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BioRxiv : the Preprint Server For... May 2024During the development of the sensory nervous system, various cell types converge to form different tissues. The trigeminal ganglion houses the cell bodies for the...
During the development of the sensory nervous system, various cell types converge to form different tissues. The trigeminal ganglion houses the cell bodies for the trigeminal nerve and exemplifies these intercellular interactions, as it is formed from the condensation of two diverse precursor cell populations, neural crest cells and placode cells. The dual origin of the trigeminal ganglion has been understood for decades, but the molecules that orchestrate this process remain relatively unknown. Assembling the trigeminal ganglion is mediated by cell adhesion molecules including the protein neural cadherin (N-cadherin), expressed first in placode-derived neurons and later in all trigeminal sensory neurons. Prior studies have shown that N-cadherin knockdown in chick trigeminal placode cells leads to early defects in trigeminal ganglion assembly by impacting the ability of placode-derived neurons to properly condense with undifferentiated neural crest cells. Later functions for N-cadherin in chick trigeminal gangliogenesis, however, are unknown. Using morpholino-mediated knockdown of N-cadherin in chick trigeminal placode cells, we examined trigeminal ganglion development at later developmental stages, when neural crest cells are differentiating into neurons. Through these experiments, we uncovered a sustained negative impact on the trigeminal ganglion, leading to decreases in ganglion size, nerve outgrowth, and branching to target tissues . Further, blocking the adhesive function of N-cadherin reveals its importance in the outgrowth ability for some, but not all, trigeminal neurons . These deficits reflect potential cell and non-cell autonomous effects on placodal and neural crest-derived neurons, respectively, and point to the importance of N-cadherin-mediated adhesion among trigeminal sensory neurons. Our findings reveal continued adhesion-dependent functions for N-cadherin in the trigeminal ganglion, which will aid in the understanding of its tissue-specific roles.
PubMed: 38826314
DOI: 10.1101/2024.05.20.594965 -
International Journal of Molecular... May 2024Sigma non-opioid intracellular receptor 1 (Sigma-1R) is an intracellular chaperone protein residing on the endoplasmic reticulum at the mitochondrial-associated membrane...
Sigma non-opioid intracellular receptor 1 (Sigma-1R) is an intracellular chaperone protein residing on the endoplasmic reticulum at the mitochondrial-associated membrane (MAM) region. Sigma-1R is abundant in the brain and is involved in several physiological processes as well as in various disease states. The role of Sigma-1R at the blood-brain barrier (BBB) is incompletely characterized. In this study, the effect of Sigma-1R activation was investigated in vitro on rat brain microvascular endothelial cells (RBMVEC), an important component of the blood-brain barrier (BBB), and in vivo on BBB permeability in rats. The Sigma-1R agonist PRE-084 produced a dose-dependent increase in mitochondrial calcium, and mitochondrial and cytosolic reactive oxygen species (ROS) in RBMVEC. PRE-084 decreased the electrical resistance of the RBMVEC monolayer, measured with the electric cell-substrate impedance sensing (ECIS) method, indicating barrier disruption. These effects were reduced by pretreatment with Sigma-1R antagonists, BD 1047 and NE 100. In vivo assessment of BBB permeability in rats indicates that PRE-084 produced a dose-dependent increase in brain extravasation of Evans Blue and sodium fluorescein brain; the effect was reduced by the Sigma-1R antagonists. Immunocytochemistry studies indicate that PRE-084 produced a disruption of tight and adherens junctions and actin cytoskeleton. The brain microcirculation was directly visualized in vivo in the prefrontal cortex of awake rats with a miniature integrated fluorescence microscope (aka, miniscope; Doric Lenses Inc.). Miniscope studies indicate that PRE-084 increased sodium fluorescein extravasation in vivo. Taken together, these results indicate that Sigma-1R activation promoted oxidative stress and increased BBB permeability.
Topics: Animals; Receptors, sigma; Blood-Brain Barrier; Sigma-1 Receptor; Rats; Reactive Oxygen Species; Endothelial Cells; Male; Mitochondria; Calcium; Morpholines; Brain; Cells, Cultured
PubMed: 38791182
DOI: 10.3390/ijms25105147 -
Biosensors Apr 2024Zebrafish larvae have emerged as a valuable model for studying heart physiology and pathophysiology, as well as for drug discovery, in part thanks to its transparency,...
