Transgenic Mice Research Articles

Mitochondrial fission is required for thermogenesis in brown adipose tissue.

Brown adipose tissue (BAT) thermogenesis is pivotal for maintaining body temperature and energy balance. Mitochondrial morphology is dynamically controlled by a balance between fusion and fission, which is crucial for cell differentiation, response to metabolic insults, and heat production. Dynamin-related protein 1 (Drp1) is a key regulator of mitochondrial fission. This study investigates the role of Drp1 in BAT development and thermogenesis by generating Drp1-deficient mice. These mice were created by crossing Drp1 floxed mice with fatty acid-binding protein 4-Cre (aP2-Cre) transgenic mice, resulting in aP2-Cre+/-Drp1flox/flox (aP2-Drp1f/f) mice. The aP2-Drp1f/f mice exhibited severe BAT and brain hypoplasia, with the majority dying within 48 hours postnatally, highlighting Drp1's crucial role in neonatal survival. Impaired thermogenic responses were observed in aP2-Drp1f/f mice, characterized by significantly decreased expression of thermogenic and lipogenic genes in BAT. Ultrastructural analysis revealed disrupted mitochondrial morphology and reduced lipid droplet content in BAT. The few surviving adult aP2-Drp1f/f mice also showed impaired BAT and brain development, along with BAT thermogenesis dysfunction during cold exposure. Our findings underscore the essential role of Drp1-mediated mitochondrial fission in BAT thermogenesis and neonatal survival, providing insights into potential therapeutic approaches for metabolic disorders.

Abstract A009: Genetic disruption of CAPNS1 impedes triple-negative breast cancer metastasis: A case for selective calpain-1/2 inhibitors

Abstract Calpains are a family of 15 calcium-activated cysteine proteases that have emerged as potential therapeutic targets in triple negative breast cancer (TNBC). They regulate the function of their substrates via limited proteolytic processing and are involved in both pro- and anti-apoptotic signaling pathways, as well as membrane-cytoskeletal remodeling events associated with cell migration and invasion. The most well-understood members of the calpain family are the classical calpain-1 and calpain-2 isoforms, which are ubiquitously expressed heterodimers each consisting of a large catalytic subunit, encoded by the capn1 and capn2 genes, respectively, and a common small regulatory subunit encoded by capns1. Overexpression of calpain-1 and calpain-2 has been associated with shorter survival in HER2+ breast cancer and TNBC, respectively. We hypothesize that inhibition of calpain-1 and calpain-2 may be exploited to improve tumor response to cytotoxic chemotherapy and suppress metastasis. CRISPR-Cas9 mediated knockout of capns1 in AC2M2 mouse mammary carcinoma cells, which results in a loss of both calpain-1 and calpain-2 activity, disrupts migration and invasion in vitro and impedes lung metastasis in tail vein and orthotopic engraftment mouse models. Calpain knockout also sensitizes AC2M2 cells to the microtubule stabilizing drug paclitaxel, and preliminary results suggest that cancer cell intrinsic calpain depletion may prevent neoadjuvant chemotherapy-induced metastasis. To explore potential off-target effects associated with systemic calpain inhibition, we have established a novel transgenic mouse with conditional deletion of capns1 in its hematopoietic stem cell lineage. By comparing the immune profiles of wild-type and capns1 knockout mice, we aim to better understand how calpain inhibitors will affect various immune cell populations and their respective functions. We plan to extend this understanding to anticancer immunity by conducting tumor immunophenotyping experiments in syngeneic orthotopic engraftment models using wild-type and capns1 knockout mice. At present, no clinically relevant calpain inhibitors are available. However, using genetic approaches, we have validated calpain-1 and calpain-2 as relevant therapeutic targets in TNBC, providing rationale for the development of selective calpain inhibitors. Citation Format: Danielle Harper, Ivan Shapovalov, Samantha Cockburn, Jenny Min, Yan Gao, Peter A. Greer. Genetic disruption of CAPNS1 impedes triple-negative breast cancer metastasis: A case for selective calpain-1/2 inhibitors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Optimizing Therapeutic Efficacy and Tolerability through Cancer Chemistry; 2024 Dec 9-11; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(12_Suppl):Abstract nr A009.

