Tg Mice Research Articles

Serum biomarkers indicate sex differences in response to pathology inhibition in aged mice with tau pathology

AbstractBackgroundThe failure of some clinical trials using drugs that remove brain amyloid to prevent progression of dementia suggest that neurodegenerative disease may not progress in a linear fashion and threshold levels of one pathology may initiate alterations that are unable to be reversed upon removal of the initiating pathological process. We used the inducible Tg4510 mouse tauopathy model to determine whether neurodegeneration could be halted following removal of transgene expression in mice aged to 18 monthsMethodPathology was induced in mice from 8 weeks until 18 months of age (n = 20 equal male/female; both tau transgenic and non‐transgenic) by removal of doxycycline (dox) diet. Dox diet was returned to half the animals from 18‐23 months to inhibit pathology. Levels of serum Nfl were measured using SIMOA assay throughout disease progression and p‐tau181 along with other serum biomarkers were measured at endpoint. Immunohistochemical assays were performed to quantitate numbers of neurons (NeuN) and tau load (PHF‐tau) in hippocampus.ResultSerum Nfl increased over aging in all mice and was significantly higher in tau transgenics relative to non‐transgenics at 18 months (p<0.05). Between 18 and 23 months, the rate of serum Nfl increase was significantly(p<0.05) reduced in male transgenic mice on dox compared to female transgenic mice, so that by 23 months levels were not different from non‐transgenic mice. Similarly, the numbers of neurons in the hippocampus of male mice on dox at 23 months was significantly higher than their female counterparts (p<0.05). When looking at levels of serum p‐tau181 at 23 months, there was significantly (p<0.05) higher levels in male mice on dox than female miceConclusionThese data suggest that aged female mice have ongoing neurodegeneration following removal of pathology induction that is similar to mice with continued pathology induction. The differences in serum p‐tau‐181 may indicate that female mice fail to clear pathological tau from the brain. Further studies will examine the relationship between histopathology and blood biomarkers and determine whether female mice on dox have a higher tau pathology load than their male counterparts.

The immune cytokine granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) is an effective treatment for both Alzheimer’s disease and the viral diseases caused by West Nile Virus and SARS‐CoV‐2 infection, indicating that inflammation and viral infection may be partners and therapeutic co‐targets in many brain disorders

AbstractBackgroundGM‐CSF has been considered a potential therapeutic target in many disorders associated with inflammation because it is upregulated and upregulates other inflammatory molecules such as IL‐1, IL‐6, and TNF‐a. However, its ability to increase the number and activation of phagocytic cells in the periphery and brain suggested to us that its upregulation in rheumatoid arthritis (RA) might underlie the finding that RA protects against another inflammatory disorder, Alzheimer’s disease (AD) by aiding phagocytosis of amyloid deposits. Treatment of mouse models of AD with GM‐CSF reverses amyloid deposition and normalizes cognition. A double‐blind placebo‐controlled phase II trial showed that three weeks of treatment with human recombinant GM‐CSF (sargramostim) in mild‐to‐moderate AD participants led to improved cognition (MMSE) and blood biomarkers of amyloid, Tau, and neurodegeneration (A+T+N). GM‐CSF/sargramostim treatment is also efficacious in humans and/or animal models with other inflammation‐associated diseases/disorders (Parkinson’s disease, Down syndrome, chemo‐brain, and normal aging).MethodBased on findings that innate immunity and microglia underlie viral control, we investigated GM‐CSF in a mouse model of West Nile Virus encephalopathy and a mouse model of SARS‐CoV‐2/COVID‐19. C57BL/6 mice were infected by footpad injection of 1,000 pfu of WNV (TX2002‐HC strain). ACE2 receptor Tg mice were infected with 4,000 pfu of SARS‐CoV‐2 by nasal instillation. Mice were treated IP daily with 5 mg of recombinant GM‐CSF starting the day after infection and analyzed for pathology, morbidity, and mortality at death or at sacrifice at 2 weeks. The SARS‐CoV2‐infected mice were also analyzed for viral titers in brain and lung.ResultGM‐CSF treatment improved survival in both the WNV and SARS‐CoV‐2 models. SARS‐CoV‐2 viral titers in affected tissues, including brain, were greatly reduced by GM‐CSF treatment.ConclusionBy activating both the innate and adaptive immune systems, GM‐CSF may be an effective therapy for viral diseases and may protect against viral re‐infection more effectively than antiviral drugs or monoclonal antibodies, a serious concern during the COVID‐19 pandemic. These results also challenge the belief that inflammation in these disorders contributes essentially to the disease. It is possible that the anti‐viral activity of GM‐CSF may also underlie some of its efficacy in AD.

The Effect of Fat Intake with Increased Omega-6-to-Omega-3 Polyunsaturated Fatty Acid Ratio in Animal Models of Early and Late Alzheimer’s Disease-like Pathogenesis

This work aims to clarify the effect of dietary polyunsaturated fatty acid (PUFA) intake on the adult brain affected by amyloid pathology. McGill-R-Thy1-APP transgenic (Tg) rat and 5xFAD Tg mouse models that represent earlier or later disease stages were employed. The animals were exposed to a control diet (CD) or an HFD based on corn oil, from young (rats) or adult (mice) ages for 24 or 10 weeks, respectively. In rats and mice, the HFD impaired reference memory in wild-type (WT) animals but did not worsen it in Tg, did not cause obesity, and did not increase triglycerides or glucose levels. Conversely, the HFD promoted stronger microglial activation in Tg vs. WT rats but had no effect on cerebral amyloid deposition. IFN-γ, IL-1β, and IL-6 plasma levels were increased in Tg rats, regardless of diet, while CXCL1 chemokine levels were increased in HFD-fed mice, regardless of genotype. Hippocampal 3-nitrotyrosine levels tended to increase in HFD-fed Tg rats but not in mice. Overall, an HFD with an elevated omega-6-to-omega-3 ratio as compared to the CD (25:1 vs. 8.4:1) did not aggravate the outcome of AD regardless of the stage of amyloid pathology, suggesting that many neurobiological processes relevant to AD are not directly dependent on PUFA intake.

