An inducible hACE2 transgenic mouse model recapitulates SARS-CoV-2 infection and pathogenesis in vivo.

The classical manifestation of COVID-19 is pulmonary infection. After host cell entry via human angiotensin-converting enzyme II (hACE2), the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus can infect pulmonary epithelial cells, especially the AT2 (alveolar type II) cells that are crucial for maintaining normal lung function. However, previous hACE2 transgenic models have failed to specifically and efficiently target the cell types that express hACE2 in humans, especially AT2 cells. In this study, we report an inducible, transgenic hACE2 mouse line and showcase three examples for specifically expressing hACE2 in three different lung epithelial cells, including AT2 cells, club cells, and ciliated cells. Moreover, all these mice models develop severe pneumonia after SARS-CoV-2 infection. This study demonstrates that the hACE2 model can be used to precisely study any cell type of interest with regard to COVID-19-related pathologies.

Speeding up Glioblastoma Cancer Research: Highlighting the Zebrafish Xenograft Model.

Glioblastoma multiforme (GBM) is a very aggressive and lethal primary brain cancer in adults. The multifaceted nature of GBM pathogenesis, rising from complex interactions between cells and the tumor microenvironment (TME), has posed great treatment challenges. Despite significant scientific efforts, the prognosis for GBM remains very poor, even after intensive treatment with surgery, radiation, and chemotherapy. Efficient GBM management still requires the invention of innovative treatment strategies. There is a strong necessity to complete cancer in vitro studies and in vivo studies to properly evaluate the mechanisms of tumor progression within the complex TME. In recent years, the animal models used to study GBM tumors have evolved, achieving highly invasive GBM models able to provide key information on the molecular mechanisms of GBM onset. At present, the most commonly used animal models in GBM research are represented by mammalian models, such as mouse and canine ones. However, the latter present several limitations, such as high cost and time-consuming management, making them inappropriate for large-scale anticancer drug evaluation. In recent years, the zebrafish (Danio rerio) model has emerged as a valuable tool for studying GBM. It has shown great promise in preclinical studies due to numerous advantages, such as its small size, its ability to generate a large cohort of genetically identical offspring, and its rapid development, permitting more time- and cost-effective management and high-throughput drug screening when compared to mammalian models. Moreover, due to its transparent nature in early developmental stages and genetic and anatomical similarities with humans, it allows for translatable brain cancer research and related genetic screening and drug discovery. For this reason, the aim of the present review is to highlight the potential of relevant transgenic and xenograft zebrafish models and to compare them to the traditionally used animal models in GBM research.

Noncoupled Mitochondrial Respiration as Therapeutic Approach for the Treatment of Metabolic Diseases: Focus on Transgenic Animal Models.

Mitochondrial dysfunction contributes to numerous chronic diseases, and mitochondria are targets for various toxins and xenobiotics. Therefore, the development of drugs or therapeutic strategies targeting mitochondria is an important task in modern medicine. It is well known that the primary, although not the sole, function of mitochondria is ATP generation, which is achieved by coupled respiration. However, a high membrane potential can lead to uncontrolled reactive oxygen species (ROS) production and associated dysfunction. For over 50 years, scientists have been studying various synthetic uncouplers, and for more than 30 years, uncoupling proteins that are responsible for uncoupled respiration in mitochondria. Additionally, the proteins of the mitochondrial alternative respiratory pathway exist in plant mitochondria, allowing noncoupled respiration, in which electron flow is not associated with membrane potential formation. Over the past two decades, advances in genetic engineering have facilitated the creation of various cellular and animal models that simulate the effects of uncoupled and noncoupled respiration in different tissues under various disease conditions. In this review, we summarize and discuss the findings obtained from these transgenic models. We focus on the advantages and limitations of transgenic organisms, the observed physiological and biochemical changes, and the therapeutic potential of uncoupled and noncoupled respiration.

A murine Skint6 W168X allele contributes to autoimmune disease in a transgenic model.