Delta opioid receptor agonists activate PI3K-mTORC1 signaling in parvalbumin-positive interneurons in mouse infralimbic prefrontal cortex to exert acute antidepressant-lie effects.

The delta opioid receptor (DOP) is a promising target for novel antidepressants due to its potential for rapid action with minimal adverse effects; however, the functional mechanism underlying acute antidepressant actions remains elusive. We report that subcutaneous injection of the selective DOP agonist KNT-127 reduced immobility in the forced swimming test, and that this antidepressant-like response was reversed by intracerebroventricular injection of the selective mechanistic (or mammalian) target of rapamycin (mTOR) inhibitor rapamycin or the phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002. KNT-127 also alleviated social avoidance and reduced sucrose consumption (anhedonia) among chronic vicarious social defeat stress model mice, which were similarly reversed by PI3K and mTOR inhibitors. In addition, KNT-127 increased phosphorylation levels of the mTOR signaling-related proteins Akt and p70S6 kinase in medial prefrontal cortex as revealed by immunoblotting. In the forced swimming test, a microinfusion of KNT-127 and another DOP agonist SNC80 in the infralimbic prefrontal cortex (IL-PFC) attenuated the immobility, which were blocked by rapamycin and LY294002. Perfusion of KNT-127 onto IL-PFC slices increased miniature excitatory postsynaptic current frequency and reduced miniature inhibitory postsynaptic current frequency in pyramidal neurons as measured by whole-cell patch-clamping, and both responses were reversed by rapamycin. Imaging of brain slices from transgenic mice with DOP-promoter-driven green fluorescent protein revealed that most DOPs were expressed in parvalbumin-positive interneurons in the IL-PFC. These findings suggest that DOP agonists exert antidepressant-like actions by suppressing GABA release from parvalbumin-positive interneurons via the PI3K-Akt-mTORC1-p70S6 kinase pathway, thereby enhancing IL-PFC pyramidal neuron excitation.

Elucidating Sex-Specific Immune Profiles in a Breast Cancer Model

Breast cancer is commonly thought of as a “women’s disease”. However, men are increasingly diagnosed with the disease, and their mortality rates are disparately higher than those of female patients. The abundance and composition of the immune microenvironment are determinants of breast cancer progression and survival. It is well documented that there are sex-specific differences in the immune response to several diseases, including various cancers. However, the effects of these differences in the context of breast cancer remain to be explored. This study demonstrates sex differences in the hormonal and immune landscape of the MMTV-PyMT transgenic murine model of female and male ER+ breast cancer using single-cell RNA sequencing (scRNA-Seq), whole-slide immunohistochemistry, and flow cytometry. Mammary tumors of transgenic male mice had increased estrogen receptor alpha expression and enriched nuclear binding signatures compared to female tumors. In the tumor immune compartment, male mice had lower intratumoral leukocyte infiltration. Yet, scRNA-Seq analysis reveals a more immunostimulatory microenvironment and increased antitumor immune populations in the primary and metastatic lungs as compared to transgenic females. Despite a more favorable innate immune profile, the metastatic burden was increased in male mice. Our data support a sex-dependent immune response in mammary carcinoma associated with the tumor, and likely host, hormonal environment. With emerging therapeutics targeting the tumor immune microenvironment, characterizing immune profiles is critical for optimizing their use in all breast cancer patients.

Epitope-focused immunogens targeting the hepatitis C virus glycoproteins induce broadly neutralizing antibodies.

Hepatitis C virus (HCV) infection causes ~290,000 annual human deaths despite the highly effective antiviral treatment available. Several viral immune evasion mechanisms have hampered the development of an effective vaccine against HCV, among them the remarkable conformational flexibility within neutralization epitopes in the HCV antigens. Here, we report the design of epitope-focused immunogens displaying two distinct HCV cross-neutralization epitopes. We show that these immunogens induce a pronounced, broadly neutralizing antibody response in laboratory and transgenic human antibody mice. Monoclonal human antibodies isolated from immunized human antibody mice specifically recognized the grafted epitopes and neutralized four diverse HCV strains. Our results highlight a promising strategy for developing HCV immunogens and provide an encouraging paradigm for targeting structurally flexible epitopes to improve the induction of neutralizing antibodies.