A c- MPL Splice Variant up-Regulated in Myeloproliferative Neoplasms Modulates Thrombopoietin Signaling and Induces Cycling of Hematopoietic Stem Cells and Inflammatory Progenitors

Introduction: Myeloproliferative neoplasms (MPN) are clonal disorders caused by mutations that activate JAK/STAT signaling through the thrombopoietin receptor, MPL, leading to overproduction of myeloid lineages. MPL is the only hematopoietic growth factor receptor expressed on the surface of hematopoietic stem cells (HSC) where it maintains their quiescence by tethering HSCs to osteoblasts in the endosteal niche. MPL is also expressed on megakaryocytes and platelets. Individuals born with loss of function mutations in the distal MPL cytokine receptor homology domain (CRHD) develop thrombocytopenia and progressive bone marrow failure. How MPL functions in MPN remains unclear. Here, we describe a novel MPL splice variant ( MPL-SV) that is present in CD34 + cells and platelets from healthy controls, but overexpressed in the MPN. We therefore sought to: 1) define MPL-SV function in hematopoiesis and MPN, and, 2) elucidate mechanisms underlying MPL-SV. Methods: After identifying a novel MPL-SV transcript from JAK2 V617F MPNplatelets, we compared its expression to MPL transcripts lacking the deletion in MPN patients and healthy controls. To assess its function, we generated transgenic (Tg) mice expressing varied levels of MPL-SV in HSC. Next, we examined responses of the Tg mice to thrombopoietin (THPO), hematopoietic stem progenitor cell (HSPC) frequency by flow cytometry, and HSPC function by colony-forming assays (CFA). To elucidate mechanisms, we performed single cell RNA sequencing (scRNAseq) of bone marrow (BM)-derived Lin -, Sca +, c-kit + (LSK) cells followed by pathway analysis. Results: Using primers to amplify the MPL CRHD in platelets from patients with JAK2 V617F MPN, we discovered an MPL- SV lacking 7 amino acids in the MPL amino-terminal domain, a common hotspot for activating MPN mutations. MPL-SV is present in CD34 + cells and platelets of healthy controls, but overexpressed in MPN relative to MPL transcripts lacking the deletion. To examine its function, we generated transgenic (Tg) mice expressing MPL-SV in HSC. Tg miceexpressing a single copy of MPL-SV have unperturbed hematopoiesis, whereas Tg mice expressing 6 copies of the transgene ( MPL-SV-6) develop marked thrombocytopenia, mild splenomegaly, and a modest increase in red cells by 9-12 weeks of age. Baseline serum THPO levels are elevated in Tg MPL-SV-6 mice and they fail to increase platelet counts following exogenous THPO administration, suggesting defective function of MPL-SV in binding and/or internalizing THPO. To further assess MPL-SV function, we compared CFA from HSPC of Tg MPL-SV-6 and wildtype mice, which show a decrease in total colony forming units (CFU) from BM or spleen-derived MPL-SV-6 HSPC. Accordingly, there was a decrease in LSK cells, multi-potent progenitors (MPPs), and megakaryocytes (Mk) in BM by flow cytometry. By contrast, spleens show increases in LK cells and short-term HSC. Further analysis by scRNAseq of BM-derived LSK cells reveal a marked decrease in both quiescent HSC and HSC with a Mk-bias associated with a relative expansion in more proliferative HSC, including those with a bias towards erythroid and mature myeloid differentiation. Pathway analysis reveal that MPL-SV activates transcriptional networks involved in TNF-α signaling via NF-κB and oxidative phosphorylation. Analysis of cell-cell interactions by CellChat show decreased signaling through intercellular adhesion molecule 1 (ICAM-1) in MPL-SV-6 HSC compared to controls. Importantly, ICAM1 maintains quiescent HSC within their BM niche. Similarly, cell cycle analysis from scRNAseq indicate that quiescent HSC are stimulated to cycle and generate HSC primed to differentiate into erythroid and myeloid progeny. Conclusions: We discovered a novel MPL-SV that is up-regulated in MPN. In Tg mice, MPL-SV impairs responses to THPO and disrupts hematopoiesis by depleting quiescent HSC and BM megakaryocytes. Single cell transcriptomics suggest that MPL-SV drives expansion of cycling HSC and HSC primed to differentiate into progenitors involved in inflammation, including myeloid cells. Mechanistically, HSPC expressing MPL-SV-6 activate transcriptional networks involved in TNFα signaling via NF-κB pathways. Our results highlight the central role for MPL in hematopoiesis and suggest that aberrant MPL splicing contributes to MPN pathogenesis.

Effects of B Cell Depletion by CD19-Targeted Chimeric Antigen Receptor T Cells in a Murine Model of Systemic Sclerosis.