BACKGROUND: The genetic factor is a great driver of systemic lupus erythematosus. A Skint6 W168X allele was previously identified in the murine lupus susceptibility rec1d1 sublocus. The purpose of this study is to investigate the pathogenic role and mechanism of the Skint6 W168X allele in lupus autoimmune disease. METHODS: The gene-editing CRISPR/Cas9 system was used to generate transgenic models with the Skint6 W168X allele. PCR and Sanger's sequencing techniques were applied to mRNA quantification and DNA sequence detection. Flow cytometry was adopted for immunophenotyping. Pathological evaluation of kidneys and lungs was performed using several immunopathological approaches. RESULTS: The transgenic models with the Skint6 W168X allele were created, including B6.Skint6X/X and B6.lpr.Skint6X/X strains. The B6.lpr.Skint6X/X mice showed bigger spleen and lymph nodes, more lymphocytes and effector T cell populations, more severe nephritis with more IgG and C3 deposit in glomeruli as well as worse proteinuria, and more severe lung inflammation than control B6.lpr mice. In addition, a skint6 receptor binding Skint6 peptide was identified from T and B lymphocytes. B6.Skint6X/X mice have lower percentages of skint6 receptor+ T and B cells in spleen than B6 mice. CONCLUSION: The Skint6 W168X allele in murine lupus rec1d1 sublocus was validated to be a pathogenic mutant gene and contributes to autoimmune disease through producing a truncated Skint6 peptide of binding the skint6 receptors on lymphocytes.

Identification of Impacted Pathways and Transcriptomic Markers as Potential Mediators of Pulmonary Fibrosis in Transgenic Mice Expressing Human IGFBP5.

Pulmonary fibrosis is a serious disease characterized by extracellular matrix (ECM) component overproduction and remodeling. Insulin-like growth factor-binding protein 5 (IGFBP5) is a conserved member of the IGFBP family of proteins that is overexpressed in fibrotic tissues and promotes fibrosis. We used RNA sequencing (RNAseq) to identify differentially expressed genes (DEGs) between primary lung fibroblasts (pFBs) of homozygous (HOMO) transgenic mice expressing human IGFBP5 (hIGFBP5) and wild type mice (WT). The results of the differential expression analysis showed 2819 DEGs in hIGFBP5 pFBs. Functional enrichment analysis confirmed the pro-fibrotic character of IGFBP5 and revealed its impact on fundamental signaling pathways, including cytokine-cytokine receptor interaction, focal adhesion, AGE-RAGE signaling, calcium signaling, and neuroactive ligand-receptor interactions, to name a few. Noticeably, 7% of the DEGs in hIGFBP5-expressing pFBs are receptors and integrins. Furthermore, hub gene analysis revealed 12 hub genes including Fpr1, Bdkrb2, Mchr1, Nmur1, Cnr2, P2ry14, and Ptger3. Validation assays were performed to complement the RNAseq data. They confirmed significant differences in the levels of the corresponding proteins in cultured pFBs. Our study provides new insights into the molecular mechanism(s) of IGFBP5-associated pulmonary fibrosis through possible receptor interactions that drive fibrosis and tissue remodeling.

Apnea Associated with Brainstem Seizures in Cacna1aS218L Mice Is Caused by Medullary Spreading Depolarization.

