Transgenic Mice Research Articles

Dl-3-n-butylphthalide promotes microglial phagocytosis and inhibits microglial inflammation via regulating AGE-RAGE pathway in APP/PS1 mice

Alzheimer's disease (AD) stands as the most prevalent neurodegenerative condition worldwide, and its correlation with microglial function is notably significant. Dl-3-n-butylphthalide (NBP), derived from the seeds of Apium graveolens L. (Chinese celery), has demonstrated the capacity to diminish Aβ levels in the brain tissue of Alzheimer’s transgenic mice. Despite this, its connection to neuroinflammation and microglial phagocytosis, along with the specific molecular mechanism involved, remains undefined. In this study, NBP treatment exhibited a substantial improvement in learning deficits observed in AD transgenic mice (APP/PS1 transgenic mice).Furthermore, NBP treatment significantly mitigated the total cerebral Aβ plaque deposition. This effect was attributed to the heightened presence of activated microglia surrounding Aβ plaques and an increase in microglial phagocytosis of Aβ plaques. Transcriptome sequencing analysis unveiled the potential involvement of the AGE (advanced glycation end products) -RAGE (receptor for AGE) signaling pathway in NBP’s impact on APP/PS1 mice. Subsequent investigation disclosed a reduction in the secretion of AGEs, RAGE, and proinflammatory factors within the hippocampus and cortex of NBP-treated APP/PS1 mice.In summary, NBP alleviates cognitive impairment by augmenting the number of activated microglia around Aβ plaques and ameliorating AGE-RAGE-mediated neuroinflammation. These findings underscore the related mechanism of the crucial neuroprotective roles of microglial phagocytosis and anti-inflammation in NBP treatment for AD, offering a potential therapeutic target for the disease.

Decreased proteasome function increases oxidative stress in the early stage of pressure ulcer development

The aging process in the elderly results in heightened skin fragility associated with various disorders, including pressure ulcers (PUs). Despite the high incidence of PUs in the elderly population, there is a limited body of research specifically examining the impact of aging on the development of pressure ulcers. Therefore, investigating age-related physiological abnormalities is essential to elucidate the pathogenesis of PUs. Ischemia-reperfusion (I/R) injury and the subsequent oxidative stress caused by reactive oxygen species (ROS) play essential roles in the early stage of PUs. In this study, we used a mouse model of proteasomal dysfunction with an age-related phenotype to examine the role of proteasome activity in cutaneous I/R injury in vivo. Decreased proteasome function did not affect the expression of inflammatory cytokines and adhesion molecules in the I/R area in transgenic mice; however, proteasome inhibition increased oxidative stress that was not attenuated by activation of the oxidative stress response mediated by NF-E2-related factor 2 (Nrf2). In dermal fibroblasts (FCs) subjected to hypoxia-reoxygenation (H/R), proteasome inhibition induced oxidative stress and ROS production, and Nrf2 activation did not adequately upregulate antioxidant enzyme expression, possibly leading to antioxidant/oxidant imbalance. The free radical scavenger edaravone had protective effects against I/R injury in vivo and decreased oxidative stress in FCs treated with a proteasome inhibitor and subjected to H/R in vitro. The results suggest that the age-related decline in proteasome activity promotes cutaneous I/R injury-induced oxidative stress, and free radical scavengers may exert protective effects by preventing oxidative stress in the early stage of PUs in elderly patients with impaired proteasome function.

Measuring the Replicability of our own Research

In the study of transgenic mouse models of neurodevelopmental and neurodegenerative disorders, we use batteries of tests to measure deficits in behaviour and from the results of these tests, we make inferences about the mental states of the mice that we interpret as deficits in "learning", "memory", "anxiety", "depression", etc. This paper discusses the problems of determining whether a particular transgenic mouse is a valid mouse model of disease X, the problem of background strains, and the question of whether our behavioural tests are measuring what we say they are. The problem of the reliability of results is then discussed: are they replicable between labs and can we replicate our results in our own lab? This involves the study of intra- and inter- experimenter reliability. The variables that influence replicability and the importance of conducting a complete behavioural phenotype: sensory, motor, cognitive and social emotional behaviour are discussed. Then the thorny question of failure to replicate is examined: Is it a curse or a blessing? Finally, the role of failure in research and what it tells us about our research paradigms is examined.

