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International Journal of Molecular... Mar 2024Research on GM1 ganglioside and its neuroprotective role in Parkinson's disease (PD), particularly in mitigating the aggregation of α-Synuclein (aSyn), is well...
Research on GM1 ganglioside and its neuroprotective role in Parkinson's disease (PD), particularly in mitigating the aggregation of α-Synuclein (aSyn), is well established across various model organisms. This essential molecule, GM1, is intimately linked to preventing aSyn aggregation, and its deficiency is believed to play a key role in the initiation of PD. In our current study, we attempted to shed light on the cytosolic interactions between GM1 and aSyn based on previous reports demonstrating gangliosides and monomeric aSyn to be present in neuronal cytosol. Native-PAGE and Western blot analysis of neuronal cytosol from mouse brains demonstrated the presence of both GM1 and monomeric aSyn in the neuronal cytosol of normal mouse brain. To demonstrate that an adequate level of GM1 prevents the aggregation of aSyn, we used NG108-15 and SH-SY5Y cells with and without treatment of 1-phenyl-2-palmitoyl-3-morpholino-1-propanol (PPMP), which inhibits the synthesis/expression of GM1. Cells treated with PPMP to reduce GM1 expression showed a significant increase in the formation of aggregated aSyn compared to untreated cells. We thus demonstrated that sufficient GM1 prevents the aggregation of aSyn. For this to occur, aSyn and GM1 must show proximity within the neuron. The present study provides evidence for such co-localization in neuronal cytosol, which also facilitates the inverse interaction revealed in studies with the two cell types above. This adds to the explanation of how GM1 prevents the aggregation of aSyn and onset of Parkinson's disease.
Topics: Animals; Humans; Mice; alpha-Synuclein; Cytosol; G(M1) Ganglioside; Neuroblastoma; Neurons; Parkinson Disease
PubMed: 38542297
DOI: 10.3390/ijms25063323 -
Frontiers in Cell and Developmental... 2024LncRNA-based control affects cardiac pathophysiologies like myocardial infarction, coronary artery disease, hypertrophy, and myotonic muscular dystrophy. This study used...
LncRNA-based control affects cardiac pathophysiologies like myocardial infarction, coronary artery disease, hypertrophy, and myotonic muscular dystrophy. This study used a gene-break transposon (GBT) to screen zebrafish () for insertional mutagenesis. We identified three insertional mutants where the GBT captured a cardiac gene. One of the adult viable GBT mutants had bradycardia (heart arrhythmia) and enlarged cardiac chambers or hypertrophy; we named it "bigheart." Bigheart mutant insertion maps to grin2bb or N-methyl D-aspartate receptor (NMDAR2B) gene intron 2 in reverse orientation. Rapid amplification of adjacent cDNA ends analysis suggested a new insertion site transcript in the intron 2 of grin2bb. Analysis of the RNA sequencing of wild-type zebrafish heart chambers revealed a possible new transcript at the insertion site. As this putative lncRNA transcript satisfies the canonical signatures, we called this transcript . Using hybridization, we confirmed localized expression in the heart, central nervous system, and muscles in the developing embryos and wild-type adult zebrafish atrium and bulbus arteriosus. The bigheart mutant had reduced Grin2bbART expression. We showed that bigheart gene trap insertion excision reversed cardiac-specific arrhythmia and atrial hypertrophy and restored expression. Morpholino-mediated antisense downregulation of in wild-type zebrafish embryos mimicked bigheart mutants; this suggests is linked to bigheart. Cardiovascular tissues use Grin2bb as a calcium-permeable ion channel. Calcium imaging experiments performed on bigheart mutants indicated calcium mishandling in the heart. The bigheart cardiac transcriptome showed differential expression of calcium homeostasis, cardiac remodeling, and contraction genes. Western blot analysis highlighted Camk2d1 and Hdac1 overexpression. We propose that altered calcium activity due to disruption of , a putative lncRNA in bigheart, altered the Camk2d-Hdac pathway, causing heart arrhythmia and hypertrophy in zebrafish.
PubMed: 38533084
DOI: 10.3389/fcell.2024.1339292 -
Heliyon Mar 2024Polycomb group RING finger (PCGF) proteins, a crucial subunits of the Polycomb complex, plays an important role in regulating gene expression, embryonic development, and...