Our goal was to study the tolerance and efficacy of two B cell depletion strategies, including one with CD19-targeted chimeric antigen receptor (CAR) T cells, in a preclinical model mimicking the severe lung damages observed in systemic sclerosis. B cell depletion strategies were evaluated in the Fra-2 transgenic (Tg) mouse model. We considered a first group of 16 untreated mice, a second group of 15 mice receiving a single dose of anti-CD20 monoclonal antibody (mAb), and a third group of 8 mice receiving CD19-targeted CAR-T cells in combination with anti-CD20 monoclonal antibody. After six weeks of clinical evaluation, different validated markers of inflammation, lung fibrosis, and pulmonary vascular remodeling were assessed. CD19-targeted CAR-T cells infusion in combination with anti-CD20 mAb resulted in a deeper B cell depletion than anti-CD20 mAb alone in the peripheral blood and lesional lungs of Fra-2 Tg mice. CAR-T cell infusion worsened the clinical score and increased mortality in Fra-2 Tg mice. In line with the above findings, CAR-T cell infusion significantly increased lung collagen content, the histological fibrosis score, and right ventricular systolic pressure. CAR-T cells accumulated in lesional lungs and promoted T activation and inflammatory cytokine production. Treatment with anti-CD20 mAb in monotherapy had no impact on lung inflammation-driven fibrosis and pulmonary hypertension. B cell therapies failed to show efficacy in the Fra2 Tg mice. The exacerbated Fra-2 lung inflammatory burden stimulated accumulation and expansion of activated CD19-targeted CAR-T cells, secondarily inducing T cell activation and systemic inflammation, finally leading to disease worsening.

Icariin Inhibits Overexpression and Aggregation of α-Synuclein in A53T α-Synuclein Transgenic Mice by Regulating Parkin and PLK2.

Synucleinopathies, which are major pathological features of Parkinson's disease (PD), are characterized by misfolded aggregates of α-synuclein in the peripheral and central nervous system. Icariin (ICA) is the main active component of Epimedium flavonoids. Our previous study found that ICA decreases α-synuclein expression in APPV717I transgenic mice. The aim of the present study was to examine the potential applications and mechanisms of ICA in PD using A53T α-synuclein transgenic (A53T Tg) mice. After 3 months of intragastric ICA administration, rotarod and pole tests were used to assess behavioral changes in A53T Tg mice at 8 and 13 months of age. SH-SY5Y cells over-expressing wild-type α-synuclein were used to further examine the pharmacological effect and underlying mechanism of ICA. Western blotting and immunocytochemistry were used to detect the expression levels of α-synuclein and its related proteins. ICA significantly improved the impaired motor function and coordination in A53T Tg mice. It also decreased the expression, Ser129 phosphorylation, and aggregation of α-synuclein in SH-SY5Y cells transfected with α-synuclein and the striatum of A53T Tg mice. Moreover, ICA increased the expression of parkin, which is associated with the ubiquitin-proteasome system (UPS), and decreased the level of polo-like kinase 2 (PLK2), an enzyme that phosphorylates α-synuclein. ICA alleviated motor impairments in A53T mice, an effect which may be associated with the decreased phosphorylation and aggregation of α-synuclein through PLK2 and parkin regulation.

Local perfusion of capillaries reveals disrupted beta-amyloid homeostasis at the blood-brain barrier in Tg2576 murine Alzheimer’s model

BackgroundParenchymal accumulation of beta-amyloid (Aβ) characterizes Alzheimer’s disease (AD). Aβ homeostasis is maintained by two ATP-binding cassette (ABC) transporters (ABCC1 and ABCB1) mediating efflux, and the receptor for advanced glycation end products (RAGE) mediating influx across the blood-brain barrier (BBB). Altered transporter levels and disruption of tight junctions (TJ) were linked to AD. However, Aβ transport and the activity of ABCC1, ABCB1 and RAGE as well as the functionality of TJ in AD are unclear.MethodsISMICAP, a BBB model involving microperfusion of capillaries, was used to assess BBB properties in acute cortical brain slices from Tg2576 mice compared to wild-type (WT) controls using two-photon microscopy. TJ integrity was tested by vascularly perfusing biocytin-tetramethylrhodamine (TMR) and quantifying its extravascular diffusion as well as the diffusion of FM1-43 from luminal to abluminal membranes of endothelial cells (ECs). To assess ABCC1 and ABCB1 activity, calcein-AM was perfused, which is converted to fluorescent calcein in ECs and gets actively extruded by both transporters. To probe which transporter is involved, probenecid or Elacridar were applied, individually or combined, to block ABCC1 and ABCB1, respectively. To assess RAGE activity, the binding of 5-FAM-tagged Aβ by ECs was quantified with or without applying FPS-ZM1, a RAGE antagonist.ResultsIn Tg2576 mouse brain, extravascular TMR was 1.8-fold that in WT mice, indicating increased paracellular leakage. FM1-43 staining of abluminal membranes in Tg2576 capillaries was 1.7-fold that in WT mice, indicating reduced TJ integrity in AD. While calcein was undetectable in WT mice, its accumulation was significant in Tg2576 mice, suggesting lower calcein extrusion in AD. Incubation with probenecid or Elacridar in WT mice resulted in a marked calcein accumulation, yet probenecid alone had no effect in Tg2576 mice, implying the absence of probenecid-sensitive ABC transporters. In WT mice, Aβ accumulated along the luminal membranes, which was undetectable after applying FPS-ZM1. In contrast, marginal Aβ fluorescence was observed in Tg2576 vessels, and FPS-ZM1 was without effect, suggesting reduced RAGE binding activity.ConclusionsDisrupted TJ integrity, reduced ABCC1 functionality and decreased RAGE binding were identified as BBB alterations in Tg2576 mice, with the latter finding challenging the current concepts. Our results suggest to manage AD by including modulation of TJ proteins and Aβ-RAGE binding.