Developing a Novel Murine Meningococcal Meningitis Model Using a Capsule-Null Bacterial Strain

Background: Neisseria meningitidis (meningococcus) is a Gram-negative bacterium that colonises the nasopharynx of about 10% of the healthy human population. Under certain conditions, it spreads into the body, causing infections with high morbidity and mortality rates. Although the capsule is the key virulence factor, unencapsulated strains have proved to possess significant clinical implications as well. Meningococcal meningitis is a primarily human infection, with limited animal models that are dependent on a variety of parameters such as bacterial virulence and mouse strain. In this study, we aimed to develop a murine Neisseria meningitidis meningitis model to be used in the study of various antimicrobial compounds. Method: We used a capsule-deficient Neisseria meningitidis strain that was thoroughly analysed through various methods. The bacterial strain was incubated for 48 h in brain–heart infusion (BHI) broth before being concentrated and injected intracisternally to bypass the blood–brain barrier in CD-1 mice. This prolonged incubation time was a key factor in increasing the virulence of the bacterial strain. A total of three more differently prepared inoculums were tested to further solidify the importance of the protocol (a 24-h incubated inoculum, a diluted inoculum, and an inactivated inoculum). Antibiotic treatment groups were also established. The clinical parameters and number of deaths were recorded over a period of 5 days, and comatose mice with no chance of recovery were euthanised. Results: The bacterial strain was confirmed to have no capsule but was found to harbour a total of 56 genes coding virulence factors, and its antibiotic susceptibility was established. Meningitis was confirmed through positive tissue culture and histological evaluation, where specific lesions were observed, such as perivascular sheaths with inflammatory infiltrate. In the treatment groups, survival rates were significantly higher (up to 81.25% in one of the treatment groups compared to 18.75% in the control group). Conclusion: We managed to successfully develop a cost-efficient murine (using simple CD-1 mice instead of expensive transgenic mice) meningococcal meningitis model using an unencapsulated strain with a novel method of preparation.

Targeting STING in dendritic cells alleviates psoriatic inflammation by suppressing IL-17A production.

Psoriasis is a common chronic inflammatory skin disease driven by the aberrant activation of dendritic cells (DCs) and T cells, ultimately leading to increased production of cytokines such as interleukin (IL)-23 and IL-17A. It is established that the cGAS-STING pathway is essential for psoriatic inflammation, however, the specific role of cGAS-STING signaling in DCs within this context remains unclear. In this study, we demonstrated the upregulation of cGAS-STING signaling in psoriatic lesions by analyzing samples from both clinical patients and imiquimod (IMQ)-treated mice. Using a conditional Sting-knockout transgenic mouse model, we elucidated the impact of cGAS-STING signaling in DCs on the activation of IL-17- and IFN-γ-producing T cells in psoriatic inflammation. Ablation of the Sting hampers DC activation leads to decreased numbers of IL-17-producing T cells and Th1 cells, and thus subsequently attenuates psoriatic inflammation in the IMQ-induced mouse model. Furthermore, we explored the therapeutic potential of the STING inhibitor C-176, which reduces psoriatic inflammation and enhances the anti-IL-17A therapeutic response. Our results underscore the critical role of cGAS-STING signaling in DCs in driving psoriatic inflammation and highlight a promising psoriasis treatment.

Sequential Infection with Influenza A Virus Followed by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Leads to More Severe Disease and Encephalitis in a Mouse Model of COVID-19

COVID-19 is a spectrum of clinical symptoms in humans caused by infection with SARS-CoV-2. The coalescence of SARS-CoV-2 with seasonal respiratory viruses, particularly influenza viruses, is a global health concern. To understand this, transgenic mice expressing the human ACE2 receptor (K18-hACE2) were infected with influenza A virus (IAV) followed by SARS-CoV-2 and the host response and effect on virus biology was compared to K18-hACE2 mice infected with IAV or SARS-CoV-2 alone. The sequentially infected mice showed reduced SARS-CoV-2 RNA synthesis, yet exhibited more rapid weight loss, more severe lung damage and a prolongation of the innate response compared to the singly infected or control mice. Sequential infection also exacerbated the extrapulmonary encephalitic manifestations associated with SARS-CoV-2 infection. Conversely, prior infection with a commercially available, multivalent live-attenuated influenza vaccine (Fluenz Tetra) elicited the same reduction in SARS-CoV-2 RNA synthesis, albeit without the associated increase in disease severity. This suggests that the innate immune response stimulated by IAV inhibits SARS-CoV-2. Interestingly, infection with an attenuated, apathogenic influenza vaccine does not result in an aberrant immune response and enhanced disease severity. Taken together, the data suggest coinfection (‘twinfection’) is deleterious and mitigation steps should be instituted as part of the comprehensive public health and management strategy of COVID-19.