Polycomb group RING finger (PCGF) proteins, a crucial subunits of the Polycomb complex, plays an important role in regulating gene expression, embryonic development, and cell fate determination. In our research, we investigated Pcgf5, one of the six PCGF homologs, and its impact on the differentiation of P19 cells into neural stem cells. Our findings revealed that knockdown of Pcgf5 resulted in a significant decrease in the expression levels of the neuronal markers Sox2, Zfp521, and Pax6, while the expression levels of the pluripotent markers Oct4 and Nanog increased. Conversely, Pcgf5 overexpression upregulated the expression of Sox2 and Pax6, while downregulating the expression of Oct4 and Nanog. Additionally, our analysis revealed that Pcgf5 suppresses Wnt3 expression via the activation of Notch1/Hes1, and ultimately governs the differentiation fate of neural stem cells. To further validate our findings, we conducted experiments in zebrafish. We found that knockdown of pcgf5a using morpholino resulted in the downregulated expression of neurodevelopmental genes such as sox2, sox3, and foxg1 in zebrafish embryos. Consequently, these changes led to neurodevelopmental defects. In conclusion, our study highlights the important role of Pcgf5 in neural induction and the determination of neural cell fate.
PubMed: 38533065
DOI: 10.1016/j.heliyon.2024.e27634 -
Disease Models & Mechanisms Jun 2024De novo truncating variants in fibrosin-like 1 (FBRSL1), a member of the AUTS2 gene family, cause a disability syndrome, including organ malformations such as heart...
De novo truncating variants in fibrosin-like 1 (FBRSL1), a member of the AUTS2 gene family, cause a disability syndrome, including organ malformations such as heart defects. Here, we use Xenopus laevis to investigate whether Fbrsl1 plays a role in heart development. Xenopus laevis fbrsl1 is expressed in tissues relevant for heart development, and morpholino-mediated knockdown of Fbrsl1 results in severely hypoplastic hearts. Our data suggest that Fbrsl1 is required for the development of the first heart field, which contributes to the ventricle and the atria, but not for the second heart field, which gives rise to the outflow tract. The morphant heart phenotype could be rescued using a human N-terminal FBRSL1 isoform that contains an alternative exon, but lacks the AUTS2 domain. N-terminal isoforms carrying patient variants failed to rescue. Interestingly, a long human FBRSL1 isoform, harboring the AUTS2 domain, also did not rescue the morphant heart defects. Thus, our data suggest that different FBRSL1 isoforms may have distinct functions and that only the short N-terminal isoform, appears to be critical for heart development.
Topics: Animals; Xenopus laevis; Humans; Xenopus Proteins; Heart; Heart Defects, Congenital; Protein Isoforms; Phenotype; Gene Knockdown Techniques; Gene Expression Regulation, Developmental
PubMed: 38501224
DOI: 10.1242/dmm.050507 -
BioRxiv : the Preprint Server For... Mar 2024CD47 is a ubiquitous and pleiotropic cell-surface receptor. Disrupting CD47 enhances injury repair in various tissues but the role of CD47 has not been studied in bone...
CD47 is a ubiquitous and pleiotropic cell-surface receptor. Disrupting CD47 enhances injury repair in various tissues but the role of CD47 has not been studied in bone injuries. In a murine closed-fracture model, CD47-null mice showed decreased callus bone volume, bone mineral content, and tissue mineral content as assessed by microcomputed tomography 10 days post-fracture, and increased fibrous volume as determined by histology. To understand the cellular basis for this phenotype, mesenchymal progenitors (MSC) were harvested from bone marrow. CD47-null MSC showed decreased large fibroblast colony formation (CFU-F), significantly less proliferation, and fewer cells in S-phase, although osteoblast differentiation was unaffected. However, consistent with prior research, CD47-null endothelial cells showed increased proliferation relative to WT cells. Similarly, in a murine ischemic fracture model, CD47-null mice showed reduced fracture callus bone volume and bone mineral content relative to WT. Consistent with our results, EdU labeling showed decreased cell proliferation in the callus of CD47-null mice, while staining for CD31 and endomucin demonstrated increased endothelial cell mass. Finally, WT mice administered a CD47 morpholino, which blocks CD47 protein production, showed a callus phenotype similar to that of non-ischemic and ischemic fractures in CD47-null mice, suggesting the phenotype was not due to developmental changes in the knockout mice. Thus, inhibition of CD47 during bone healing reduces both non-ischemic and ischemic fracture healing, in part, by decreasing MSC proliferation. Furthermore, the increase in endothelial cell proliferation and early blood vessel density caused by CD47 disruption is not sufficient to overcome MSC dysfunction.