Mouse models of primary sclerosing cholangitis: we just can’t get enough

Primary sclerosing cholangitis (PSC) is a rare but devastating disease affecting the intra- and extrahepatic bile ducts, frequently progressing to end-stage liver disease. Patients develop peribiliary inflammation and fibrosis, leading to multifocal biliary strictures that evolve to biliary cirrhosis. PSC is frequently associated with inflammatory bowel disease and a high risk of cholangiocarcinoma development. The pathogenesis of this disease is not completely understood, and currently, there are no effective therapies beyond liver transplantation. The available experimental models of PSC do not fully reproduce the phenotype of the disease, and this is a major limitation for unraveling its pathogenic mechanisms and evaluating novel therapies. A recent study by Lukasova et al. proposed a new hypothesis on the pathogenesis of PSC. The relevance of their work is two-fold: (1) the authors provide preliminary evidence suggesting that the disruption of tight junctions in mouse biliary epithelium leads to a PSC-like phenotype; and (2) they provide the research community with a novel transgenic mouse model of the disease. Follow-up studies on this new mouse model are eagerly awaited.

SARS-CoV-2 N protein recruits G3BP to double membrane vesicles to promote translation of viral mRNAs

Ras-GTPase-activating protein SH3-domain-binding proteins (G3BP) are critical for the formation of stress granules (SGs) through their RNA- and ribosome-binding properties. SARS-CoV-2 nucleocapsid (N) protein exhibits strong binding affinity for G3BP and inhibits infection-induced SG formation soon after infection. To study the impact of the G3BP-N interaction on viral replication and pathogenesis in detail, we generated a mutant SARS-CoV-2 (RATA) that specifically lacks the G3BP-binding motif in the N protein. RATA triggers a stronger and more persistent SG response in infected cells, showing reduced replication across various cell lines, and greatly reduced pathogenesis in K18-hACE2 transgenic mice. At early times of infection, G3BP and WT N protein strongly colocalise with dsRNA and with non-structural protein 3 (nsp3), a component of the pore complex in double membrane vesicles (DMVs) from which nascent viral RNA emerges. Furthermore, G3BP-N complexes promote highly localized translation of viral mRNAs in the immediate vicinity of the DMVs and thus contribute to efficient viral gene expression and replication. In contrast, G3BP is absent from the DMVs in cells infected with RATA and translation of viral mRNAs is less efficient. This work provides a fuller understanding of the multifunctional roles of G3BP in SARS-CoV-2 infection.

Potent AMA1-specific human monoclonal antibody against Plasmodium vivax Pre-erythrocytic and Blood Stages

New therapeutics are necessary for preventing Plasmodium vivax malaria due to easy transmissibility and dormancy in the liver that increases the clinical burden due to recurrent relapse. In this manuscript we characterize 12 Pv Apical Membrane Antigen 1 (PvAMA1) specific human monoclonal antibodies from Peripheral Blood Mononuclear Cells of a Pv-exposed individual. PvAMA1 is essential for sporozoite and merozoite invasion, making it a unique therapeutic target. We show that humAb 826827 blocks the invasion of human reticulocytes using Pv clinical isolates and inhibits sporozoite invasion of human hepatocytes in vitro (IC50 of 0.3 – 3.7 µg/mL). Inoculation of human liver transgenic (FRG-humHep) female mice with humAb 826827 significantly reduces liver infection in vivo. The crystal structure of rPvAMA1 bound to 826827 shows that 826827 partially occupies the highly conserved hydrophobic groove in PvAMA1 that binds its known receptor, RON2. We have isolated a potent humAb that is isolate-transcendent, blocks both pre-erythrocytic and blood stage infection, and could be a potential therapy for Pv.