An Optimized Protocol for the Generation of Alveolospheres from Wild-Type Mice

Organoid models have become an integral part of the research methodology in the lung field. These systems allow for the study of progenitor and stem cell self-renewal, self-organization, and differentiation. Distinct models of lung organoids mimicking various anatomical regions of mature lungs have emerged in parallel to the increased gain of knowledge regarding epithelial stem and progenitor cell populations and the corresponding mesenchymal cells that populate the in vivo niche. In the distal lung, type 2 alveolar epithelial cells (AEC2s) represent a stem cell population that is engaged in regenerative mechanisms in response to various insults. These cells self-renew and give rise to AEC1s that carry out gas exchange. Multiple experimental protocols allowing the generation of alveolar organoids, or alveolospheres, from murine lungs have been described. Among the drawbacks have been the requirement of transgenic mice allowing the isolation of AEC2s with high viability and purity, and the occasional emergence of bronchiolar and bronchioalveolar organoids. Here, we provide a refined gating strategy and an optimized protocol for the generation of alveolospheres from wild-type mice. Our approach not only overcomes the need for transgenic mice to generate such organoids, but also yields a pure culture of alveolospheres that is devoid of bronchiolar and bronchioalveolar organoids. Our protocol contributes to the standardization of this important research tool.

A novel combinatorial approach usingsulforaphane- and withaferin A-rich extracts for prevention of estrogen receptor-negative breast cancer through epigenetic and gut microbial mechanisms.

Estrogen receptor-negative [ER(-)] mammary cancer is the most aggressive type of breast cancer (BC) with higher rate of metastasis and recurrence. In recent years, dietary prevention of BC with epigenetically active phytochemicals has received increased attention due to its feasibility, effectiveness, and ease of implementation. In this regard, combinatorial phytochemical intervention enables more efficacious BC inhibition by simultaneously targeting multiple tumorigenic pathways. We, therefore, focused on investigation of the effect of sulforaphane (SFN)-rich broccoli sprouts (BSp) and withaferin A (WA)-rich Ashwagandha (Ash) combination on BC prevention in estrogen receptor-negative [ER(-)] mammary cancer using transgenic mice. Our results indicated that combinatorial BSp + Ash treatment significantly reduced tumor incidence and tumor growth (~ 75%) as well as delayed (~ 21%) tumor latency when compared to the control treatment and combinatorial BSp + Ash treatment was statistically more effective in suppressing BC compared to single BSp or Ash intervention. At the molecular level, the BSp and Ash combination upregulated tumor suppressors (p53, p57) along with apoptosis associated proteins (BAX, PUMA) and BAX:BCL-2 ratio. Furthermore, our result indicated an expressional decline of epigenetic machinery HDAC1 and DNMT3A in mammary tumor tissue because of combinatorial treatment. Interestingly, we have reported multiple synergistic interactions between BSp and Ash that have impacted both tumor phenotype and molecular expression due to combinatorial BSp and Ash treatment. Our RNA-seq analysis results also demonstrated a transcriptome-wide expressional reshuffling of genes associated with multiple cell-signaling pathways, transcription factor activity and epigenetic regulations due to combined BSp and Ash administration. In addition, we discovered an alteration of gut microbial composition change because of combinatorial treatment. Overall, combinatorial BSp and Ash supplementation can prevent ER(-) BC through enhanced tumor suppression, apoptosis induction and transcriptome-wide reshuffling of gene expression possibly influencing multiple cell signaling pathways, epigenetic regulation and reshaping gut microbiota.

Megakaryocytic IGF1 coordinates activation and ferroptosis to safeguard hematopoietic stem cell regeneration after radiation injury.

