Molecular Medicine最新文献

Background: The neuromuscular junction (NMJ) is the synapse between motor neurons and skeletal muscle and controlls movement. Impaired synaptic transmission and NMJ degeneration has been observed during healthy ageing and is also implicated in several neuromuscular diseases. On account of the high energy demands of being distally located and large sized, NMJs are enriched with mitochondria. This enrichment is dependent on transport of mitochondria across the axon to the NMJ.

Methods: We first established a human 3D neuromuscular assembloid model to study in-vitro NMJs, by fusing human stem cell derived spinal cord organoids and primary skeletal muscle organoids. To determine whether enhancing axonal mitochondrial transport modulates NMJ formation and maintenance, we generated a CRISPR-Cas9 meditated knock-out of syntaphilin in human stem cells.

Results: Firstly, we characterised the neuromuscular assembloid model which showed functional innervated NMJs as measured by juxtaposed neurofilament⁺ axons and α-bungarotoxin⁺ acetylcholine receptors. Secondly, we showed that spinal cord selective genetic ablation of syntaphilin - an axonally localised mitochondrial anchor protein - resulted in increased mitochondrial motility in motor neurons, and consequently increased axonal density and NMJ formation.

Conclusion: This proof-of-concept study demonstrated that enhancing mitochondrial mobility could provide a therapeutic target to prevent NMJ degeneration.

Background: High Mobility Group Box 1 (HMGB1) is a nuclear protein that upon extracellular release acts as an alarmin to initiate and amplify inflammation. HMGB1 release from nociceptors contributes to both inflammation and pain; however, the mechanisms for its regulation remain incompletely understood. The cholinergic anti-inflammatory pathway, mediated by α7 nicotinic acetylcholine receptor (α7nAChR) activation, inhibits HMGB1 release from immune cells and reduces inflammation. This study investigates whether α7nAChR signaling similarly inhibits HMGB1 release from nociceptors, thereby affecting pain and inflammation.

Methods: Dorsal root ganglia (DRG) neurons were isolated from C57BL/6 or VGlut2-Cre/ChR2-YFP mice (expressing ChR2 in sensory neurons for optogenetic stimulation at 470 nm). HMGB1 release in vitro was triggered by optogenetic stimulation or exposure to capsaicin (5 µM), in the presence or absence of cholinergic agonists (acetylcholine, GTS-21, PNU-282987), and subsequently measured by ELISA. Immunohistochemistry was used to visualize cellular HMGB1 localization. In vivo models, including optogenetic stimulation and formalin-induced pain-like behavior, were used to evaluate the effects of cholinergic agonists on pain-like behavior, mechanical allodynia and inflammation. α7nAChR knockout (KO) mice served to determine receptor-specific effects. Levels of proinflammatory mediators calcitonin gene-related peptide (CGRP), substance P, HMGB1, and IL-6 were also measured.

Results: Optogenetic stimulation of cultured DRG neurons significantly increased HMGB1 release, which was markedly inhibited by cholinergic agonists. Similarly, capsaicin-induced HMGB1 release was suppressed by acetylcholine, GTS-21, and PNU-282987, promoting HMGB1 retention within the nucleus; this effect was abolished in α7nAChR KO neurons. In contrast, the release of CGRP and substance P following optogenetic or capsaicin stimulation of DRG neurons from wild-type mice was not influenced by cholinergic agonists. In vivo, GTS-21 reduced pain-like behaviors and mechanical allodynia in both the formalin-induced and optogenetically-stimulated nociceptive behavior models, as demonstrated by reduced mechanical allodynia and extracellular HMGB1 levels. These effects were absent in α7nAChR KO mice, confirming the critical role of α7nAChR in mediating these responses.

Conclusion: This study reveals a novel α7nAChR-dependent cholinergic mechanism that reduces nociceptive behavior and inflammation by retaining nuclear HMGB1 in nociceptors. Cholinergic agonists may serve as promising therapeutic agents to mitigate nociceptive behavior and inflammation by targeting α7nAChR in sensory neurons.

Background: Coactivator-associated arginine methyltransferase 1 (CARM1) regulates diverse cellular processes-including transcription, cell cycle progression, metabolism, and autophagy-through asymmetric dimethylation of both histone and non-histone substrates. Although TP-064 and EZM2302 both inhibit CARM1, they may elicit distinct biological effects.

Methods: We employed immunoblotting, subcellular fractionation, histone extraction, chromatin immunoprecipitation assay, quantitative PCR, and confocal microscopy to compare the effects of TP-064 and EZM2302. Substrate methylation and autophagic responses were evaluated under nutrient-deprived conditions.