RANKL treatment restores thymic function and improves T cell-mediated immune responses in aged mice.

Age-related thymic involution, leading to reduced T cell production, is one of the major causes of immunosenescence. This results in an increased susceptibility to cancers, infections, and autoimmunity and in reduced vaccine efficacy. Here, we identified that the receptor activator of nuclear factor κB (RANK)-RANK ligand (RANKL) axis in the thymus is altered during aging. Using a conditional transgenic mouse model, we demonstrated that endothelial cells depend on RANK signaling for their cellularity and functional maturation. Decreased RANKL availability during aging resulted in a decline in cellularity and function of both endothelial cells and thymic epithelial cells, contributing to thymic involution. We then found that, whereas RANKL neutralization in young mice mimicked thymic involution, exogenous RANKL treatment in aged mice restored thymic architecture as well as endothelial cell and epithelial cell abundance and functional properties. Consequently, RANKL improved T cell progenitor homing to the thymus and boosted T cell production. This cascade of events resulted in peripheral T cell renewal and effective antitumor and vaccine responses in aged mice. Furthermore, we conducted a proof-of-concept study that showed that RANKL stimulates endothelial cells and epithelial cells in human thymic organocultures. Overall, our findings suggest that targeting the RANK-RANKL axis through exogenous RANKL administration could represent a therapeutic strategy to rejuvenate thymic function and improve T cell immunity during aging.

Enhanced prefrontal nicotinic signaling as evidence of active compensation in Alzheimer’s disease models

BackgroundCognitive reserve allows for resilience to neuropathology, potentially through active compensation. Here, we examine ex vivo electrophysiological evidence for active compensation in Alzheimer’s disease (AD) focusing on the cholinergic innervation of layer 6 in prefrontal cortex. Cholinergic pathways are vulnerable to neuropathology in AD and its preclinical models, and their modulation of deep layer prefrontal cortex is essential for attention and executive function.MethodsWe functionally interrogated cholinergic modulation of prefrontal layer 6 pyramidal neurons in two preclinical models: a compound transgenic AD mouse model that permits optogenetically-triggered release of endogenous acetylcholine and a transgenic AD rat model that closely recapitulates the human trajectory of AD. We then tested the impact of therapeutic interventions to further amplify the compensated responses and preserve the typical kinetic profile of cholinergic signaling.ResultsIn two AD models, we found potentially compensatory upregulation of functional cholinergic responses above non-transgenic controls after onset of pathology. To identify the locus of this enhanced cholinergic signal, we dissected key pre- and post-synaptic components with pharmacological strategies. We identified a significant and selective increase in post-synaptic nicotinic receptor signalling on prefrontal cortical neurons. To probe the additional impact of therapeutic intervention on the adapted circuit, we tested cholinergic and nicotinic-selective pro-cognitive treatments. Inhibition of acetylcholinesterase further enhanced endogenous cholinergic responses but greatly distorted their kinetics. Positive allosteric modulation of nicotinic receptors, by contrast, enhanced endogenous cholinergic responses and retained their rapid kinetics.ConclusionsWe demonstrate that functional nicotinic upregulation occurs within the prefrontal cortex in two AD models. Promisingly, this nicotinic signal can be further enhanced while preserving its rapid kinetic signature. Taken together, our work suggests that compensatory mechanisms are active within the prefrontal cortex that can be harnessed by nicotinic receptor positive allosteric modulation, highlighting a new direction for cognitive treatment in AD neuropathology.