Hematopoietic stem cell (HSC) regeneration underlies hematopoietic recovery from myelosuppression, which is a life-threatening side effect of cytotoxicity. HSC niche is profoundly disrupted after myelosuppressive injury, while if and how the niche is reshaped and regulates HSC regeneration are poorly understood. A mouse model of radiation injury-induced myelosuppression was built by exposing mice to a sublethal dose of ionizing radiation. The dynamic changes in the number, distribution and functionality of HSCs and megakaryocytes were determined by flow cytometry, immunofluorescence, colony assay and bone marrow transplantation, in combination with transcriptomic analysis. The communication between HSCs and megakaryocytes was determined using a coculture system and adoptive transfer. The signaling mechanism was investigated both in vivo and in vitro, and was consolidated using megakaryocyte-specific knockout mice and transgenic mice. Megakaryocytes become a predominant component of HSC niche and localize closer to HSCs after radiation injury. Meanwhile, transient insulin-like growth factor 1 (IGF1) hypersecretion is predominantly provoked in megakaryocytes after radiation injury, whereas HSCs regenerate paralleling megakaryocytic IGF1 hypersecretion. Mechanistically, HSCs are particularly susceptible to megakaryocytic IGF1 hypersecretion, and mTOR downstream of IGF1 signaling not only promotes activation including proliferation and mitochondrial oxidative metabolism of HSCs, but also inhibits ferritinophagy to restrict HSC ferroptosis. Consequently, the delicate coordination between proliferation, mitochondrial oxidative metabolism and ferroptosis ensures functional HSC expansion after radiation injury. Importantly, punctual IGF1 administration simultaneously promotes HSC regeneration and hematopoietic recovery after radiation injury, representing a superior therapeutic approach for myelosuppression. Our study identifies megakaryocytes as a last line of defense against myelosuppressive injury and megakaryocytic IGF1 as a novel niche signal safeguarding HSC regeneration.

Euonymus hamiltonianus Extract Improves Amnesia in APPswe/Tau Transgenic and Scopolamine-Induced Dementia Models.

Dementia is a syndrome exhibiting progressive impairments on cognition and behavior beyond the normal course of aging, and Alzheimer's disease (AD) is one of the neurodegenerative diseases known to cause dementia. We investigated the effect of KGC07EH, the 30% ethanol extract of Euonymus hamiltonianus, against amyloid-β (Aβ) production and cognitive dysfunction in dementia models. KGC07EH was treated on Hela cells expressing the Swedish mutant form of amyloid precursor protein (APP), and the AD triple transgenic (3× TG) mice were given KGC07EH orally during 11-14 months of age (100 and 300mg/kg/day). SH-SY5Y cell line was used to test KGC07EH on scopolamine-induced elevation of acetylcholinesterase (AChE) activity. ICR mice were intraperitoneally injected with scopolamine, and KGC07EH was administered orally (50, 100, and 200mg/kg/day) for 4 weeks. KGC07EH treatment decreased Aβ, sAPPβ-sw, and sAPPβ-wt levels and APP protein expressions while sAPPα was increased in Swedish mutant-transfected HeLa cells. KGC07EH treatment also significantly reduced the accumulation of Aβ plaques and tau tangles in the brain of 3× TG mice as well as improving the cognitive function. In SH-SY5Y cells cultured with scopolamine, KGC07EH dose-dependently attenuated the increase of AChE activity. KGC07EH also improved scopolamine-induced learning and memory impairment in scopolamine-injected mice, and in their cerebral cortex and hippocampus, the expression levels of p-ERK, p-CREB, p-Akt, and BDNF were attenuated. KGC07EH inhibits APP processing and Aβ production both in vitro and in vivo, while enhancing acetylcholine signaling and cognitive dysfunction which are the major symptoms of dementia.

Mature astrocytes as source for astrocyte repopulation after deletion in the medial prefrontal cortex: Implications for depression.