Seizure-related apnea is common and can be lethal. Its mechanisms however remain unclear and preventive strategies are lacking. We postulate that brainstem spreading depolarization (SD), previously associated with lethal seizures in animal models, initiates apnea upon invasion of brainstem respiratory centers. To study this, we assessed effects of brainstem seizures on brainstem function and respiration in male and female mice carrying a homozygous S218L missense mutation that leads to gain-of-function of voltage-gated CaV2.1 Ca2+ channels and high risk for fatal seizures. Recordings of brainstem DC potential and neuronal activity, cardiorespiratory activity and local tissue oxygen were performed in freely behaving animals. Brainstem SD occurred during all spontaneous fatal seizures and, unexpectedly, during a subset of nonfatal seizures. Seizure-related SDs in the ventrolateral medulla correlated with respiratory suppression. Seizures induced by stimulation of the inferior colliculus could evoke SD that spread in a rostrocaudal direction, preceding local tissue hypoxia and apnea, indicating that invasion of SD into medullary respiratory centers initiated apnea and hypoxia rather than vice versa Fatal outcome was prevented by timely resuscitation. Moreover, NMDA receptor antagonists MK-801 and memantine prevented seizure-related SD and apnea, which supports brainstem SD as a prerequisite for brainstem seizure-related apnea in this animal model and has translational value for developing strategies that prevent fatal ictal apnea.SIGNIFICANCE STATEMENT Apnea during and following seizures is common, but also likely implicated in sudden unexpected death in epilepsy (SUDEP). This underlines the need to understand mechanisms for potentially lethal seizure-related apnea. In the present work we show, in freely behaving SUDEP-prone transgenic mice, that apnea is induced when spontaneous brainstem seizure-related spreading depolarization (SD) reaches respiratory nuclei in the ventrolateral medulla. We show that brainstem seizure-related medullary SD is followed by local hypoxia and recovers during nonfatal seizures, but not during fatal events. NMDA receptor antagonists prevented medullary SD and apnea, which may be of translational value.

A cardiomyocyte show of force: A fluorescent alpha-actinin reporter line sheds light on human cardiomyocyte contractility versus substrate stiffness.

Cardiovascular disease is often associated with cardiac remodeling, including cardiac fibrosis, which may lead to increased stiffness of the heart wall. This stiffness in turn may cause subsequent failure of cardiac myocytes, however the response of these cells to increased substrate stiffness is largely unknown. To investigate the contractile response of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) to increased substrate stiffness, we generated a stable transgenic human pluripotent stem cell line expressing a fusion protein of α-Actinin and fluorescent mRubyII in a previously characterized NKX2.5-GFP reporter line. Cardiomyocytes differentiated from this line were subjected to a substrate with stiffness ranging from 4 kPa to 101 kPa, while contraction of sarcomeres and bead displacement in the substrate were measured for each single cardiomyocyte. We found that sarcomere dynamics in hPSC-CMs on polyacrylamide gels of increasing stiffness are not affected above physiological levels (21 kPa), but that contractile force increases up to a stiffness of 90 kPa, at which cell shortening, deducted from bead displacement, is significantly reduced compared to physiological stiffness. We therefore hypothesize that this discrepancy may be the cause of intracellular stress that leads to hypertrophy and consequent heart failure in vivo.

Overexpression of Spock2 in mice leads to altered lung alveolar development and worsens lesions induced by hyperoxia.

SPARC/osteonectin, cwcv and kazal-like domains proteoglycan 2 (SPOCK2) was previously associated with genetic susceptibility to bronchopulmonary dysplasia in a French population of very preterm neonates. Its expression increases during lung development and is increased after exposure of rat pups to hyperoxia compared with controls bred in room air. To further investigate the role of SPOCK2 during lung development, we designed two mouse models, one that uses a specific anti-Spock2 antibody and one that reproduces the hyperoxia-induced Spock2 expression with a transgenic mouse model resulting in a conditional and lung-targeted overexpression of Spock2. When mice were bred under hyperoxic conditions, treatment with anti-Spock2 antibodies significantly improved alveolarization. Lung overexpression of Spock2 altered alveolar development in pups bred in room air and worsened hyperoxia-induced lesions. Neither treatment with anti-Spock2 antibody nor overexpression of Spock2 was associated with abnormal activation of matrix metalloproteinase-2. These two models did not alter the expression of known players in alveolar development. This study brings strong arguments for the deleterious role of SPOCK2 on lung alveolar development especially after lung injury, suggesting its role in bronchopulmonary dysplasia susceptibility. These effects are not mediated by a deregulation in metalloproteases activity and in expression of factors essential to normal alveolarization. The balance between types 1 and 2 epithelial alveolar cells may be involved.