Compositional changes in fecal microbiota in a new Parkinson's disease model: C57BL/6-Tg(NSE-haSyn) mice

BackgroundThe gut–brain axis (GBA) in Parkinson's disease (PD) has only been investigated in limited mice models despite dysbiosis of the gut microbiota being considered one of the major treatment targets for neurodegenerative disease. Therefore, this study examined the compositional changes of fecal microbiota in novel transgenic (Tg) mice overexpressing human α-synuclein (hαSyn) proteins under the neuron-specific enolase (NSE) to analyze the potential as GBA model.ResultsThe expression level of the αSyn proteins was significantly higher in the substantia nigra and striatum of NSE-hαSyn Tg mice than the Non-Tg mice, while those of tyrosine hydroxylase (TH) were decreased in the same group. In addition, a decrease of 72.7% in the fall times and a 3.8-fold increase in the fall number was detected in NSE-hαSyn Tg mice. The villus thickness and crypt length on the histological structure of the gastrointestinal (GI) tract decreased in NSE-hαSyn Tg mice. Furthermore, the NSE-hαSyn Tg mice exhibited a significant increase in 11 genera, including Scatolibacter, Clostridium, Feifania, Lachnoclostridium, and Acetatifactor population, and a decrease in only two genera in Ligilactobacillus and Sangeribacter population during enhancement of microbiota richness and diversity.ConclusionsThe motor coordination and balance dysfunction of NSE-hαSyn Tg mice may be associated with compositional changes in gut microbiota. In addition, these mice have potential as a GBA model.

EXTH-18. DEVELOPMENT OF A TRANSGENIC MOUSE MODEL ENABLING COMPARATIVE ANALYSIS OF CHIMERIC ANTIGEN RECEPTOR (CAR)-EXPRESSING IMMUNE CELL POPULATIONS FOR CANCER IMMUNOTHERAPY

Abstract We reported the creation of C57BL/6J-background transgenic (Tg) mice carrying the anti-epidermal growth factor receptor (EGFR)vIII-CAR with CD3z-CD28-4-1BB downstream of a Lox-Stop-Lox cassette in the Rosa26 locus. Breeding these mice with CD4-Cre Tg allows rigorous and reproducible evaluation of CAR-T cell therapy in syngeneic models (Chuntova et al. Neuro-Onc 2022). As the extension of the approach, we crossed the EGFRvIII-CAR Tg mice with mice carrying Vav-Cre, which allowed the expression of anti-EGFRvIII CAR in all hematopoietic cells. In particular, we evaluated CAR-T, CAR-NKT, CAR-NK cells, and CAR-macrophages. CAR-NK cells displayed a robust antigen-specific cytolytic activity against EGFRvIII-expressing SB28 cells, comparable to CAR-T cells in vitro and mice bearing intracerebral EGFRvIII-expressing SB28 gliomas. Remarkably, CAR-NKT cells produced higher levels of inflammatory cytokines when co-cultured with EGFRvIII-expressing SB28 cells, such as IFN-g, Perforin, IL4, and IL17. On the other hand, despite their in vitro cytotoxic effects against SB28 cells, CAR-NK cells failed to mediate a therapeutic response in mice bearing intracerebral glioma. Future studies will be directed at improving the in vivo persistence of CAR-NK cells using engineered cytokines, such as IL-2 superkine. In addition, we extensively investigated CAR-macrophages in vitro and in vivo. Unfortunately, we did not observe significant CAR-specific anti-tumor effects. Consequently, we modified the intracellular domain of CAR from CD3z-CD28-4-1BB to CD3z alone. This alteration led to increased TNF-α production through CAR signaling. This transgenic mouse model offers rigorous and reproducible evaluations of CAR-expressing hematopoietic cells. Furthermore, the model provides a platform for developing combination therapies, such as CAR-T plus CAR-NKT or CAR-T plus CAR-NK cells, with potential implications for enhanced cancer treatment strategies.

IMMU-04. LOW-INTENSITY PULSED ULTRASOUND ENHANCES CAR T CELL THERAPY BY INDUCING TWO-WAY IMMUNE CELL TRAFFICKING – T CELL INTO TUMOR AND MYELOID CELL INTO PERIPHERY IN A SYNGENEIC MOUSE GLIOBLASTOMA MODEL