Role of inducible nitric oxide (iNOS) and nitrosative stress in regulating sex differences in secondary lymphedema

Secondary lymphedema is a common complication following surgical treatment of solid tumors. Although more prevalent in women due to higher breast cancer rates, men also develop lymphedema, often with more severe manifestations. Despite these differences in clinical presentation, the cellular mechanisms underlying sex differences are poorly understood. Previous studies have shown that inducible nitric oxide synthase (iNOS) expression by inflammatory cells is an important regulator of lymphatic pumping and leakiness in lymphedema and that lymphatic endothelial cells are highly sensitive to nitrosative stress. Based on this rationale, we used a mouse tail model of lymphedema to study the role of nitric oxide in sex-related differences in disease severity. Consistent with clinical findings, we found that male mice have significantly worse tail edema and higher rates of tail necrosis compared with female mice following tail skin/lymphatic excision (p = 0.001). Our findings correlated with increased tissue infiltration of iNOS + inflammatory cells, increased iNOS protein expression, and increased nitrosative stress in male mouse lymphedematous skin tissues (p < 0.05). Importantly, transgenic male mice lacking the iNOS gene (iNOS-KO) displayed markedly reduced swelling, inflammation, and tissue necrosis rates, whereas no differences were observed between wild-type and iNOS-KO female mice. Overall, our results indicate that iNOS-mediated nitric oxide production contributes to sex-based differences in secondary lymphedema severity, emphasizing the need to consider sex as a biological variable in lymphedema research.

Parasympathetic Airway Hyperreactivity Is Enhanced in Acute but Not Chronic Eosinophilic Asthma Mouse Models.

Airway hyperreactivity in asthma is mediated by airway nerves, including sensory nerves in airway epithelium and parasympathetic nerves innervating airway smooth muscle. Isolating the function of these two nerve populations in vivo, to distinguish how each is affected by inflammatory processes and contributes to hyperreactivity in asthma, has been challenging. In this study, we used optogenetic acti-vation of airway nerves in vivo to study parasympathetic contributions to airway hyperreactivity in two mouse models of asthma: 1) acute challenge with house dust mite antigen, and 2) chronic airway hy-pereosinophilia due to genetic IL-5 overexpression in airways. Overall airway hyperreactivity, as meas-ured by bronchoconstriction to an inhaled agonist, was increased in both models. In contrast, optoge-netic stimulation of isolated efferent parasympathetic nerves induced bronchoconstriction only in the acute house dust mite antigen challenge group. Using whole mount tissue immunofluorescence and modeling software, we then measured, in three dimensions, the interactions between eosinophils and parasympathetic nerves in both models and found that eosinophils were more numerous and more proximal to airway parasympathetic nerves in antigen challenged and IL-5 transgenic mice than in their respective controls, but were not significantly different between the two asthma models. Thus even though eosinophils were increased around nerves in both models, parasympathetic nerves only mediated airway hyperreactivity in the antigen challenged mice. This study demonstrates divergent effects of acute versus chronic eosinophilia on parasympathetic airway nerve activity and points to eo-sinophil-nerve interactions as a key regulator of airway hyperreactivity in antigen challenged mice.

Comparative analysis of gut microbiota in hormone-sensitive and castration-resistant prostate cancer in Japanese men.

Gut microbiota plays a crucial role in the development and progression of prostate cancer, with previous studies indicating that certain bacterial taxa are more abundant in castration-resistant prostate cancer (CRPC) compared to hormone-sensitive prostate cancer (HSPC). Notably, the composition of gut microbiota can vary significantly by geographic region, and Japanese individuals have a distinct microbial profile. However, research exploring these differences within Japanese populations remains limited. This study investigated the gut microbiota differences between Japanese men with HSPC and CRPC and further validated these findings using a transgenic mouse model. Rectal swab samples were collected from 140 Japanese men diagnosed with HSPC (n = 84) or CRPC (n = 56) between September 2020 and July 2022. Gut microbiota composition was analyzed using 16S rRNA gene sequencing. Additionally, Pten-KO mice, which model the progression from HSPC to CRPC, underwent similar microbiota analysis. Results revealed significant differences in gut microbiota composition between HSPC and CRPC patients. Specifically, the CRPC group showed a higher abundance of Firmicutes, including Gemella and Lactobacillus, compared to the HSPC group. These differences were mirrored in the mouse model, where CRPC mice also showed an increase in these bacteria. This study identifies distinct microbial differences between HSPC and CRPC in Japanese men, suggesting that Gemella and Lactobacillus may be associated with the progression to castration resistance in prostate cancer. These findings suggest that gut microbiota differences may be associated with prostate cancer progression. Further research is needed to explore the potential of targeting the microbiota as a therapeutic strategy.