Zebrafish larvae have emerged as a valuable model for studying heart physiology and pathophysiology, as well as for drug discovery, in part thanks to its transparency, which simplifies microscopy. However, in fluorescence-based optical mapping, the beating of the heart results in motion artifacts. Two approaches have been employed to eliminate heart motion during calcium or voltage mapping in zebrafish larvae: the knockdown of cardiac troponin T2A and the use of myosin inhibitors. However, these methods disrupt the mechano-electric and mechano-mechanic coupling mechanisms. We have used ratiometric genetically encoded biosensors to image calcium in the beating heart of intact zebrafish larvae because ratiometric quantification corrects for motion artifacts. In this study, we found that halting heart motion by genetic means (injection of morpholino) or chemical tools (incubation with -aminoblebbistatin) leads to bradycardia, and increases calcium levels and the size of the calcium transients, likely by abolishing a feedback mechanism that connects contraction with calcium regulation. These outcomes were not influenced by the calcium-binding domain of the gene-encoded biosensors employed, as biosensors with a modified troponin C (Twitch-4), calmodulin (mCyRFP1-GCaMP6f), or the photoprotein aequorin (GFP-aequorin) all yielded similar results. Cardiac contraction appears to be an important regulator of systolic and diastolic Ca levels, and of the heart rate.
Topics: Animals; Zebrafish; Calcium; Biosensing Techniques; Myocardial Contraction; Larva; Heart; Troponin T; Zebrafish Proteins; Troponin C
PubMed: 38785693
DOI: 10.3390/bios14050219 -
Nature Communications May 2024Hypomyelinating leukodystrophy (HLD) is an autosomal recessive disorder characterized by defective central nervous system myelination. Exome sequencing of two siblings...
Hypomyelinating leukodystrophy (HLD) is an autosomal recessive disorder characterized by defective central nervous system myelination. Exome sequencing of two siblings with severe cognitive and motor impairment and progressive hypomyelination characteristic of HLD revealed homozygosity for a missense single-nucleotide variant (SNV) in EPRS1 (c.4444 C > A; p.Pro1482Thr), encoding glutamyl-prolyl-tRNA synthetase, consistent with HLD15. Patient lymphoblastoid cell lines express markedly reduced EPRS1 protein due to dual defects in nuclear export and cytoplasmic translation of variant EPRS1 mRNA. Variant mRNA exhibits reduced METTL3 methyltransferase-mediated writing of N-methyladenosine (mA) and reduced reading by YTHDC1 and YTHDF1/3 required for efficient mRNA nuclear export and translation, respectively. In contrast to current models, the variant does not alter the sequence of mA target sites, but instead reduces their accessibility for modification. The defect was rescued by antisense morpholinos predicted to expose mA sites on target EPRS1 mRNA, or by mA modification of the mRNA by METTL3-dCas13b, a targeted RNA methylation editor. Our bioinformatic analysis predicts widespread occurrence of SNVs associated with human health and disease that similarly alter accessibility of distal mRNA mA sites. These results reveal a new RNA-dependent etiologic mechanism by which SNVs can influence gene expression and disease, consequently generating opportunities for personalized, RNA-based therapeutics targeting these disorders.
Topics: Female; Humans; Male; Adenosine; Hereditary Central Nervous System Demyelinating Diseases; Homozygote; Methyltransferases; Mutation, Missense; Nerve Tissue Proteins; RNA Splicing Factors; RNA, Messenger; RNA-Binding Proteins
PubMed: 38769304
DOI: 10.1038/s41467-024-48549-x -
Frontiers in Cell and Developmental... 2024Prohibitin (PHB) is an essential scaffold protein that modulates signaling pathways controlling cell survival, metabolism, inflammation, and bone formation. However,...
Prohibitin (PHB) is an essential scaffold protein that modulates signaling pathways controlling cell survival, metabolism, inflammation, and bone formation. However, its specific role in periodontium development remains less understood. This study aims to elucidate the expression pattern and function of PHB in periodontium development and its involvement in alveolar bone formation. Immunolocalization of PHB in the periodontium of postnatal (PN) mice were examined. morpholino was micro-injected into the right-side mandible at PN5, corresponding to the position where the alveolar bone process forms in relation to the lower first molar. The micro-injection with a scramble control (PF-127) and the left-side mandibles were used as control groups. Five days post-micro-injection, immunohistochemical analysis and micro-CT evaluation were conducted to assess bone mass and morphological changes. Additionally, expression patterns of signaling molecules were examined following downregulation using 24-h cultivation of developing dental mesenchyme at E14.5. The immunostaining of PHB showed its localization in the periodontium at PN5, PN8, and PN10. The cultivation of dental mesenchyme resulted in alterations in Bmps, Runx2, and Wnt signalings after knock-down. At 5 days post-micro-injection, knocking down showed weak immunolocalizations of runt-related transcription factor (RUNX2) and osteocalcin (OCN). However, knocking down led to histological alterations characterized by decreased bone mass and stronger localizations of Ki67 and PERIOSTIN in the periodontium compared 1 to control groups. The micro-CT evaluation showed decreased bone volume and increased PDL space in the knock-down specimens, suggesting its regulatory role in bone formation. The region-specific localization of PHB in the margin where alveolar bone forms suggests its involvement in alveolar bone formation and the differentiation of the periodontal ligament. Overall, our findings suggest that plays a modulatory role in alveolar bone formation by harmoniously regulating bone-forming-related signaling molecules during periodontium development.