Abstract Chimeric Antigen Receptor (CAR) T-cell therapy has been successful against hematological malignancies. However, it has not established efficacy in glioblastoma (GBM) patients. Significant challenges currently limiting the effectiveness of CAR T-cell therapy for patients with GBM include poor CAR-T cell trafficking to GBM due to the blood-brain barrier, and the presence of highly immunosuppressive and pro-tumoral tumor-associated macrophages (TAMs) in the tumor microenvironment. Furthermore, the heterogeneous expression of target antigens in GBM can lead to antigen escape and therapeutic resistance. Low-intensity pulsed ultrasound with microbubbles (LIPU/MB) has been shown to enhance CAR T-cell trafficking and improve survival in immunocompromised xenograft mice GBM models. In this study, we investigated whether multiple rounds of LIPU/MB can improve not only CAR T-cell but also endogenous T-cell infiltration into the tumors and induce myeloid cell trafficking into lymph nodes (LNs), thus enhancing CAR T-cell therapy against GBM in the immunocompetent syngeneic mice model. We recently developed a novel immunocompetent transgenic (Tg) mouse that expresses an anti-EGFRvIII CAR on T-cells and enables us to investigate the interaction between CAR T-cells and the endogenous immune environment. C57BL6/J mice bearing intracerebral EGFRvIII+ GBM received an intravenous administration of anti-EGFRvIII CAR T-cells derived from Tg-mice. The mice were stratified to receive single-LIPU (1 min before CAR T-cells administration) or triple-LIPU/MB (1 min before CAR T-cells administration, then two and four days later). Triple-LIPU/MB significantly suppressed tumor growth and improved survival and was associated with increased CAR T-cells and endogenous T-cells infiltration into the tumors compared with single-LIPU/MB or without LIPU. Furthermore, draining cervical LNs showed increased macrophages trafficking after triple-LIPU/MB. Moreover, TAMs isolated from triple-LIPU/MB mice brains showed higher levels of MHC-II expression. These results suggest that LIPU/MB may promote the tumor antigen presentation to endogenous T-cells and enhance the efficacy of CAR T-cell therapy for GBM.

Fangchinoline inhibits SARS-CoV-2 and MERS-CoV entry

The COVID-19 pandemic caused by SARS-CoV-2, lead to mild to severe respiratory illness and resulted in 6.9 million deaths worldwide. Although vaccines are effective in preventing COVID-19, they may not be sufficient to protect immunocompromised individuals from this respiratory illness. Moreover, novel emerging variants of SARS-CoV-2 pose a risk of new COVID-19 waves. Therefore, identification of effective antivirals is critical in controlling SARS and other coronaviruses, such as MERS-CoV. We show that Fangchinoline (Fcn), a bisbenzylisoquinoline alkaloid, inhibits replication of SARS-CoV, SARS-CoV-2, and MERS-CoV in a range of in vitro assays, by blocking entry. Therapeutic use of Fcn inhibited viral loads in the lungs, and suppressed associated airway inflammation in hACE2. Tg mice and Syrian hamster infected with SARS-CoV-2. Combination of Fcn with remdesivir (RDV) or an anti-leprosy drug, Clofazimine, exhibited synergistic antiviral activity. Compared to Fcn, its synthetic derivative, MK-04-003, more effectively inhibited SARS-CoV-2 and its variants B.1.617.2 and BA.5 in mice. Taken together these data demonstrate that Fcn is a pan beta coronavirus inhibitor, which possibly can be used to combat novel emerging coronavirus diseases.

Abstract 17015: Mitophagy Facilitates Cytosolic Proteostasis to Preserve Cardiac Function

Introduction: Protein quality control (PQC) is critical for maintaining sarcomere structure and function in cardiac myocytes. Mutations in PQC pathway proteins, such as CRYAB (arginine to glycine at position 120, R120G) and BAG3 (proline to lysine at position 209, P209L), induce protein aggregates with cardiomyopathy in humans. Novel observations in yeast and mammalian cells demonstrate mitochondrial uptake of cytosolic protein aggregates. The functional relevance of this pathway in myocardial proteostasis is unknown. Hypothesis: We hypothesize that mitophagy removes cytosolic protein aggregates in cardiac myocytes. Methods: Mice with inducible cardiac myocyte specific ablation of TRAF2 (TRAF2icKO), which impairs mitophagy, were assayed for protein aggregates with biochemical fractionation and super-resolution imaging in comparison to floxed controls. Transgenic mice expressing R120G CRYAB (R120G TG) were subjected to both TRAF2 gain-of-function (with AAV9-cardiac Troponin T promoter-driven TRAF2 transduction) and TRAF2 loss-of-function (with tamoxifen-inducible ablation of one Traf2 allele) in cardiac myocytes to determine the effect of mitophagy modulation on cardiac structure, function, and protein aggregate pathology. Results: Poly-ubiquitinated protein-rich aggregates accumulate in the cytosol and within mitochondria with desmin mislocalization in TRAF2icKO myocardium. CRYAB R120G and BAG3 P209L mutant proteins are taken up by isolated mitochondria. CRYAB R120G protein aggregates co-localize with damaged mitochondria and lysosomes by Coupa imaging, and accumulate in the mitochondrial fraction in R120G TG myocardium with upregulation of TRAF2 and induction of mitophagy. Adult-onset haplo-insufficiency of Traf2 accelerated mortality (P=0.003), impaired left ventricular (LV) fractional shortening (P<0.001) and worsened mitochondrial morphology in R120G TG mice. AAV9-cTnT-TRAF2 transduction delayed mortality (P=0.027), rescued LV fractional shortening (P=0.039), reduced mitochondrial protein aggregates and improved desmin localization in R120G TG mice. Conclusions: Mitophagy facilitates removal of cytosolic protein aggregates and can be stimulated to ameliorate proteotoxic cardiomyopathy.

Exploratory Study of Gastrointestinal Redox Biomarkers in the Presymptomatic and Symptomatic Tg2576 Mouse Model of Familial Alzheimer's Disease: Phenotypic Correlates and Effects of Chronic Oral d-Galactose.