The adult brain retains a high repopulation capacity of astrocytes after deletion, and both mature astrocytes in the neocortex and neural stem cells in neurogenic regions possess the potential to generate astrocytes. However, the origin and the repopulation dynamics of the repopulating astrocytes after deletion remain largely unclear. The number of astrocytes is reduced in the medial prefrontal cortex (mPFC) of patients with depression, and selective elimination of mPFC astrocytes is sufficient to induce depression-like behaviors in rodents. However, whether astrocyte repopulation capacity is impaired in depression is unknown. In this study, we used different transgenic mouse lines to genetically label different cell types and demonstrated that in the mPFC of normal adult mice of both sexes, mature astrocytes were a major source of the repopulating astrocytes after acute deletion induced by an astrocyte-specific toxin, L-alpha-aminoadipic acid (L-AAA), and astrocyte regeneration was accomplished within two weeks accompanied by reversal of depression-like behaviors. Furthermore, re-ablation of mPFC astrocytes post repopulation led to reappearance of depression-like behaviors. In adult male mice subjected to 14-day chronic restraint stress, a well-validated mouse model of depression, the number of mPFC astrocytes was reduced; however, the ability of mPFC astrocytes to repopulate after L-AAA-induced deletion was largely unaltered. Our study highlights a potentially beneficial role for repopulating astrocytes in depression and provides novel therapeutic insights into enhancing local mature astrocyte generation in depression.

Research on Alzheimer's Disease (AD) Involving the Use of In vivo and In vitro Models and Mechanisms.

Alzheimer's Disease (AD) is a neurodegenerative disorder characterized by the progressive formation of extracellular amyloid plaques, intracellular neurofibrillary tangles, inflammation, and impaired antioxidant systems. Early detection and intervention are vital for managing AD effectively. This review scrutinizes both in-vivo and in-vitro screening models employed in Alzheimer's disease research. In-vivo models, including transgenic mice expressing AD-related mutations, offer profound insights into disease progression and potential therapeutic targets. A thorough understanding of these models and mechanisms will facilitate the development of novel therapies and interventions for Alzheimer's disease. This review aims to provide an overview of the current experimental models in AD research, assess their strengths and weaknesses as model systems, and underscore the future prospects of experimental AD modeling. We conducted a systematic literature search across multiple databases, such as Pub- Med, Bentham Science, Elsevier, Springer Nature, Wiley, and Research Gate. The search strategy incorporated pertinent keywords related to Alzheimer's disease, in-vivo models, in-vitro models, and screening mechanisms. Inclusion criteria were established to identify studies focused on in-vivo and in-vitro screening models and their mechanisms in Alzheimer's disease research. Studies not meeting the predefined criteria were excluded from the review. A well-structured experimental animal model can yield significant insights into the neurobiology of AD, enhancing our comprehension of its pathogenesis and the potential for cutting-edge therapeutic strategies. Given the limited efficacy of current AD medications, there is a pressing need for the development of experimental models that can mimic the disease, particularly in pre-symptomatic stages, to investigate prevention and treatment approaches. To address this requirement, numerous experimental models replicating human AD pathology have been established, serving as invaluable tools for assessing potential treatments. In summary, this comprehensive review underscores the pivotal role of in-vivo and in-vitro screening models in advancing our understanding of Alzheimer's disease. These models offer invaluable insights into disease progression, pathological mechanisms, and potential therapeutic targets. By conducting a rigorous investigation and evaluation of these models and mechanisms, effective screening and treatment methods for Alzheimer's disease can be devised. The review also outlines future research directions and areas for enhancing AD screening models.

Alzheimer's disease associated isoforms of human CD33 distinctively modulate microglial cell responses in 5XFAD mice.

Microglia play diverse pathophysiological roles in Alzheimer's disease (AD), with genetic susceptibility factors skewing microglial cell function to influence AD risk. CD33 is an immunomodulatory receptor associated with AD susceptibility through a single nucleotide polymorphism that modulates mRNA splicing, skewing protein expression from a long protein isoform (CD33M) to a short isoform (CD33m). Understanding how human CD33 isoforms differentially impact microglial cell function in vivo has been challenging due to functional divergence of CD33 between mice and humans. We address this challenge by studying transgenic mice expressing either of the human CD33 isoforms crossed with the 5XFAD mouse model of amyloidosis and find that human CD33 isoforms have opposing effects on the response of microglia to amyloid-β (Aβ) deposition. Mice expressing CD33M have increased Aβ levels, more diffuse plaques, fewer disease-associated microglia, and more dystrophic neurites compared to 5XFAD control mice. Conversely, CD33m promotes plaque compaction and microglia-plaque contacts, and minimizes neuritic plaque pathology, highlighting an AD protective role for this isoform. Protective phenotypes driven by CD33m are detected at an earlier timepoint compared to the more aggressive pathology in CD33M mice that appears at a later timepoint, suggesting that CD33m has a more prominent impact on microglia cell function at earlier stages of disease progression. In addition to divergent roles in modulating phagocytosis, scRNAseq and proteomics analyses demonstrate that CD33m+ microglia upregulate nestin, an intermediate filament involved in cell migration, at plaque contact sites. Overall, our work provides new functional insights into how CD33, as a top genetic susceptibility factor for AD, modulates microglial cell function.