PubMed: 38496546
DOI: 10.1101/2024.03.06.583756 -
International Journal of Molecular... Mar 2024Mutations of coding for polycystin-1 (PC1) account for most cases of autosomal-dominant polycystic kidney disease (ADPKD). The extracellular region of PC1 contains many...
Mutations of coding for polycystin-1 (PC1) account for most cases of autosomal-dominant polycystic kidney disease (ADPKD). The extracellular region of PC1 contains many evolutionarily conserved domains for ligand interactions. Among these are the leucine-rich repeats (LRRs) in the far N-terminus of PC1. Using zebrafish () as an in vivo model system, we explored the role of LRRs in the function of PC1. Zebrafish expresses two human paralogs, and . Knockdown of both genes in zebrafish by morpholino antisense oligonucleotides produced phenotypes of dorsal-axis curvature and pronephric cyst formation. We found that overexpression of LRRs suppressed both phenotypes in -morphant zebrafish. Purified recombinant LRR domain inhibited proliferation of HEK cells in culture and interacted with the heterotrimeric basement membrane protein laminin-511 (α5β1γ1) in vitro. Mutations of amino acid residues in LRRs structurally predicted to bind laminin-511 disrupted LRR-laminin interaction in vitro and neutralized the ability of LRRs to inhibit cell proliferation and cystogenesis. Our data support the hypothesis that the extracellular region of PC1 plays a role in modulating PC1 interaction with the extracellular matrix and contributes to cystogenesis of PC1 deficiency.
Topics: Animals; Humans; Polycystic Kidney, Autosomal Dominant; Zebrafish; Leucine; TRPP Cation Channels; Polycystic Kidney Diseases; Laminin; Kidney
PubMed: 38474131
DOI: 10.3390/ijms25052886 -
TUBA4A downregulation as observed in ALS motor cortex causes ALS-related abnormalities in zebrafish.Frontiers in Cellular Neuroscience 2024Disease-associated variants of (alpha-tubulin 4A) have recently been identified in familial ALS. Interestingly, a downregulation of TUBA4A protein expression was...
Disease-associated variants of (alpha-tubulin 4A) have recently been identified in familial ALS. Interestingly, a downregulation of TUBA4A protein expression was observed in familial as well as sporadic ALS brain tissue. To investigate whether a decreased TUBA4A expression could be a driving factor in ALS pathogenesis, we assessed whether knockdown in zebrafish could recapitulate an ALS-like phenotype. For this, we injected an antisense oligonucleotide morpholino in zebrafish embryos targeting the zebrafish orthologue. An antibody against synaptic vesicle 2 was used to visualize motor axons in the spinal cord, allowing the analysis of embryonic ventral root projections. Motor behavior was assessed using the touch-evoked escape response. In ALS motor cortex, we observed reduced TUBA4A levels. The knockdown of the zebrafish orthologue induced a motor axonopathy and a significantly disturbed motor behavior. Both phenotypes were dose-dependent and could be rescued by the addition of human wild-type mRNA. Thus, downregulation as observed in ALS motor cortex could be modeled in zebrafish and induced a motor axonopathy and motor behavior defects reflecting a motor neuron disease phenotype, as previously described in embryonic zebrafish models of ALS. The rescue with human wild-type mRNA suggests functional conservation and strengthens the causal relation between TUBA4A protein levels and phenotype severity. Furthermore, the loss of TUBA4A induces significant changes in post-translational modifications of tubulin, such as acetylation, detyrosination and polyglutamylation. Our data unveil an important role for in ALS pathogenesis, and extend the relevance of to the majority of ALS patients, in addition to cases bearing mutations.
PubMed: 38463699
DOI: 10.3389/fncel.2024.1340240 -
EMBO Molecular Medicine Apr 2024Clinical deployment of oligonucleotides requires delivery technologies that improve stability, target tissue accumulation and cellular internalization. Exosomes show...