The gut might play an important role in the etiopathogenesis of Alzheimer's disease (AD) as gastrointestinal alterations often precede the development of neuropathological changes in the brain and correlate with disease progression in animal models. The gut has an immense capacity to generate free radicals whose role in the etiopathogenesis of AD is well-known; however, it remains to be clarified whether gastrointestinal redox homeostasis is associated with the development of AD. The aim was to (i) examine gastrointestinal redox homeostasis in the presymptomatic and symptomatic Tg2576 mouse model of AD; (ii) investigate the effects of oral d-galactose previously shown to alleviate cognitive deficits and metabolic changes in animal models of AD and reduce gastrointestinal oxidative stress; and (iii) investigate the association between gastrointestinal redox biomarkers and behavioral alterations in Tg2576 mice. In the presymptomatic stage, Tg2576 mice displayed an increased gastrointestinal electrophilic tone, characterized by higher lipid peroxidation and elevated Mn/Fe-SOD activity. In the symptomatic stage, these alterations are rectified, but the total antioxidant capacity is decreased. Chronic oral d-galactose increased the antioxidant capacity and reduced lipid peroxidation in the Tg2576 but had the opposite effects in the wild-type animals. The total antioxidant capacity of the gastrointestinal tract was associated with greater spatial memory. Gut redox homeostasis might be involved in the development and progression of AD pathophysiology and should be further explored in this context.

HIV-1 gp120-Induced Cognitive Disorder Is Concomitant with Altered Gut Microbiota

Background HIV-associated neurocognitive disorders (HAND) is an essential factor affecting the quality of life of people living with HIV (PLWH), and the prevalence of HAND in PLWH can reach 19% to 64%. More than 50% of HAND patients treated with highly active antiretroviral therapy (HAART) did not show improvement in symptoms of cognitive impairment. HIV-1 envelope protein gp120 may be a major contributor to HAND pathology. HIV-1 gp120 enters neurons by binding CXCR4 and CCR5 and can also bind neuronal tubulin to cause direct neurotoxicity and neuronal death. HIV-1 gp120 transgenic mice (Tgm) are commonly used as HAND animal models, and Tgm brain pathological changes and differential gene expression are highly consistent with HAND patients. Intestinal flora affects the progression of diseases by regulatory the intestinal barrier, and several studies have confirmed that the normalization of abnormal intestinal flora reduces the damage to the nervous system in a variety of neurodegenerative diseases. Studies reported that Akkermansia muciniphila has a downward trend in PLWH, which may cause intestinal barrier damage. There is still a lack of relevant evidence to confirm the association between abnormal changes in gut microbiota and HAND induced by HIV-1 gp120. Therefore, in this study, we examined the gut microbiota changes in 6, 9, and 12 months Tgm to explore the relationship between age-dependent significantly different bacterial genera, intestinal barrier and neuronal damage. Methods The fresh feces were collected from HIV-1 gp120 transgenic mice (Tgm) and C57BL/6J mice (wild type, WT) at ages 6, 9, and 12 months using perianal stimulation at the same time (n=5). Total bacterial DNA was extracted from each fecal sample and the V3-V4 region of 16S rRNA was sequenced by the Illumina MiSeq platform. 12 months Tg and WT mice were sacrificed, and serum was collected to detect lipopolysaccharide (LPS) concentration by ELISA, and brain tissue of mice in each group was collected for Map2 immunohistochemical staining (n=5). SPSS 20.0 software was used for statistical analysis. P <0.05 were considered statistically significant. Results 16S rRNA analysis showed that increased age was significantly associated with increased abundance of Blautia ( P <0.01) and Prevotellaceae_NK3B31_group ( P <0.0001) genus and decreased abundance of Akkermansia genus ( P <0.05) in Tgm. Notably, the increasing age of WT was associated with the opposite trend of Blautia, Prevotellaceae_NK3B31_group and Akkermansia genus abundance, indicating those genus changes with age and gp120 induced. To further explore the effect of gut microbiota on the gut barrier, serum LPS concentrations were detected for 12 months Tgm and WT. The results showed that serum LPS level was significantly higher in Tgm than in WT ( P <0.01), indicating that the intestinal barrier may be impaired in Tgm. Finally, we collected 12 months Tgm and WT brain tissues for Map2 immunohistochemical staining to evaluate neuronal synaptic length and integrity. Compared with WT, the expression of map2 in the hippocampus and cerebral cortex was significantly decreased ( P <0.05), indicating neuronal damage in Tgm. Conclusion At the genus level, the abundance of Akkermansia was negatively correlated and the Blautia and Prevotellaceae_NK3B31_group abundance was positively correlated with increasing age in Tgm but showed an opposite trend in WT. The serum LPS level increased dramatically in the 12 months Tgm, indicating Tgm with intestinal barrier damage. Map2 expression was reduced considerably hippocampus and cortex in Tgm brain, which means neuronal synaptic integrity was impaired. ( Acknowledgements: National Natural Science Foundation of China, No. 82172259 to H.C.; Corresponding author: Hong Cao, gzhcao@smu.edu.cn.)