The triphenyltin carboxylate derivative triphenylstannyl 2-(benzylcarbamoyl)benzoate impedes prostate cancer progression via modulation of Akt/FOXO3a signaling.

Prostate cancer (PCa) incidence is surging in United States and other parts of the world. Synthetic and natural compounds have been explored as potential modulators of PI3K/Akt signaling that is known to drive PCa growth. Here, we evaluated the efficacy of a series of triphenyltin (IV) carboxylate derivatives against PCa. From this library, triphenylstannyl 2-(benzylcarbamoyl)benzoate (Ch-319) resulted in reduced viability and induction of cell cycle arrest in PTEN-/- PC3M and PTEN+/- DU145 cells. In parallel, downregulation of PI3K p85/p110 subunits, dephosphorylation of Akt-1 and increase in FOXO3a expression were observed. In silico studies indicated binding interactions of Ch-319 within the ATP binding site of Akt-1 at Met281, Phe442 and Glu234 residues. Elevated po-pulation of apoptotic cells, activation of Bax and reduced Bcl2 expression indicated apoptosis by Ch-319. Pre-sensitization of PCa cells with Ch-319 augmented the effect of cabazitaxel, a commonly used taxane in patients with castration-resistant PCa. Next, in a prostate-specific PTENp-/- mice, Ch-319 showed reduced weights of genitourinary apparatus as compared to DMSO treated controls. Histological studies indicated absence of neoplastic foci in Ch-319 treated prostates. Consistently, dephosphorylation of Akt-1, reduced expression of PRAS40 and androgen receptor and increase in FOXO3a were observed in treated group. Notably, no overt organ toxicity was noted in Ch-319 treated animals. Our studies identify Akt/FOXO3a signaling as a target of triphenyltin (IV) carboxylate Ch-319 and provide a molecular basis of its growth inhibitory effect in PCa cells. We propose that Ch-319 has the potential to be developed as an anticancer agent against PCa.

ß-Catenin haploinsufficiency promotes mammary tumorigenesis in an ErbB2-positive basal breast cancer model.

Aberrant activation of ß-catenin through its activity as a transcription factor has been observed in a large proportion of human malignancies. Despite the improved understanding of the ß-catenin signaling pathway over the past three decades, attempts to develop therapies targeting ß-catenin remain challenging, and none of these targeted therapies have advanced to the clinic. In this study, we show that part of the challenge in antagonizing ß-catenin is caused by its dual functionality as a cell adhesion molecule and a signaling molecule. In a mouse model of basal ErbB2 receptor tyrosine kinase 2 (ErbB2)-positive breast cancer (ErbB2KI), which exhibits aberrant ß-catenin nuclear signaling, ß-catenin haploinsufficiency induced aggressive tumor formation and metastasis by promoting the disruption of adherens junctions, dedifferentiation, and an epithelial to mesenchymal transition (EMT) transcriptional program. In contrast to the accelerated tumor onset observed in the haploid-insufficient ErbB2 tumors, deletion of both ß-catenin alleles in the ErbB2KI model had only a minor impact on tumor onset that further correlated with the retention of normal adherens junctions. We further showed that retention of adherens junctional integrity was caused by the up-regulation of the closely related family member plakoglobin (γ-catenin) that maintained both adherens junctions and the activation of Wnt target genes. In contrast to the ErbB2KI basal tumor model, modulation of ß-catenin levels had no appreciable impact on tumor onset in an ErbB2-driven model of luminal breast cancer [murine mammary tumor virus promoter (MMTV-NIC)]. These observations argue that the balance of junctional and nuclear ß-catenin activity has a profound impact on tumor progression in this basal model of ErbB2-positive breast cancer.

Monoclonal glomerulopathy with features of cryoglobulinemic glomerulopathy in murine multiple myeloma model.