PubMed: 38756696
DOI: 10.3389/fcell.2024.1369634 -
Biomedicine & Pharmacotherapy =... Jun 2024Antisense oligonucleotide (ASO) has emerged as a promising therapeutic approach for treating central nervous system (CNS) disorders by modulating gene expression with...
Antisense oligonucleotide (ASO) has emerged as a promising therapeutic approach for treating central nervous system (CNS) disorders by modulating gene expression with high selectivity and specificity. However, the poor permeability of ASO across the blood-brain barrier (BBB) diminishes its therapeutic success. Here, we designed and synthesized a series of BBB-penetrating peptides (BPP) derived from either the receptor-binding domain of apolipoprotein E (ApoE) or a transferrin receptor-binding peptide (THR). The BPPs were conjugated to phosphorodiamidate morpholino oligomers (PMO) that are chemically analogous to the 2'-O-(2-methoxyethyl) (MOE)-modified ASO approved by the FDA for treating spinal muscular atrophy (SMA). The BPP-PMO conjugates significantly increased the level of full-length SMN2 in the patient-derived SMA fibroblasts in a concentration-dependent manner with minimal to no toxicity. Furthermore, the systemic administration of the most potent BPP-PMO conjugates significantly increased the expression of full-length SMN2 in the brain and spinal cord of SMN2 transgenic adult mice. Notably, BPP8-PMO conjugate showed a 1.25-fold increase in the expression of full-length functional SMN2 in the brain. Fluorescence imaging studies confirmed that 78% of the fluorescently (Cy7)-labelled BPP8-PMO reached brain parenchyma, with 11% uptake in neuronal cells. Additionally, the BPP-PMO conjugates containing retro-inverso (RI) D-BPPs were found to possess extended half-lives compared to their L-counterparts, indicating increased stability against protease degradation while preserving the bioactivity. This delivery platform based on BPP enhances the CNS bioavailability of PMO targeting the SMN2 gene, paving the way for the development of systemically administered neurotherapeutics for CNS disorders.
Topics: Animals; Blood-Brain Barrier; Oligonucleotides, Antisense; Humans; Apolipoproteins E; Mice, Transgenic; Mice; Morpholinos; Survival of Motor Neuron 2 Protein; Muscular Atrophy, Spinal; Drug Delivery Systems; Fibroblasts; Brain; Peptides; Cell-Penetrating Peptides
PubMed: 38749176
DOI: 10.1016/j.biopha.2024.116737 -
BioRxiv : the Preprint Server For... May 2024Propranolol reduces experimental murine cerebral cavernous malformations (CCMs) and prevents embryonic caudal venous plexus (CVP) lesions in zebrafish that follow mosaic...
Propranolol reduces experimental murine cerebral cavernous malformations (CCMs) and prevents embryonic caudal venous plexus (CVP) lesions in zebrafish that follow mosaic inactivation of . Because morpholino silencing of the β1 adrenergic receptor () prevents the embryonic CVP lesion, we proposed that plays a role in CCM pathogenesis. Here we report that zebrafish exhibited 86% fewer CVP lesions and 87% reduction of CCM lesion volume relative to wild type brood mates at 2dpf and 8-10 weeks stage, respectively. Treatment with metoprolol, a β1 selective antagonist, yielded a similar reduction in CCM lesion volume. zebrafish embryos exhibited reduced heart rate and contractility and reduced CVP blood flow. Similarly, slowing the heart and eliminating the blood flow in CVP by administration of 2,3-BDM suppressed the CVP lesion. In sum, our findings provide genetic and pharmacological evidence that the therapeutic effect of propranolol on CCM is achieved through β1 receptor antagonism.
PubMed: 38746306
DOI: 10.1101/2024.05.05.592554 -
Molecular Pharmacology May 2024DNA topoisomerase IIα (TOP2α, 170kDa, TOP2α/170) is an essential enzyme for proper chromosome dysjunction by producing transient DNA double-stranded breaks and is a...