T cell-intrinsic PKD3 fine-tunes differentiation into CD8+ central memory T cells and CD8 single positive thymocyte development.

Members of the Protein kinases D (PKD) family are described as regulators of T cell responses. From the two T cell-expressed isoforms PKD2 and PKD3, so far mainly the former was thoroughly investigated and is well understood. Recently, we have investigated also PKD3 using conventional as well as conditional T cell-specific knockout models. These studies suggested PKD3 to be a T cell-extrinsic regulator of the cells' fate. However, these former model systems did not take into account possible redundancies with the highly homologous PKD2. To overcome this issue and thus properly unravel PKD3's T cell-intrinsic functions, here we additionally used a mouse model overexpressing a constitutively active isoform of PKD3 specifically in the T cell compartment. These transgenic mice showed a slightly higher proportion of central memory T cells in secondary lymphoid organs and blood. This effect could not be explained via differences upon polyclonal stimulation in vitro, however, may be connected to the observed developmental aberrances in the CD8 single positive compartment during thymic development. Lastly, the observed alterations in the CD8+ T cell compartment did not impact proper immune response upon immunization with ovalbumin or in a subcutaneous tumour model suggesting only a small to absent biological relevance. Taking together the knowledge of all our published studies on PKD3 in the T cell compartment, we now conclude that T cell-intrinsic PKD3 is a fine-tuner of central memory T cell as well as CD8 single positive thymocyte development.

Human tau N-terminal domain-specific monoclonal antibodies: screening and application in blood detection

The antibodies to the microtubule-associated protein tau play a role in basic and clinical studies of Alzheimer's disease (AD) and other tauopathies. With the recombinant human tau441 as the immunogen, the hybridoma cell strains secreting the anti-human tau N-terminal domain (NTD-tau) monoclonal antibodies were generated by cell fusion and screened by limiting dilution. The purified monoclonal antibodies were obtained by inducing the mouse ascites and affinity chromatography. The sensitivity and specificity of the monoclonal antibodies were examined by indirect ELISA and Western blotting, respectively. A double antibody sandwich ELISA method for detecting human tau protein was established and optimized. The results showed that the positive cloning rate of hybridoma cells was 83.6%. A stable cell line producing ZD8F7 antibodies was established, and the antibody titer in the supernatant of the cell line was 1:16 000. The antibody titer in the ascitic fluid was higher than 1:256 000; and the titer of purified ZD8F7 monoclonal antibodies was higher than 1:128 000. The epitope analysis showed that the ZD8F7 antibody recognized tau21-37 amino acid in the N-terminal domain. The Western blotting results showed that the ZD8F7 antibody recognized the recombinant human tau protein of 50-70 kDa and the human tau protein of 50 kDa in the brain tissue of transgenic AD model mice (APP/PS1/tau). With ZD8F7 as a capture antibody, a quantitative detection method for human tau protein was established, which showed a linear range of 7.8-500.0 pg/mL and could identify human tau protein in the brain tissue of AD transgenic mice and human plasma but not recognize the mouse tau protein. In conclusion, the human NTD-tau-specific monoclonal antibody and the double antibody sandwich ELISA method established in this study are highly sensitive and can serve as a powerful tool for the detection of tau protein in neurodegenerative diseases.