In vivo and animal models of monoclonal light chain-associated renal diseases are limited. The Vk*MYC transgenic model with multiple myeloma in 50-70 weeks old mice with renal involvement has been reported before, but detailed renal pathologic changes have not been well documented. This study fully investigated pathologic changes in the kidneys of Vk*MYC transgenic model using light microscopy, immunofluorescence stains for kappa and lambda light chains, and electron microscopy. Compared to the kidneys of wild-type mice, the kidneys of transgenic mice showed either mesangial segmental expansion, some with associated hypercellularity, and/or thrombotic obstruction of glomerular capillaries. The glomeruli revealed stronger lambda staining than kappa light chain staining. Six out of 12 kidneys from transgenic mice showed abundant electron-dense deposits when examined ultrastructurally. The deposits were located in glomerular capillary lumina in three cases. Large luminal and subendothelial deposits were characterized by randomly disposed microtubular structures measuring up to 16 nm in diameter, with overall features most consistent with cryoglobulins. In summary, about 50% of kidneys from the Vk*MYC mice with myeloma had features most consistent with monoclonal cryoglobulinemic glomerulopathy.

Phenotypic Characterization of Transgenic Mice Expressing Human IGFBP-5.

Pulmonary fibrosis is one of the important causes of morbidity and mortality in fibroproliferative disorders such as systemic sclerosis (SSc) and idiopathic pulmonary fibrosis (IPF). Insulin-like growth factor binding protein-5 (IGFBP-5) is a conserved member of the IGFBP family of proteins that is overexpressed in SSc and IPF lung tissues. In this study, we investigated the functional role of IGFBP-5 in the development of fibrosis in vivo using a transgenic model. We generated transgenic mice ubiquitously expressing human IGFBP-5 using CRISPR/Cas9 knock-in. Our data show that the heterozygous and homozygous mice are viable and express human IGFBP-5 (hIGFBP-5). Transgenic mice had increased expression of extracellular matrix (ECM) genes, especially Col3a1, Fn, and Lox in lung and skin tissues of mice expressing higher transgene levels. Histologic analysis of the skin tissues showed increased dermal thickness, and the lung histology showed subtle changes in the heterozygous and homozygous mice as compared with the wild-type mice. These changes were more pronounced in animals expressing higher levels of hIGFBP-5. Bleomycin increased ECM gene expression in wild-type mice and accentuated an increase in ECM gene expression in transgenic mice, suggesting that transgene expression exacerbated bleomycin-induced pulmonary fibrosis. Primary lung fibroblasts cultured from lung tissues of homozygous transgenic mice showed significant increases in ECM gene expression and protein levels, further supporting the observation that IGFBP-5 resulted in a fibrotic phenotype in fibroblasts. In summary, transgenic mice expressing human IGFBP-5 could serve as a useful animal model for examining the function of IGFBP-5 in vivo.

The Effect of Ionizing Radiation on Cognitive Functions in Mouse Models of Alzheimer's Disease.

The present study demonstrates the effect of combined ionizing radiation (γ rays, 0.24 Gy, 661.7 keV, whole body and 12C, 0.18 Gy, 450 MeV, head region) on the behavior of animals in mouse transgenic models of Alzheimer's disease. Significant improvement of spatial learning and stimulation of locomotor and exploratory behavior were observed in wild-type mice after irradiation. However, an anxiolytic effect and stimulation of locomotor and exploratory behavior were revealed in irradiated mice with tauopathy. Mice with cerebral amyloidosis also exhibited improved learning in the odor recognition test. No negative effects of irradiation were detected.

GFP-Mutant Human Tau Transgenic Mice Develop Tauopathy Following CNS Injections of Alzheimer's Brain-Derived Pathological Tau or Synthetic Mutant Human Tau Fibrils.