Late B Cell-Specific Dis3-Knockout Mice Do Not Develop Plasma Cell Neoplasm

Recent advances in next-generation sequencing have unveiled genetic abnormalities associated with multiple myeloma (MM). Of note, DIS3 mutations have been observed in ~10% of MM patients, and 13q deletion including the DIS3 gene locus are present in ~40% of MM patients. Furthermore, DIS3 mutations/deletions have been shown to be associated with poorer prognosis in MM. Regardless of high incidence of DIS3 mutations/deletions and their relation to adverse outcome, the roles of DIS3 in hematopoiesis and myelomagenesis remains incompletely understood. Here we show that Dis3 is required for normal hematopoiesis and Dis3 deficiency is not sufficient for the development of plasma cell neoplasm in mice. We first explored DIS3 functions in hematopoiesis by generating Dis3 conditional knockout (KO) mice (Dis3 fl/fl;Mx-Cre). To avoid possible effects of Dis3 deficiency on nonhematopoietic cells, we transplanted bone marrow (BM) cells from Dis3 fl/fl;Mx-Cre mice into lethally irradiated CD45.1 + recipient mice. Four weeks after transplantation, we knocked out Dis3 by inducing Cre recombinase via an intraperitoneal injection of Poly(I:C). The deletion of Dis3 in BM cells was confirmed by genotyping and quantitative real time PCR. Importantly, loss of Dis3 in hematopoietic cells resulted in severe pancytopenia at 2 weeks after Dis3 deletion. Consistent with this observation, Dis3 KO mice showed reduced BM counts with relative preservation of mature lymphocytes. A flow cytometric analysis of the BM exhibited lower frequencies of Lin -Sca-2 +c-Kit + (LSK), hematopoietic stem, and multipotent progenitor cells in Dis3 KO mice compared with wild-type (WT) mice. Dis3 KO mice also exhibited lower frequencies of common myeloid progenitors, granulocyte/macrophage progenitors, and megakaryocyte/erythroid progenitors compared with WT mice. A frequency of common lymphoid progenitors (CLPs) was comparable between WT and Dis3 KO mice, whereas absolute numbers of CLPs were significantly reduced in Dis3 KO mice than in WT mice. These results indicate that Dis3 is indispensable for normal hematopoiesis, and LSK and myeloid progenitor cells are more dependent on Dis3 than lymphoid progenitor cells. We next examined whether loss-of-function of Dis3 drives plasma cell neoplasm. To do so, we crossed Dis3 fl/wt or Dis3 fl/fl mice with Cgamma1-Cre mice and generated late B cell-specific Dis3 KO mice by activating Cre recombinase via immunizing with NP-CGG in these compound mice. However, these mice did not develop any B cell malignancies and shorten the survival compared with WT mice. In the samples of MM patients, the frequency of DIS3 mutations is significantly higher in t(14;16) samples than in those without t(14;16) (Walker et al. Blood 2018). We therefore utilized t(14;16) model mice (c-Maf TG) (Morito et al. Cancer Res 2011) and generated c-Maf TG; Dis3 fl/wt;Cgamma1-Cre and c-Maf TG; Dis3 flfl;Cgamma1-Cre mice. As reported (Morito et al. Cancer Res 2011), a small fraction of mice with c-Maf TG developed B cell lymphoma after a long latency. However, the addition of Dis3 KO did not increase the frequency of B cell lymphoma and shorten the survival compared with c-MAF TG mice. These results indicate that only Dis3 deficiency and the combination of c-Maf overexpression and Dis3 deficiency do not develop plasma cell neoplasm and suggest that additional oncogenic events are necessary for myelomagenesis. Further investigations are required for elucidating the mechanisms of how loss-of-function of DIS3 is involved in the development of MM.

Mobility gene expression differences among wild-type, Mmp20 null and Mmp20 over-expresser mice plus visualization of 3D mouse ameloblast directional movement

Enamel forming ameloblasts move away from the dentino-enamel junction and also move relative to each other to establish enamel shape during the secretory stage of enamel development. Matrix metalloproteinase-20 (MMP20) is a tooth specific proteinase essential for proper enamel formation. We previously reported that MMP20 cleaves cadherins and may regulate ameloblast movement. Here, we used an Amelx promoter driven tdTomato reporter to label mouse ameloblasts. With these transgenic mice, we assessed ameloblast mobility group dynamics and gene expression. Three-dimensional imaging of mouse ameloblasts were observed in hemi-mandibles by using a tissue clearing technique. The three-dimensional ameloblast layer in Tg(Amelx-Mmp20) mice that overexpress MMP20 was uneven and the ameloblasts migrated away from this layer. Mouse ameloblast movement toward incisal tips was monitored by ex vivo time-lapse imaging. Gene expression related to cell migration and adhesion was analyzed in ameloblasts from wild-type mice, Mmp20−/− mice with no functional MMP20 and from Tg(Amelx-Mmp20) overexpressing mice. Gene expression was altered in Mmp20−/− and Tg(Amelx-Mmp20) mice compared to wild type. Among the genes assessed, those encoding laminins and a gap junction protein were upregulated in Mmp20−/− mice. New techniques and findings described in this study may lead to an improved understanding of ameloblast movement during enamel formation.

Alcoholic Hericium erinaceus mycelia extract induces SIRT-1/ERK-1/2 mediated autophagy to prevent accumulation and spreading of alpha-synuclein and amyloid-beta peptide on the A53T transgenic mice

Alpha-synuclein (α-syn) and amyloid-beta peptide (Aβ) accumulation cause neurodegenerative diseases. The accumulation of neurotoxins, like Aβ, tau, and α-syn is related to aging therefore studying aging mediated neurodegenerative disease is important. In this study, we used A53T tg mice in this mice behavioral and motor abnormalities could be promoted by Aβ and α-syn interaction, and we decided to prevent this interaction and their associated accumulation in the SN brain region in the A53T tg mice by Hericium erinaceus treatment. Three-month-old A53T mice were separated into three groups A53T, A53T+ low dose alcohol extracted mycelium (3.2 mg/30 g BW/day), and A53T+ high dose alcohol extracted mycelium (6.4 mg/30 g BW/day). Age-matched normal mice were used as a control. Our results confirmed that Hericium erinaceus mycelia extract prevented the formation of Aβ-mediated α-syn accumulation in the SN brain region of A53T tg mice by activating SIRT-1/ERK-1/2 mediated autophagy. We confirmed that SIRT-1/ERK-1/2 mediated autophagy via Hericium erinaceus treatment prevents Aβ and α-syn accumulation and regulates dopamine neuron survival. Our study makes significant contribution because we reported for the first time that Hericium erinaceus mycelia prevents the formation and accumulation of Aβ and α-syn in A53T tg mice by regulating SIRT-1/ERK-1/2 mediated autophagy.