Tyrosine Hydroxylase-positive Nucleus Accumbens Neurons Influence Delay Discounting in a Mouse T-maze Task.

Delay discounting (DD) is a phenomenon where individuals devalue a reward associated with a temporal delay, with the rate of devaluation being representative of impulsive-like behavior. Here we first sought to develop and validate a mouse DD task to study brain circuits involved in DD decision-making within short developmental time windows, given widespread evidence of developmental regulation of impulse control and risk-taking. We optimized a T-maze DD task for mice that enables training and DD trials within two weeks. Mice learned to choose between a large and a small reward located at opposite arms of a T-maze. Once training criteria were met, mice underwent DD whereby the large reward choice was associated with a temporal delay. Task validation showed that adolescent C57BL/6J mice display increased preference for the small reward upon a temporal delay, confirming increased impulsivity compared to adults. We next used this DD task to explore the neural basis of decision-making. We used tyrosine hydroxylase transgenic mice (TH-Cre) to target TH-positive neurons in the nucleus accumbens (NAc) and ventral tegmental area (VTA) with Cre-dependent excitatory or inhibitory Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). Inhibition of transduced neurons in the NAc decreased preference for the small but immediate reward during DD. Inhibition of TH+ neurons in the ventral tegmental area (VTA) did not affect impulsive choice in this DD task. These results uncover a novel role for NAc TH-positive neurons in DD behavior and expand the repertoire of behavioral tasks available for studying decision-making across the lifespan.Significance Statement Delay discounting (DD) tasks are used in rodents to study impulsive choice, whereby subjects display a preference for an immediate, smaller reward when access to a larger reward is contingent on a temporal delay. Research implicates the nucleus accumbens (NAc) brain region in impulsive behavior, with recent evidence of specialization among NAc neuronal subtypes in impulsive choice. Here we interrogated the neural requirements of impulsive choice in mice. We found that inhibition of a subset of NAc neurons expressing tyrosine hydroxylase decreases impulsive choice. We also saw increased impulsive choice in adolescent mice compared to adults, consistent with reported developmental changes in impulsivity. Together, our data identify cell-specific NAc regulation of impulsive choice with important implications for neurodevelopment.

Quantitative Proteomic Analysis of APP/PS1 Transgenic Mice.

Alzheimer's disease (AD) is a prevalent neurodegenerative disorder affecting the central nervous system (CNS), with its etiology still shrouded in uncertainty. The interplay of extracellular amyloid-β (Aβ) deposition, intracellular neurofibrillary tangles (NFTs) composed of tau protein, cholinergic neuronal impairment, and other pathogenic factors is implicated in the progression of AD. The current study endeavors to delineate the proteomic landscape alterations in the hippocampus of an AD murine model, utilizing proteomic analysis to identify key physiological and pathological shifts induced by the disease. This endeavor aims to shed light on the underlying pathogenic mechanisms, which could facilitate early diagnosis and pave the way for novel therapeutic interventions for AD. To dissect the proteomic perturbations induced by Aβ and Presenilin-1 (PS1) in the AD pathogenesis, we undertook a label-free quantitative (LFQ) proteomic analysis focusing on the hippocampal proteome of the APP/PS1 transgenic mouse model. Employing a multi-faceted approach that included differential protein functional enrichment, cluster analysis, and protein-protein interaction (PPI) network analysis, we conducted a comprehensive comparative proteomic study between APP/PS1 transgenic mice and their wild-type C57BL/6 counterparts. Mass spectrometry identified a total of 4817 proteins in the samples, with 2762 proteins being quantifiable. Comparative analysis revealed 396 proteins with differential expression between the APP/PS1 and control groups. Notably, 35 proteins exhibited consistent temporal regulation trends in the hippocampus, with concomitant alterations in biological pathways and PPI networks. This study presents a comparative proteomic profile of transgenic (APP/PS1) and wild-type mice, highlighting the proteomic divergences. Furthermore, it charts the trajectory of proteomic changes in the AD mouse model across the developmental stages from 2 to 12 months, providing insights into the physiological and pathological implications of the disease-associated genetic mutations.