Neurodegenerative proteinopathies characterized by intracellular aggregates of tau proteins, termed tauopathies, include Alzheimer's disease (AD), frontotemporal lobar degeneration (FTLD) with tau pathology (FTLD-tau), and related disorders. Pathological tau proteins derived from human AD brains (AD-tau) act as proteopathic seeds that initiate the templated aggregation of soluble tau upon intracerebral injection into tau transgenic (Tg) and wild-type mice, thereby modeling human tau pathology. In this study, we found that aged Tg mice of both sexes expressing human tau proteins harboring a pathogenic P301L MAPT mutation labeled with green fluorescent protein (T40PL-GFP Tg mouse line) exhibited hyperphosphorylated tau mislocalized to the somatodentritic domain of neurons, but these mice did not develop de novo insoluble tau aggregates, which are characteristic of human AD and related tauopathies. However, intracerebral injections of either T40PL preformed fibrils (PFFs) or AD-tau seeds into T40PL-GFP mice induced abundant intraneuronal pathological inclusions of hyperphosphorylated T40PL-GFP. These injections of pathological tau resulted in the propagation of tau pathology from the injection site to neuroanatomically connected brain regions, and these tau inclusions consisted of both T40PL-GFP and WT endogenous mouse tau. Primary neurons cultured from the brains of neonatal T40PL-GFP mice provided an informative in vitro model for examining the uptake and localization of tau PFFs. These findings demonstrate the seeded aggregation of T40PL-GFP in vivo by synthetic PFFs and human AD-tau and the utility of this system to study the neuropathological spread of tau aggregates.SIGNIFICANCE STATEMENT The stereotypical spread of pathological tau protein aggregates have recently been attributed to the transmission of proteopathic seeds. Despite the extensive use of transgenic mouse models to investigate the propagation of tau pathology in vivo, details of the aggregation process such as the early seeding events leading to new tau pathology have remained elusive. This study validates the use of GFP-labeled tau expressed by neurons in vivo and in vitro as models for investigating mechanisms underlying the seeded transmission of tau pathology as well as tau-focused drug discovery to identify disease-modifying therapies for AD and related tauopathies.

Aromatase/Seladin-1 Interactions in Human Neuronal Cell Culture, the Hippocampus of Healthy Rats and Transgenic Alzheimer's Disease Mice.

BACKGROUND/AIMS: Decreasing levels of aromatase and seladin-1 could be one of the molecular mechanisms of Alzheimer's disease (AD). Aromatase is an enzyme that catalyzes estrogen biosynthesis from androgen precursors, and seladin-1 is an enzyme that converts desmosterol to cholesterol, which is the precursor of all hormones. Verifying the potential relationship between these proteins and accordingly determining new therapeutic targets constitute the aims of this study. METHODS: Changes in protein levels were compared in vitro in aromatase and seladin-1 inhibitor-administered human neuroblastoma (SH-SY5Y) cells in vivo in intracerebroventricular (icv) aromatase or seladin-1 inhibitor-administered rats, as well as in transgenic AD mice in which the genes encoding these proteins were knocked out. RESULTS AND CONCLUSIONS: In the cell cultures, we observed that seladin-1 protein levels increased after aromatase enzyme inhibition. The hippocampal aromatase protein levels decreased following chronic seladin-1 inhibition in icv inhibitor-administered rats; however, the aromatase levels in the dentate gyrus of seladin-1 knockout (SelKO) AD male mice increased. These findings indicate a partial relationship between these proteins and their roles in AD pathology.

Glutaminase C overexpression in the brain induces learning deficits, synaptic dysfunctions, and neuroinflammation in mice.