Temporal dynamics of muscle mitochondrial uncoupling-induced integrated stress response and ferroptosis defense.

Mitochondria play multifaceted roles in cellular function, and impairments across domains of mitochondrial biology are known to promote cellular integrated stress response (ISR) pathways as well as systemic metabolic adaptations. However, the temporal dynamics of specific mitochondrial ISR related to physiological variations in tissue-specific energy demands remains unknown. Here, we conducted a comprehensive 24-hour muscle and plasma profiling of male and female mice with ectopic mitochondrial respiratory uncoupling in skeletal muscle (mUcp1-transgenic, TG). TG mice are characterized by increased muscle ISR, elevated oxidative stress defense, and increased secretion of FGF21 and GDF15 as ISR-induced myokines. We observed a temporal signature of both cell-autonomous and systemic ISR in the context of endocrine myokine signaling and cellular redox balance, but not of ferroptotic signature which was also increased in TG muscle. We show a progressive increase of muscle ISR on transcriptional level during the active phase (night time), with a subsequent peak in circulating FGF21 and GDF15 in the early resting phase. Moreover, we found highest levels of muscle oxidative defense (GPX and NQO1 activity) between the late active to early resting phase, which could aim to counteract excessive iron-dependent lipid peroxidation and ferroptosis in muscle of TG mice. These findings highlight the temporal dynamics of cell-autonomous and endocrine ISR signaling under skeletal muscle mitochondrial uncoupling, emphasizing the importance of considering such dissociation in translational strategies and sample collection for diagnostic biomarker analysis.

Leucine-rich repeat kinase 2 promotes the development of experimental severe acute pancreatitis.

Translocation of gut bacteria into the pancreas promotes the development of severe acute pancreatitis (SAP). Recent clinical studies have also highlighted the association between fungal infections and SAP. The sensing of gut bacteria by pattern recognition receptors promotes the development of SAP via the production of proinflammatory cytokines; however, the mechanism by which gut fungi mediate SAP remains largely unknown. Leucine-rich repeat kinase 2 (LRRK2) is a multifunctional protein that regulates innate immunity against fungi via Dectin-1 activation. Here, we investigated the role of LRRK2 in SAP development and observed that administration of LRRK2 inhibitors attenuated SAP development. The degree of SAP was greater in Lrrk2 transgenic (Tg) mice than in control mice and was accompanied by an increased production of nuclear factor-kappaB-dependent proinflammatory cytokines. Ablation of the fungal mycobiome by anti-fungal drugs inhibited SAP development in Lrrk2 Tg mice, whereas the degree of SAP was comparable in Lrrk2 Tg mice with or without gut sterilization by a broad range of antibiotics. Pancreatic mononuclear cells from Lrrk2 Tg mice produced large amounts of IL-6 and TNF-α upon stimulation with Dectin-1 ligands, and inhibition of the Dectin-1 pathway by a spleen tyrosine kinase inhibitor protected Lrrk2 Tg mice from SAP. These data indicate that LRRK2 activation is involved in the development of SAP through proinflammatory cytokine responses upon fungal exposure.

Resolvin D1 improves airway inflammation and exercise capacity in cystic fibrosis lung disease.

Mucus plugging and non-resolving inflammation are inherent features of cystic fibrosis (CF) that may lead to progressive lung disease and exercise intolerance, which are the main causes of morbidity and mortality for people with CF. Therefore, understanding the influence of mucus on basic mechanisms underlying the inflammatory response and identifying strategies to resolve mucus-driven airway inflammation and consequent morbidity in CF are of wide interest. Here, we investigated the effects of the proresolving lipid mediator resolvin (Rv) D1 on mucus-related inflammation as a proof-of-concept to alleviate the burden of lung disease and restore exercise intolerance in CF. We tested the effects of RvD1 on inflammatory responses of human organotypic airways and leukocytes to CF mucus and of humanized mice expressing the epithelial Na + channel (βENaC-Tg) having CF-like mucus obstruction, lung disease, and physical exercise intolerance. RvD1 reduced pathogenic phenotypes of CF-airway supernatant (ASN)-stimulated human neutrophils, including loss of L-selectin shedding and CD16. RNASeq analysis identified select transcripts and pathways regulated by RvD1 in ASN-stimulated CF bronchial epithelial cells that are involved in sugar metabolism, NF-κB activation and inflammation, and response to stress. In in vivo inflammation using βENaC TG mice, RvD1 reduced total leukocytes, PMN, and interstitial Siglec-MΦ when given at 6-8 weeks of age, and in older mice at 10-12 weeks of age, along with the decrease of pro-inflammatory chemokines and increase of anti-inflammatory IL-10. Furthermore, RvD1 treatment promoted the resolution of pulmonary exacerbation caused by Pseudomonas aeruginosa infection and significantly enhanced physical activity and energy expenditure associated with mucus obstruction, which was impaired in βENaC-Tg mice compared with wild-type. These results demonstrate that RvD1 can rectify features of CF and offer proof-of-concept for its therapeutic application in this and other muco-obstructive lung diseases.