Platelet-Derived Growth Factor Subunit A Strengthens the Neurovascular Unit and Inhibits Retinal Vascular Regression Under Hyperoxic Conditions

Retinopathy of prematurity (ROP) is primarily caused by the exposure of preterm infants with underdeveloped blood vessels to high oxygen concentrations. This damages the astrocytes that promote normal vascular development, leading to avascularity, pathological neovascularization, and retinal detachment, and even blindness as the disease progresses. In this study, the aim was to investigate the differences in the characteristics of astrocytes and blood vessels between wild-type (WT) and genetically modified mice overexpressing platelet-derived growth factor subunit A (PDGF-A) in the retina immediately after high oxygen exposure, a protocol in the oxygen-induced retinopathy (OIR) model of ROP. Our results showed that PDGF-A mice exhibited an increased population of astrocytes and higher vascular density than WT mice and that PDGF-A strengthened the resistance to hyperoxic conditions. In the OIR model, PDGF-A mice had reduced avascular zone areas following hyperoxia exposure. Furthermore, immunostaining for NG2 and CD31 showed that pericytes tended to regress earlier than endothelial cells, particularly at the vessel edges in both WT and transgenic mice, indicating relatively higher susceptibility to hyperoxia-induced damage. These findings suggest that PDGF-A plays a crucial role in stabilizing retinal vessels and may serve as a novel therapeutic target for ROP, highlighting the potential significance of PDGF-A in the pathological mechanisms of retinal diseases.

Protective Effects of Antcin H Isolated from Antrodia cinnamomea Against Neuroinflammation in Huntington's Disease via NLRP3 Inflammasome Inhibition.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat in the huntingtin (HTT) gene. When the CAG repeat exceeds 36, it results in the accumulation of the mutant HTT (mHTT) protein in neurons and glial cells. Key pathological mechanisms in HD include excitotoxicity, energy dysfunction, impaired mitochondrial function, increased oxidative stress, and neuroinflammation. The NLRP3 inflammasome is a multimeric protein complex element of NLRP3, ASC, and caspase-1, which regulates interleukin (IL)-1β and IL-18 secretion. The NLRP3 inflammasome plays an important role in inflammatory reactions and is involved in the pathogenesis of several neurodegenerative diseases. We have previously demonstrated high NLRP3 inflammasome expression levels in the striatum of R6/2 mice (a transgenic HD mouse model). Systematic administration of an NLRP3 inhibitor (MCC950) to R6/2 mice suppressed the NLRP3 inflammasome, decreased IL-1β and reactive oxygen species production, and reduced neuronal toxicity, suggesting protective effects against HD. Antrodia cinnamomea is an indigenous medicinal fungus in Taiwan, which shows diverse medicinal and pharmacological activities, but its effects in HD are not well understood. Herein, we report that systematic administration of Antcin-H isolated from A. cinnamomea to R6/2 mice suppressed the NLRP3 inflammasome, IL-1β production, and reduced neuronal toxicity. Most importantly, oral administration of Antcin-H reduced disease progression by increasing neuronal survival, reducing neuroinflammation during an extended lifespan, and improving motor dysfunction in R6/2 mice. Taken together, our data suggest that Antcin-H has therapeutic potential for treating HD.

Adipsin improves diabetic hindlimb ischemia through SERPINE1 dependent angiogenesis