Glutaminolysis, a metabolic process that converts glutamine to glutamate, is particularly important for the central nervous system since glutamate is the major transmitter of excitatory synapses. Glutaminase is the mitochondrial enzyme that catalyzes the first step of glutaminolysis. Two genes encode at least four isoforms of glutaminase in humans. Gls1 gene encodes isoforms kidney-type glutaminase (KGA) and glutaminase C (GAC) through alternative splicing, whereas Gls2 gene encodes liver-type glutaminase isoforms. KGA and GAC have been associated with several neurological diseases. However, it remains unclear whether changes in their expressions can directly cause brain abnormalities. Using a transgenic approach, we generated mice that overexpressed GAC in the brain. The resulting transgenic mice had severe impairments in spatial and fear learning compared with littermate controls. The learning deficits were consistent with diminished hippocampal long-term potentiation in the hippocampal slices of the GAC transgenic mice. Furthermore, we found increases in astrocyte and microglia markers, inflammatory factors, and a decrease in synapse marker synaptophysin, suggesting neuroinflammation and synaptic changes in the GAC transgenic mouse brains. In conclusion, these findings provide the first evidence that GAC overexpression in the brain has deleterious effects on learning and synaptic integrity in vivo.

Neuregulin-1 controls an endogenous repair mechanism after spinal cord injury.

Following traumatic spinal cord injury, acute demyelination of spinal axons is followed by a period of spontaneous remyelination. However, this endogenous repair response is suboptimal and may account for the persistently compromised function of surviving axons. Spontaneous remyelination is largely mediated by Schwann cells, where demyelinated central axons, particularly in the dorsal columns, become associated with peripheral myelin. The molecular control, functional role and origin of these central remyelinating Schwann cells is currently unknown. The growth factor neuregulin-1 (Nrg1, encoded by NRG1) is a key signalling factor controlling myelination in the peripheral nervous system, via signalling through ErbB tyrosine kinase receptors. Here we examined whether Nrg1 is required for Schwann cell-mediated remyelination of central dorsal column axons and whether Nrg1 ablation influences the degree of spontaneous remyelination and functional recovery following spinal cord injury. In contused adult mice with conditional ablation of Nrg1, we found an absence of Schwann cells within the spinal cord and profound demyelination of dorsal column axons. There was no compensatory increase in oligodendrocyte remyelination. Removal of peripheral input to the spinal cord and proliferation studies demonstrated that the majority of remyelinating Schwann cells originated within the injured spinal cord. We also examined the role of specific Nrg1 isoforms, using mutant mice in which only the immunoglobulin-containing isoforms of Nrg1 (types I and II) were conditionally ablated, leaving the type III Nrg1 intact. We found that the immunoglobulin Nrg1 isoforms were dispensable for Schwann cell-mediated remyelination of central axons after spinal cord injury. When functional effects were examined, both global Nrg1 and immunoglobulin-specific Nrg1 mutants demonstrated reduced spontaneous locomotor recovery compared to injured controls, although global Nrg1 mutants were more impaired in tests requiring co-ordination, balance and proprioception. Furthermore, electrophysiological assessments revealed severely impaired axonal conduction in the dorsal columns of global Nrg1 mutants (where Schwann cell-mediated remyelination is prevented), but not immunoglobulin-specific mutants (where Schwann cell-mediated remyelination remains intact), providing robust evidence that the profound demyelinating phenotype observed in the dorsal columns of Nrg1 mutant mice is related to conduction failure. Our data provide novel mechanistic insight into endogenous regenerative processes after spinal cord injury, demonstrating that Nrg1 signalling regulates central axon remyelination and functional repair and drives the trans-differentiation of central precursor cells into peripheral nervous system-like Schwann cells that remyelinate spinal axons after injury. Manipulation of the Nrg1 system could therefore be exploited to enhance spontaneous repair after spinal cord injury and other central nervous system disorders with a demyelinating pathology.media-1vid110.1093/brain/aww039_video_abstractaww039_video_abstract.

A53T human α-synuclein overexpression in transgenic mice induces pervasive mitochondria macroautophagy defects preceding dopamine neuron degeneration.

