Molecular Medicine最新文献

Background: Giant cell arteritis (GCA) is a chronic granulomatous inflammatory disease involving large- and medium-sized arteries. The disease spectrum comprises cranial (C-GCA), extracranial (EC-GCA) and mixed phenotypes. Toll-like receptors (TLRs) in the affected arteries may play an important role in GCA pathogenesis. However, data on TLR and TLR-ligands expression pattern in GCA arteries are lacking.

Objective: To investigate the expression of TLRs and putative ligands in temporal artery biopsies (TAB) from C-GCA, EC-GCA and isolated polymyalgia rheumatica (PMR) patients to establish a link between TLRs, antigen expression, and disease stage. To correlate the plasma levels of identified TLR-ligands with standard inflammatory markers (IL-6, CRP, ESR) in these patients.

Methods: Immunofluorescence staining of TLR2/4/7/8, HMGB-1, SAA, fibrinogen, and p-glycoprotein was performed with TABs of six biopsy proven C-GCA, six EC-GCA, five PMR patients and seven age-matched controls. Association studies among plasma inflammatory markers were done with 139 PMR and 40 GCA patients.

Results: The levels of TLR2/4/7/8 and the alarmins HMGB-1, SAA, and fibrinogen were highly increased in C-GCA TABs in the sites of inflammation and less in EC-GCA TABs. P-glycoprotein was overexpressed in C-GCA TABs. Glucocorticoids or TAK1-inhibitor treatment decreased the fibrinogen- and SAA-mediated IL-6 production in control PBMCs. Plasma levels of SAA and fibrinogen associated strongly with CRP and ESR levels.

Conclusion: TLRs are overexpressed at the site of vascular inflammation in C-GCA and at a lower level in EC-GCA and PMR with negative TAB. Moreover, HMGB-1, SAA, and fibrinogen may serve as disease biomarkers of patients with C-GCA.

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the pathological accumulation of α-synuclein aggregates and the selective degeneration of dopaminergic neurons in the substantia nigra. Growing evidence implicates dysfunction of the ubiquitin-proteasome system (UPS), a critical regulator of protein homeostasis, in the pathogenesis of PD through impaired clearance of toxic protein species. As key components of the UPS, deubiquitinating enzymes (DUBs) counterbalance ubiquitin ligase activity by cleaving ubiquitin chains from substrate proteins, thereby playing pivotal roles in maintaining protein turnover and regulating cellular signaling pathways. Notably, emerging research has demonstrated that specific DUBs are intimately involved in modulating multiple PD-related pathological processes, including α-synuclein aggregation, mitochondrial oxidative stress, iron homeostasis, and neuronal survival. These findings suggest DUBs as promising therapeutic targets for PD intervention. This review comprehensively summarize the pathophysiological roles of PD-associated DUBs, their molecular mechanisms in disease progression, and recent advances in the development of DUB inhibitors as potential disease-modifying therapies for PD.

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.