BackgroundAdipsin (complement factor D, CFD), as the first described adipokine, is well-known for its regulatory effects in diabetic cardiovascular complications. However, its role in diabetic hind-limb ischemia was not clarified. This study aimed to evaluate the possible therapeutic effect of Adipsin in hind-limb ischemia in type 2 diabetic mice and to elucidate the molecular mechanisms involved.MethodsA high-fat diet and streptozotocin (HFD/STZ)-induced diabetic mouse model, and a transgenic mouse model with adipose tissue-specific overexpression of Adipsin (Adipsin-Tg) were employed. Hindlimb ischemia was established by femoral artery ligation, and blood flow recovery was monitored using Laser Doppler perfusion imaging. Molecular mechanisms underlying Adipsin-potentiated angiogenesis were examined using RNA sequencing and co-immunoprecipitation/mass spectrometry (Co-IP/MS) analyses.ResultsAdipsin expression was upregulated in non-diabetic mice following HLI, while suppressed in diabetic mice, indicating its potential role in ischemic recovery which is impaired in diabetes. Adipsin-Tg mice exhibited significantly improved blood flow recovery, increased capillary density, and enhanced muscle regeneration in comparison with non-transgenic (NTg) diabetic mice. Adipsin facilitated proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) under hyperglycemic and hypoxic conditions. Additionally, it enhanced phosphorylation of AKT, ERK, and eNOS pathways both in vivo and in vitro. RNA sequencing and co-immunoprecipitation/mass spectrometry (Co-IP/MS) analyses identified that Adipsin promoted angiogenesis by interacting with SERBP1, which disrupted the binding of SERBP1 to SERPINE1 mRNA, resulting in reduced SERPINE1 expression and the subsequent activation of the VEGFR2 signaling cascade.ConclusionsAdipsin promotes angiogenesis and facilitates blood perfusion recovery in diabetic mice with HLI by downregulating SERPINE1 through interaction with SERBP1. These findings elucidate a novel therapeutic potential for Adipsin in the management of PAD in diabetic patients, highlighting its role in enhancing angiogenesis and tissue repair.

Constructing a Metal-Organic Frameworks-Based Long-Acting Sequential Release System for the Treatment of Alzheimer's Disease.

Due to the unclear pathogenesis of Alzheimer's disease (AD) and the lack of a completely cured medication, AD patients need to take medication in order and on time every day all one's life, which is difficult for severe memory impairment patients to strictly follow on time. Traditional AD drug carriers, such as sugar coating and capsules, rely on dissolution or fragmentation to achieve drug release, which lacks the interaction between drug molecules and carriers, thus they cannot achieve sufficient long-acting and sequential drug release. Herein, Mn-MOF-74, which ligand structure is similar to two antioxidants dihydroquercetin (DHQ) and resveratrol (Res) is chosen as the carrier. Due to the differences in adsorption energy between DHQ/MOF and Res/MOF, the release speed of DHQ is much faster than Res. Therefore, Mn-MOF-74 loaded with DHQ and Res (DR@MOF) showed sequential drug release and a long-term antioxidant effect for ≈72h, with an efficacy time six times longer than that of vitamin E. In 5×FAD transgenic mice, DR@MOF exhibited excellent capacity in maintaining oxidative balance in the brain, ameliorating spatial learning and memory deficits, and showed the potential of an AD agent for long-acting and sequential treatment.

Modulating immunodominance hierarchy of immunoglobulin germline-encoded structural motifs targeting the influenza hemagglutinin stem

Antibodies targeting epitopes through germline-encoded motifs can be found in different individuals. While these public antibodies are often beneficial, they also pose hurdles for subdominant antibodies to emerge. Here, we use transgenic mice that reproduce the human IGHV1-69∗01 germline-encoded antibody response to the conserved stem epitope on group 1 hemagglutinin (HA) of influenza A virus to show that this germline-endowed response can be overridden by a subdominant yet cross-group reactive public antibody response. Immunization with a non-cognate group 2 HA stem enriched B cells harboring the IGHD3-9 gene, thereby switching from IGHV1-69- to IGHD3-9-encoded motif-dependent epitope recognition. These IGHD3-9 antibodies bound, neutralized, and conferred cross-group protection in mice against influenza A viruses. A cryoelectron microscopy (cryo-EM) structure of an IGHD3-9 antibody resembled the human broadly neutralizing antibody FI6v3, which uses IGHD3-9. Together, our findings offer insights into vaccine regimens that engage an immunoglobulin repertoire with broader cross-reactivity to influenza A viruses.