated a worse prognosis. The present study assessed SPTBN2 mRNA and protein expression levels by reverse transcription-quantitative PCR and western blotting, respectively. Cell viability, proliferation, migration and invasion were assessed with Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine incorporation, wound healing and Transwell assays, respectively. SPTBN2 expression was notably enhanced in ovarian cancer cell lines, especially in A2780 cells compared with HOSEPiC cells (P<0.001). Following transfection with small interfering (si)RNA targeting SPTBN2, the viability, proliferation, migration and invasion of A2780 cells were decreased compared with those of A2780 cells transfected with siRNA-NC (P<0.001). Gene Set Enrichment Analysis database revealed that SPTBN2 was primarily enriched in 'focal adhesion' and 'extracellular matrix (ECM)-receptor interaction', whereas SPTBN2 was significantly associated with integrin β4 (ITGB4) in the GEPIA database. In addition, rescue experiments were performed to determine the mechanism of SPTBN2 in endometroid ovarian cancer. ITGB4 overexpression reversed the inhibitory effects of the SPTBN2 knockdown on viability, proliferation, migration and invasion of A2780 cells (P<0.05). The impacts of SPTBN2 on the expression of focal adhesion and downstream ECM receptor signalling-related proteins, including Src and p-FAK/FAK, were significantly reversed by ITGB4 overexpression (P<0.01). Collectively, SPTBN2 may regulate endometroid ovarian cancer cell proliferation, invasion and migration through the ITGB4-mediated focal adhesion and ECM receptor signalling pathway.
PubMed: 37206547
DOI: 10.3892/etm.2023.11977 -
Biochemical Society Transactions Jun 2023Nesprins (nuclear envelope spectrin repeat proteins) are multi-isomeric scaffolding proteins. Giant nesprin-1 and -2 localise to the outer nuclear membrane, interact... (Review)
Review
Nesprins (nuclear envelope spectrin repeat proteins) are multi-isomeric scaffolding proteins. Giant nesprin-1 and -2 localise to the outer nuclear membrane, interact with SUN (Sad1p/UNC-84) domain-containing proteins at the inner nuclear membrane to form the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex, which, in association with lamin A/C and emerin, mechanically couples the nucleus to the cytoskeleton. Despite ubiquitous expression of nesprin giant isoforms, pathogenic mutations in nesprin-1 and -2 are associated with tissue-specific disorders, particularly related to striated muscle such as dilated cardiomyopathy and Emery-Dreifuss muscular dystrophy. Recent evidence suggests this muscle-specificity might be attributable in part, to the small muscle specific isoform, nesprin-1α2, which has a novel role in striated muscle function. Our current understanding of muscle-specific functions of nesprin-1 and its isoforms will be summarised in this review to provide insight into potential pathological mechanisms of nesprin-related muscle disease and may inform potential targets of therapeutic modulation.
Topics: Humans; Cell Nucleus; Mechanotransduction, Cellular; Muscle, Skeletal; Muscular Diseases; Nerve Tissue Proteins; Nuclear Envelope; Protein Isoforms; Animals
PubMed: 37171063
DOI: 10.1042/BST20221541 -
Experimental and Therapeutic Medicine Jun 2023The majority of low-grade gliomas (LGGs) in adults invariably progress to glioblastoma over time. Spectrin β non-erythrocytic 2 () is detected in numerous tumors and is...
The majority of low-grade gliomas (LGGs) in adults invariably progress to glioblastoma over time. Spectrin β non-erythrocytic 2 () is detected in numerous tumors and is involved in tumor occurrence and metastasis. However, the specific roles and detailed mechanisms of in LGG are largely unknown. The present study performed pan-cancer analysis for the expression and prognosis of SPTBN2 in LGG using The Cancer Genome Atlas and The Genotype-Tissue Expression. Western blotting was used to detect the amount of SPTBN2 between glioma tissues and normal brain tissues. Subsequently, based on expression, prognosis, correlation and immune infiltration, non-coding RNAs (ncRNAs) were identified that regulated expression. Finally, tumor immune infiltrates associated with and prognosis were performed. Lower expression of was correlated with an unfavorable outcome in LGG. A significant correlation between the low mRNA expression and poor clinicopathological features was observed, including wild-type isocitrate dehydrogenase status (P<0.001), 1p/19q non-codeletion (P<0.001) and elders (P=0.019). The western blotting results revealed that, compared with normal brain tissues, the amount of SPTBN2 was significantly lower in LGG tissues (P=0.0266). Higher expression of five microRNAs (miRs/miRNAs), including and , correlated with poor prognosis by targeting SPTBN2 in LGG. Subsequently, four long ncRNAs (lncRNAs) [antisense RNA 1 ] were observed in the regulation of via five miRNAs. Moreover, the expression of was significantly correlated with tumor immune infiltration, immune checkpoint expression and biomarkers of immune cells. In conclusion, was lowly expressed and correlated with an unfavorable prognosis in LGG. A total of six miRNAs and four lncRNAs were identified as being able to modulate in a lncRNA-miRNA-mRNA network of LGG. Furthermore, the current findings also indicated that SPTBN2 possessed anti-tumor roles by regulating tumor immune infiltration and immune checkpoint expression.