Results: Both TP-064 and EZM2302 inhibited CARM1-dependent methylation of non-histone substrates, including p300, GAPDH, and DRP1. However, TP-064 markedly reduced nuclear histone methylation marks H3R17me2a and H3R26me2a, whereas EZM2302 had minimal effect on these epigenetic modifications. Reflecting this differential impact, TP-064-but not EZM2302-suppressed transcription of autophagy-related genes and impaired LC3 lipidation and puncta formation under glucose deprivation. Consequently, TP-064 sensitized cells to energy stress by disrupting autophagic flux. These findings indicate that TP-064 inhibits both nuclear and cytoplasmic functions of CARM1, while EZM2302 selectively targets non-histone methylation events.

Conclusion: Our study reveals fundamental mechanistic differences between TP-064 and EZM2302 in regulating CARM1 substrates and downstream pathways. This substrate-selective inhibition has important implications for experimental design and therapeutic development, underscoring the need for context-specific selection of CARM1 inhibitors in both basic research and precision medicine.

The 2024 FASEB Scientific Research Conference on NAD Metabolism and Signaling was held in Lisbon, Portugal and served to (1) unite researchers, clinicians, and trainees, (2) create opportunities for early-stage investigators by showcasing their work on an international stage and promote collaborations, (3) train the next generation of scientists in the field, and (4) improve human health by furthering our understanding of NAD⁺ metabolism and signaling. With the burgeoning potential of NAD⁺ as a therapeutic agent for multiple health conditions, as well as many remaining scientific questions about the NAD⁺ metabolome, an expert panel discussion titled "NAD⁺ Health Outcomes Forum: A Call to Action" was hosted on Thursday, August 29, 2024. The main objectives were to discuss and translate what is known about NAD⁺ biology into tangible actions and to identify what remains unknown into a research call to action. Given the broad and reaching impact of NAD⁺ on health, there is significant interest in NAD⁺ pathway modulation, including through precursors such as nicotinic acid, nicotinamide (NAM), nicotinamide riboside (NR), and nicotinamide mononucleotide (NMN). There is also growing research regarding the heterogeneity among individuals, as well as differences and similarities among the NAD⁺ precursors, specifically in relation to dosing, timing, and their impact on various health conditions.

Introduction: Resolution of acute gut ischemia causes reperfusion injury, resulting in the release of damage-associated molecular patterns (DAMPs) and tissue injury. A key DAMP, extracellular cold-inducible RNA-binding protein (eCIRP), exacerbates inflammation in reperfusion injury, contributing to organ failure and death. Apoptosis inhibitor of macrophage (AIM or CD5L) is a glycoprotein secreted by macrophages which can influence the activity of immune cells. We seek to investigate AIM expression in ischemia/reperfusion (I/R) and elucidate its anti-inflammatory role in macrophages and intestinal epithelial cells.

Methods: Male mice underwent occlusion of the superior mesenteric artery for 60 min, followed by reperfusion for 4 h before sample collection. AIM expression in blood and tissue was evaluated by qPCR, Western blot, and ELISA. Primary peritoneal macrophages from male mice, IEC-6 intestinal epithelial cells, and RAW 264.7 macrophages were stimulated with recombinant mouse (rm) CIRP (denoted eCIRP) and treated with rmAIM. Cytokine levels were assessed by ELISA and qPCR. Metabolic function was measured in macrophages using the Agilent Seahorse XF Pro analyzer. Interactions involving AIM, eCIRP, and eCIRP's receptors, Toll-like receptor 4 (TLR4) and triggering receptor expressed on myeloid cells-1 (TREM-1), were elucidated by in silico approaches.

Results: Pulmonary AIM mRNA expression decreased by 55.9% (p = 0.018), and protein levels decreased by 26.9% (p = 0.032) in gut I/R mice compared to sham mice. Plasma AIM concentration decreased by 22.0% (p = 0.0362) in gut I/R mice compared to sham. eCIRP treatment increased pro-inflammatory cytokine production by macrophages and intestinal epithelial cells. This increase was significantly attenuated by co-treatment with rmAIM. Macrophages also increased basal oxygen consumption rate by 66.7% and ATP production by 70.3% when treated with rmAIM compared to eCIRP stimulation alone (p < 0.0001). Computational modeling predicted strong interactions between AIM and eCIRP's receptors, TLR4 and TREM-1, and showed that the presence of AIM altered eCIRP's binding to these receptors.

Conclusion: In male mice, gut I/R decreases AIM protein levels and mRNA expression in the lungs as well as AIM plasma concentration. AIM reduces eCIRP-induced pro-inflammatory cytokine production in macrophages, potentially by inhibiting eCIRP's binding to TLR4 and TREM-1. These findings suggest AIM is a promising therapeutic candidate in males with gut I/R.