A novel mouse model for investigating α-synuclein aggregates in oligodendrocytes: implications for the glial cytoplasmic inclusions in multiple system atrophy

The aggregated alpha-synuclein (αsyn) in oligodendrocytes (OLGs) is one of the pathological hallmarks in multiple system atrophy (MSA). We have previously reported that αsyn accumulates not only in neurons but also in OLGs long after the administration of αsyn preformed fibrils (PFFs) in mice. However, detailed spatial and temporal analysis of oligodendroglial αsyn aggregates was technically difficult due to the background neuronal αsyn aggregates. The aim of this study is to create a novel mouse that easily enables sensitive and specific detection of αsyn aggregates in OLGs and the comparable analysis of the cellular tropism of αsyn aggregates in MSA brains. To this end, we generated transgenic (Tg) mice expressing human αsyn-green fluorescent protein (GFP) fusion proteins in OLGs under the control of the 2’, 3’-cyclic nucleotide 3’-phosphodiesterase (CNP) promoter (CNP-SNCAGFP Tg mice). Injection of αsyn PFFs in these mice induced distinct GFP-positive aggregates in the processes of OLGs as early as one month post-inoculation (mpi), and their number and size increased in a centripetal manner. Moreover, MSA-brain homogenates (BH) induced significantly more oligodendroglial αsyn aggregates than neuronal αsyn aggregates compared to DLB-BH in CNP-SNCAGFP Tg mice, suggestive of their potential tropism of αsyn seeds for OLGs. In conclusion, CNP-SNCAGFP Tg mice are useful for studying the development and tropism of αsyn aggregates in OLGs and could contribute to the development of therapeutics targeting αsyn aggregates in OLGs.

Analysis of the pharmacokinetics and efficacy of RBD1016 – A GalNAc-siRNA targeting Hepatitis B Virus X gene using semi-mechanistic PK/PD model

Small interference RNA (siRNA) is a class of short double-stranded RNA molecules that cause mRNA degradation through an RNA interference mechanism and is a promising therapeutic modality. RBD1016 is a siRNA drug in clinical development for the treatment of chronic Hepatitis B Virus (HBV) infection, which contains a conjugated with N-acetylglucosamine moiety that can facilitate its hepatic delivery. We aimed to construct a semi-mechanistic model of RBD1016 in pre-clinical animals, to elucidate the pharmacokinetic/pharmacodynamic (PK/PD) profiles in mice and PK profiles in monkeys, which can lay the foundation for potential future translation of RBD1016 PK and PD from the pre-clinical stage to the clinic stage. The proposed semi-mechanistic PK/PD model fitted PK and PD data in HBV transgenic mice well and described plasma and liver concentrations in the monkeys well. The simulation results showed that our model has a reasonable predictive ability for Hepatitis B surface antigen (HBsAg) levels after multiple dosing in mice. Further PK and PD data for RBD1016, including clinical data, will assist in refining the model presented here. Our current effort focused on model building for RBD1016, we anticipate that the model could apply to other GalNAc-siRNA drugs.

DEAD-box helicase 17 (DDX17) protects cardiac function by promoting mitochondrial homeostasis in heart failure

DEAD-box helicase 17 (DDX17) is a typical member of the DEAD-box family with transcriptional cofactor activity. Although DDX17 is abundantly expressed in the myocardium, its role in heart is not fully understood. We generated cardiomyocyte-specific Ddx17-knockout mice (Ddx17-cKO), cardiomyocyte-specific Ddx17 transgenic mice (Ddx17-Tg), and various models of cardiomyocyte injury and heart failure (HF). DDX17 is downregulated in the myocardium of mouse models of heart failure and cardiomyocyte injury. Cardiomyocyte-specific knockout of Ddx17 promotes autophagic flux blockage and cardiomyocyte apoptosis, leading to progressive cardiac dysfunction, maladaptive remodeling and progression to heart failure. Restoration of DDX17 expression in cardiomyocytes protects cardiac function under pathological conditions. Further studies showed that DDX17 can bind to the transcriptional repressor B-cell lymphoma 6 (BCL6) and inhibit the expression of dynamin-related protein 1 (DRP1). When DDX17 expression is reduced, transcriptional repression of BCL6 is attenuated, leading to increased DRP1 expression and mitochondrial fission, which in turn leads to impaired mitochondrial homeostasis and heart failure. We also investigated the correlation of DDX17 expression with cardiac function and DRP1 expression in myocardial biopsy samples from patients with heart failure. These findings suggest that DDX17 protects cardiac function by promoting mitochondrial homeostasis through the BCL6-DRP1 pathway in heart failure.