Clinical deployment of oligonucleotides requires delivery technologies that improve stability, target tissue accumulation and cellular internalization. Exosomes show potential as ideal delivery vehicles. However, an affordable generalizable system for efficient loading of oligonucleotides on exosomes remain lacking. Here, we identified an Exosomal Anchor DNA Aptamer (EAA) via SELEX against exosomes immobilized with our proprietary CP05 peptides. EAA shows high binding affinity to different exosomes and enables efficient loading of nucleic acid drugs on exosomes. Serum stability of thrombin inhibitor NU172 was prolonged by exosome-loading, resulting in increased blood flow after injury in vivo. Importantly, Duchenne Muscular Dystrophy PMO can be readily loaded on exosomes via EAA (EXO). EXO elicited significantly greater muscle cell uptake, tissue accumulation and dystrophin expression than PMO in vitro and in vivo. Systemic administration of EXO elicited therapeutic levels of dystrophin restoration and functional improvements in mdx mice. Altogether, our study demonstrates that EAA enables efficient loading of different nucleic acid drugs on exosomes, thus providing an easy and generalizable strategy for loading nucleic acid therapeutics on exosomes.
Topics: Animals; Mice; Dystrophin; Mice, Inbred mdx; Exosomes; Morpholinos; Muscular Dystrophy, Duchenne; Oligonucleotides
PubMed: 38448545
DOI: 10.1038/s44321-024-00049-7 -
Frontiers in Cell and Developmental... 2024Mutations in lead to a syndromic form of holoprosencephaly (HPE) known as the Galloway-Mowat syndrome (GAMOS). While a connection between PRDM15, a zinc finger...
Mutations in lead to a syndromic form of holoprosencephaly (HPE) known as the Galloway-Mowat syndrome (GAMOS). While a connection between PRDM15, a zinc finger transcription factor, and WNT/PCP signaling has been established, there is a critical need to delve deeper into their contributions to early development and GAMOS pathogenesis. We used the South African clawed frog as the vertebrate model organism and observed that was enriched in the tissues and organs affected in GAMOS. Furthermore, we generated a morpholino oligonucleotide-mediated knockdown model showing that the depletion of Prdm15 leads to abnormal eye, head, and brain development, effectively recapitulating the anterior neural features in GAMOS. An analysis of the underlying molecular basis revealed a reduced expression of key genes associated with eye, head, and brain development. Notably, this reduction could be rescued by the introduction of RNA, particularly during the induction of the respective tissues. Mechanistically, our data demonstrate that Prdm15 acts upstream of both canonical and non-canonical Wnt4 signaling during anterior neural development. Our findings describe severe ocular and anterior neural abnormalities upon Prdm15 depletion and elucidate the role of Prdm15 in canonical and non-canonical Wnt4 signaling.
PubMed: 38444828
DOI: 10.3389/fcell.2024.1316048 -
Heliyon Mar 2024The selectivity of a novel chemosensor, based on a modified nitrobenzofurazan referred to as NBD-Morph, has been investigated for the detection of heavy metal cations...
The selectivity of a novel chemosensor, based on a modified nitrobenzofurazan referred to as NBD-Morph, has been investigated for the detection of heavy metal cations (Co, Pb, Mg, Ag, Cu, Hg, Ni2+, and Zn). The ligand, 4-morpholino-7-nitrobenzofurazan (NBD-Morph), was characterized using spectroscopic techniques including FT-IR and H NMR. Vibrational frequencies obtained from FT-IR and proton NMR (H) chemical shifts were accurately predicted employing the density functional theory (DFT) at the B3LYP level of theory. Furthermore, an examination of the structural, electronic, and quantum chemical properties was conducted and discussed. DFT calculations were employed to explore the complex formation ability of the NBD-Morph ligand with Co, Pb, Mg, Ag, Cu, Hg, Ni, and Zn metal cations. The comparison of adsorption energies for all possible conformations reveals that NBD-Morph exhibits sensitivity and selectivity towards metal ions, including Pb, Cu, Ag, and Ni. However, an assessment of their reactivity using QTAIM topological parameters demonstrated the ligand's greater complexation ability toward Cu or Ni than those formed by Pb or Ag. Additionally, molecular electrostatic potential (MEP), Hirshfeld surfaces, and their associated 2D-fingerprint plots were applied to a detailed study of the inter-molecular interactions in NBD-Morph-X (X = Pb, Cu, Ag, Ni) complexes. The electron localization function (ELF) and the localized-orbital locator (LOL) were generated to investigate the charge transfer and donor-acceptor interactions within the complexes. Electrochemical analysis further corroborates the theoretical findings, supporting the prediction of NBD-Morph's sensory ability towards Ni metal cations. In conclusion, NBD-Morph stands out as a promising sensor for Ni.
PubMed: 38439845
DOI: 10.1016/j.heliyon.2024.e26709