PubMed: 37153896
DOI: 10.3892/etm.2023.11952 -
Neurobiology of Disease Jun 2023The node of Ranvier is the key element in saltatory conduction along myelinated axons, but its specific protein organization remains elusive in the human species. To...
The node of Ranvier is the key element in saltatory conduction along myelinated axons, but its specific protein organization remains elusive in the human species. To shed light on nanoscale anatomy of the human node of Ranvier in health and disease, we assessed human nerve biopsies of patients with polyneuropathy by super-resolution fluorescence microscopy. We applied direct stochastic optical reconstruction microscopy (dSTORM) and supported our data by high-content confocal imaging combined with deep learning-based analysis. As a result, we revealed a ∼ 190 nm periodic protein arrangement of cytoskeletal proteins and axoglial cell adhesion molecules in human peripheral nerves. In patients with polyneuropathy, periodic distances increased at the paranodal region of the node of Ranvier, both at the axonal cytoskeleton and at the axoglial junction. In-depth image analysis revealed a partial loss of proteins of the axoglial complex (Caspr-1, neurofascin-155) in combination with detachment from the cytoskeletal anchor protein ß2-spectrin. High content analysis showed that such paranodal disorganization occurred especially in acute and severe axonal neuropathy with ongoing Wallerian degeneration and related cytoskeletal damage. We provide nanoscale and protein-specific evidence for the prominent, but vulnerable role of the node of Ranvier for axonal integrity. Furthermore, we show that super-resolution imaging can identify, quantify and map elongated periodic protein distances and protein interaction in histopathological tissue samples. We thus introduce a promising tool for further translational applications of super resolution microscopy.
Topics: Humans; Ranvier's Nodes; Axons; Cytoskeletal Proteins; Peripheral Nerves; Polyneuropathies
PubMed: 37146836
DOI: 10.1016/j.nbd.2023.106139 -
Molecular Biology of the Cell Jun 2023β-Spectrin is a membrane cytoskeletal protein with specialized roles in the nervous system and heart. Recent evidence also indicates a fundamental role for β-spectrin...
β-Spectrin is a membrane cytoskeletal protein with specialized roles in the nervous system and heart. Recent evidence also indicates a fundamental role for β-spectrin in angiogenesis as its endothelial-specific gene deletion in mice enhances embryonic lethality due to hypervascularization and hemorrhagic defects. During early vascular sprouting, β-spectrin is believed to inhibit tip cell sprouting in favor of the stalk cell phenotype by mediating VEGFR2 internalization and degradation. Despite these essential roles, mechanisms governing β-spectrin expression remain unknown. Here we identify bone morphogenetic protein 9 (BMP9) as a major inducer of β-spectrin gene expression in the vascular system. We show that BMP9 signals through the ALK1/Smad1 pathway to induce β-spectrin expression, which then recruits CaMKII to the cell membrane to induce phosphorylation-dependent VEGFR2 turnover. Although BMP9 signaling promotes stalk cell behavior through activation of hallmark stalk cell genes ID-1/3 and Hes-1 and Notch signaling cross-talk, we find that β-spectrin acts upstream of these pathways as loss of β-spectrin in neonate mice leads to retinal hypervascularization due to excessive VEGFR2 levels, increased tip cell populations, and strong Notch inhibition irrespective of BMP9 treatment. These findings demonstrate β-spectrin as a BMP9 gene target critical for tip/stalk cell selection during nascent vessel sprouting.
Topics: Animals; Mice; Endothelial Cells; Growth Differentiation Factor 2; Neovascularization, Physiologic; Signal Transduction; Spectrin
PubMed: 37126382
DOI: 10.1091/mbc.E23-02-0064