In vitro evidence suggests that the inefficient removal of damaged mitochondria by macroautophagy contributes to Parkinson's disease (PD). Using a tissue-specific gene amplification strategy, we generated a transgenic mouse line with human α-synuclein A53T overexpression specifically in dopamine (DA) neurons. Transgenic mice showed profound early-onset mitochondria abnormalities, characterized by macroautophagy marker-positive cytoplasmic inclusions containing mainly mitochondrial remnants, which preceded the degeneration of DA neurons. Genetic deletion of either parkin or PINK1 in these transgenic mice significantly worsened mitochondrial pathologies, including drastically enlarged inclusions and loss of total mitochondria contents. These data suggest that mitochondria are the main targets of α-synuclein and their defective autophagic clearance plays a significant role during pathogenesis. Moreover, endogenous PINK1 or parkin is indispensable for the proper autophagic removal of damaged mitochondria. Our data for the first time establish an essential link between mitochondria macroautophagy impairments and DA neuron degeneration in an in vivo model based on known PD genetics. The model, its well-defined pathologies, and the demonstration of a main pathogenesis pathway in the present study have set the stage and direction of emphasis for future studies.

Spaceflight impairs antigen-specific tolerance induction in vivo and increases inflammatory cytokines.

The health risks of a dysregulated immune response during spaceflight are important to understand as plans emerge for humans to embark on long-term space travel to Mars. In this first-of-its-kind study, we used adoptive transfer of T-cell receptor transgenic OT-II CD4 T cells to track an in vivo antigen-specific immune response that was induced during the course of spaceflight. Experimental mice destined for spaceflight and mice that remained on the ground received transferred OT-II cells and cognate peptide stimulation with ovalbumin (OVA) 323-339 plus the inflammatory adjuvant, monophosphoryl lipid A. Control mice in both flight and ground cohorts received monophosphoryl lipid A alone without additional OVA stimulation. Numbers of OT-II cells in flight mice treated with OVA were significantly increased by 2-fold compared with ground mice treated with OVA, suggesting that tolerance induction was impaired by spaceflight. Production of proinflammatory cytokines were significantly increased in flight compared with ground mice, including a 5-fold increase in IFN-γ and a 10-fold increase in IL-17. This study is the first to show that immune tolerance may be impaired in spaceflight, leading to excessive inflammatory responses.

Ectopic expression of a small cell lung cancer transcription factor, INSM1 impairs alveologenesis in lung development.

BACKGROUND: Insulinoma associated-1 (INSM1) gene is expressed exclusively in early embryonic neuroendocrine tissues, but has been found highly re-activated in most of the neuroendocrine tumors including small cell lung carcinoma. METHODS: In order to elucidate the functional effects of INSM1 in normal lung development, we used a conditional lung-specific INSM1 transgenic mouse model. Transgenic (Tet-on system) CMV-INSM1 responder mice were bred with the lung-specific, club cell secretory protein (CCSP) promoter-rtTA activator mice to produce bi-transgenic progeny carrying both alleles, CCSP-rtTA and Tet-on-INSM1. Mice were fed with doxycycline containing food at the initial mating day to the postnatal day 21. Lung samples were collected at embryonic day 17.5, newborn, and postnatal day 21 for analyses. RESULTS: Northern blot, RT-PCR, and immunohistochemical analyses revealed that doxycycline induced respiratory epithelium-specific INSM1 expression in bi-transgenic mice. Samples from postnatal day 21 mice revealed a larger lung size in the bi-transgenic mouse as compared to the single-transgenic or wild-type littermates. The histopathology results showed that the alveolar space in the bi-transgenic mice were 4 times larger than those in the single transgenic or wild-type littermates. In contrast, the size was not significantly different in the lungs collected at E17.5 or newborn among the bi-transgenic, single transgenic, or wild type mice. The respiratory epithelium with INSM1 ectopic expression suppressed cyclin D1 signal. Further in vitro studies revealed that the ectopic expression of INSM1 suppresses cyclin D1 expression and delays cell cycle progression. CONCLUSION: The current study suggests that CCSP promoter-driven INSM1 ectopic expression impairs normal lung development especially in postnatal alveologenesis.