#1738 Renal endogenous hepcidin alleviates ferroptosis of collecting duct in ischemia/reperfusion induced AKI

Abstract Background and Aims Recent studies have demonstrated that urinary and serum hepcidin predicted the incidence of acute kidney injury (AKI) among critically ill patients. As an endogenous acute phase hormone, hepcidin is synthesized not only by the liver but also secreted by distal nephron. However, the autocrine impact of hepcidin secreted by distal nephron on collecting duct cells in AKI remains poorly understood. Method We determined the expression and location of hepcidin in murine kidney in different severity of bilateral ischemia/reperfusion (bIRI) induced AKI model with ischemic duration for 20 min and 32 min. The effect of renal endogenous hepcidin was explored in cultured M-1 (Murine Cortical collecting duct) exposed to hypoxia/reoxygenation (H/R), as well as in bIRI mice with collecting duct-specific overexpression of hepcidin. Transgenic mice were generated by genetic cross between Hepcidin floxed mice which knocked the gene fragment of CAG-LSL-mHamp-flag-WPRE-PolyA into H11 locus and Cdh16-cre mice. Mechanistically, RNA-seq was performed in M-1 cells under H/R condition. Results The expression of renal endogenous hepcidin was significantly elevated in bIRI32min mice compared to both bIRI20min and sham group, suggesting that augmented secretion of hepcidin was induced by more severe AKI. Interestingly, renal endogenous hepcidin was mainly localized to distal convoluted tubule and collecting tubule as indicated by its co-staining with AQP2 and Calbindin-D28k in the kidney. To investigate the role of hepcidin in collecting duct, M-1 cells were cultured under hypoxia condition. We observed remarkable ferroptosis in collecting duct cells other than apoptosis nor necroptosis. Subsequently, hepcidin knockdown further exacerbated ferroptosis under H/R condition, as evidenced by an upregulation of PTGS2 expression and downregulation of GXP4 expression, lower proportion of GSH/GSSG ratio as well as enhanced lipid peroxide accumulation and production of Fe2+. Then, RNA-seq data revealed that hepcidin knockdown promoted the enrichment of genes related to oxidative stress and glutathione metabolic pathway which may be responsible for the deterioration of ferroptosis. To investigate the role of hepcidin in vivo, bIRI induced AKI was established in Cdh16-cre+Hepcidinflox/+ mice. We observed remarkable overexpression of Hepcidin-Flag localized to collecting duct and interstium area. Impressively, Cdh16-cre+Hepcidinflox/+ mice showed ameliorated renal dysfunction adrenal tubule injury histologically. Ferroptosis of renal was alleviated in Cdh16-cre+Hepcidinflox/+ mice as confirmed by an upregulation of GPX4 expression and downregulation of PTGS2, ACSL4, FPN expression, higher proportion of GSH/GSSG ratio, as well as reduced lipid peroxide accumulation and production of Fe2+ by Lipofluo (lipid peroxides) and FerroOrange (Fe2+) staining on frozen section of kidney. Ferroptosis of the collecting duct was alleviated as indicated by downregulation of PTGS2 expression which co-staining with AQP2. To demonstrate the beneficial role of hepcidin, exogenous hepcidin was applied to bIRI induced AKI mice to strengthen the endogenous protective response. Conclusion Renal endogenous hepcidin is induced in the distal tubule and collecting duct after AKI as a protective response via alleviating ferroptosis of collecting duct. Hepcidin may represent a novel therapeutic approach to promote tubule repair by targeting ferroptosis for AKI.

Telocytes: Detection, Visualization, Tissue Dissociation, and Tamoxifen-Induction of Transgenic Mice.

Telocytes, distinctive interstitial cells, have recently emerged as crucial components of the stem-cell niche in the intestine. Notably, telocytes are distinguished by their extremely long cellular protrusions extending hundreds of microns from the cell body, forming an interconnected network along the intestinal crypt villus axis. Due to these unique cellular characteristics, there is a need for tailored working protocols to effectively characterize and target telocytes. Here, we outline advanced and progressive protocols for tissue fixation, dissociation, visualization, and the use of tamoxifen-induced transgenic mouse models to specifically target telocytes.