DNA topoisomerase IIα (TOP2α, 170kDa, TOP2α/170) is an essential enzyme for proper chromosome dysjunction by producing transient DNA double-stranded breaks and is a significant target for DNA damage stabilizing anti-cancer agents such as etoposide. Therapeutic effects of TOP2α poisons can be limited due to acquired drug resistance. We previously demonstrated decreased TOP2α/170 levels in an etoposide-resistant human leukemia K562 subline, designated K/VP.5, accompanied by increased expression of a C-terminal truncated TOP2α isoform (90 kDa, TOP2α/90) which heterodimerized with TOP2α/170 and was a determinant of resistance by exhibiting dominant-negative effects against etoposide activity. Based on 3'-Rapid Amplification of cDNA Ends (3'-RACE), we confirmed TOP2α/90 as the translation product of a TOP2α mRNA in which a cryptic polyadenylation site (PAS) harbored in intron 19 (I19) was utilized. We hypothesized that resultant intronic polyadenylation (IPA) can would be attenuated by blocking or mutating the I19 PAS thereby circumventing acquired drug resistance. An antisense morpholino oligonucleotide (AMO) was used to hybridize/block the PAS in TOP2α pre-mRNA in K/VP.5 cells, resulting in decreased TOP2α/90 mRNA/protein levels in K/VP.5 cells and partially circumventing drug resistance. Subsequently, CRISPR/Cas9 homology-directed repair (HDR) was used to mutate the cryptic I19 PAS (AAA-->AAA) to prevent IPA. Gene-edited clones exhibited increased TOP2α/170 and decreased TOP2α/90 mRNA/protein and demonstrated restored sensitivity to etoposide and other TOP2α-targeted drugs. Together, results indicated that blocking/mutating a cryptic I19 PAS in K/VP.5 cells reduced IPA and restored sensitivity to TOP2α-targeting drugs. Results presented here indicate that CRISPR/Cas9 gene editing of a cryptic polyadenylation site (PAS) within I19 of the TOP2α gene results in reversal of acquired resistance to etoposide and other TOP2-targeted drugs. An antisense morpholino oligonucleotide (AMO) targeting the PAS also partially circumvented resistance. Results demonstrate the importance of intronic polyadenylation (IPA) in acquired drug resistance and points to tractable strategies to overcome this form of resistance to TOP2-targeted agents.
PubMed: 38719474
DOI: 10.1124/molpharm.124.000868 -
Hereditas Apr 2024Nicotinamide phosphoribosyltransferase (Nampt) is required for recycling NAD in numerous cellular contexts. Morpholino-based knockdown of zebrafish nampt-a has been...
BACKGROUND
Nicotinamide phosphoribosyltransferase (Nampt) is required for recycling NAD in numerous cellular contexts. Morpholino-based knockdown of zebrafish nampt-a has been shown to cause abnormal development and defective hematopoiesis concomitant with decreased NAD levels. However, surprisingly, nampt-a mutant zebrafish were recently found to be viable, suggesting a discrepancy between the phenotypes in knockdown and knockout conditions. Here, we address this discrepancy by directly comparing loss-of-function approaches that result in identical defective transcripts in morphants and mutants.
RESULTS
Using CRISPR/Cas9-mediated mutagenesis, we generated nampt-a mutant lines that carry the same mis-spliced mRNA as nampt-a morphants. Despite reduced NAD levels and perturbed expression of specific blood markers, nampt-a mutants did not display obvious developmental defects and were found to be viable. In contrast, injection of nampt-a morpholinos into wild-type or mutant nampt-a embryos caused aberrant phenotypes. Moreover, nampt-a morpholinos caused additional reduction of blood-related markers in nampt-a mutants, suggesting that the defects observed in nampt-a morphants can be partially attributed to off-target effects of the morpholinos.
CONCLUSIONS
Our findings show that zebrafish nampt-a mutants are viable despite reduced NAD levels and a perturbed hematopoietic gene expression program, indicating strong robustness of primitive hematopoiesis during early embryogenesis.
Topics: Animals; Zebrafish; Nicotinamide Phosphoribosyltransferase; Hematopoiesis; Mutation; Zebrafish Proteins; Phenotype; CRISPR-Cas Systems; NAD; Gene Knockdown Techniques; Morpholinos
PubMed: 38685093
DOI: 10.1186/s41065-024-00318-y -
Biomedicines Apr 2024Casimersen (AMONDYS 45) is an antisense oligonucleotide of the phosphorodiamidate morpholino oligomer subclass developed by Sarepta therapeutics. It was approved by the... (Review)
Review
Casimersen (AMONDYS 45) is an antisense oligonucleotide of the phosphorodiamidate morpholino oligomer subclass developed by Sarepta therapeutics. It was approved by the Food and Drug Administration (FDA) in February 2021 to treat Duchenne muscular dystrophy (DMD) in patients whose gene mutation is amenable to exon 45 skipping. Administered intravenously, casimersen binds to the pre-mRNA of the gene to skip a mutated region of an exon, thereby producing an internally truncated yet functional dystrophin protein in DMD patients. This is essential in maintaining the structure of a myocyte membrane. While casimersen is currently continuing in phase III of clinical trials in various countries, it was granted approval by the FDA under the accelerated approval program due to its observed increase in dystrophin production. This article discusses the pathophysiology of DMD, summarizes available treatments thus far, and provides a full drug review of casimersen (AMONDYS 45).
PubMed: 38672266
DOI: 10.3390/biomedicines12040912