Knockdown Mouse Model Research Articles

PFKFB4 facilitates palbociclib resistance in oestrogen receptor-positive breast cancer by enhancing stemness.

ER+ breast cancer (ER+ BC) is the most common subtype of BC. Recently, CDK4/6 inhibitors combined with aromatase inhibitors have been approved by FDA as the first-line therapy for patients with ER+ BC, and showed promising therapeutic efficacy in clinical treatment. However, resistance to CDK4/6 inhibitors is frequently observed. A better understanding of the drug resistance mechanism is beneficial to improving therapeutic strategies by identifying optimal combinational treatments. Western blotting, qPCR, flow cytometry and a series of cell experiments were performed to evaluate the phenotype of MCF-7/R cells. RNA sequencing, non-targeted metabolomics, shRNA knockdown and tumour cell-bearing mouse models were used to clarify the drug resistance mechanism. Here, we found that ER+ BC cells have shown an adaptive resistance to palbociclib-induced cell cycle arrest by activating an alternative signal pathway, independent of the CDK4/6-RB signal transduction. Continuing treatment of palbociclib evoked cellular senescence of ER+ BC cells. Subsequently, the senescence-like phenotype promoted stemness of ER+ BC cells, accompanied by increased chemoresistance and tumour-initiating potential. Based on transcriptome analysis, we found that PFKFB4 played an important role in stemness transformation and drug resistance. A close correlation was determined between PFKFB4 expression by ER+ BC cells and cell senescence and stemness. Mechanistically, metabolomic profiling revealed that PFKFB4 reprogramed glucose metabolism and promoted cell stemness by enhancing glycolysis. Strikingly, diminishing PFKFB4 levels improved drug sensitivity and overcame chemoresistance during palbociclib treatment in ER+ BC. These findings not only demonstrated the novel mechanism underlying which ER+ BC cells resisted to palbociclib, but also provided a possible therapeutic strategy in the intervention of ER+ BC to overcome drug resistance.

Epithelial JAM-A is fundamental for intestinal wound repair in vivo.

Junctional adhesion molecule-A (JAM-A) is expressed in several cell types, including epithelial and endothelial cells, as well as some leukocytes. In intestinal epithelial cells (IEC), JAM-A localizes to cell junctions and plays a role in regulating barrier function. In vitro studies with model cell lines have shown that JAM-A contributes to IEC migration; however, in vivo studies investigating the role of JAM-A in cell migration–dependent processes such as mucosal wound repair have not been performed. In this study, we developed an inducible intestinal epithelial–specific JAM-A–knockdown mouse model (Jam-aERΔIEC). While acute induction of IEC-specific loss of JAM-A did not result in spontaneous colitis, such mice had significantly impaired mucosal healing after chemically induced colitis and after biopsy colonic wounding. In vitro primary cultures of JAM-A–deficient IEC demonstrated impaired migration in wound healing assays. Mechanistic studies revealed that JAM-A stabilizes formation of protein signaling complexes containing Rap1A/Talin/β1 integrin at focal adhesions of migrating IECs. Loss of JAM-A in primary IEC led to decreased Rap1A activity and protein levels of Talin and β1 integrin, and it led to a reduction in focal adhesion structures. These findings suggest that epithelial JAM-A plays a critical role in controlling mucosal repair in vivo through dynamic regulation of focal adhesions.

Effects of ambient PM2.5 on development of psoriasiform inflammation through KRT17-dependent activation of AKT/mTOR/HIF-1α pathway

Exposure to fine particulate matter (PM2.5) has significant effects on human skin health, mainly disrupting skin homeostasis and accelerating aging. To date, the effects of PM2.5 on psoriasis (PSO) have not been elucidated. An ambient particulate matter exposed and well characterized imiquimod (IMQ)-induced psoriasis mouse model was established. Thirty male C57BL/6 mice aged 8 weeks were randomly divided into three groups: filtered air (FA) group (Control group), PSO+ FA group and PSO + PM2.5 group. A KRT17 knockdown (KRT17-KD) mouse model was simultaneously established by subcutaneously injecting KRT17-KD lentivirus. Forty male C57BL/6 mice were randomly divided into four groups: PSO + FA + KRT17-RNAi negative control lentivirus (KRT17-NC) group, PSO+ FA+ KRT17-KD group, PSO + PM2.5 + KRT17-NC group and PSO + PM2.5 + KRT17-KD group. PM2.5 exposure continued for 8 weeks. Psoriasis was induced by topically applying IMQ on the dorsal skin of the mice for 6 days during week 8. Morphometric and histological analyses were performed to investigate the changes in psoriatic lesions. Differentially expressed genes and enriched pathways were explored using bioinformatics analysis and showed that KRT17 gene and the vascular endothelial growth factor receptor signaling pathway were associated with psoriasis. HaCaT cells were stimulated with interleukin-17A and infected with KRT17-KD lentivirus to establish an in vitro KRT17 knockdown psoriasis cell model. Notably, PM2.5 exposure increased the expression of KRT17 protein and activated AKT/mTOR/HIF-1α signaling pathway in vivo. Moreover, specific agonist of AKT (740Y-P) reversed the decreased neovascularization induced by KRT17 knockdown through AKT/mTOR/HIF-1α signaling pathway in vitro. Consequently, PM2.5 exposure could promote the development and progression of psoriasis through KRT17-dependent activation of AKT/mTOR/HIF-1α signaling pathway.

Septin7 is indispensable for proper skeletal muscle architecture and function.

Today septins are considered as the fourth component of the cytoskeleton, with the Septin7 isoform playing a critical role in the formation of higher-order structures. While its importance has already been confirmed in several intracellular processes of different organs, very little is known about its role in skeletal muscle. Here, using Septin7 conditional knockdown (KD) mouse model, the C2C12 cell line, and enzymatically isolated adult muscle fibers, the organization and localization of septin filaments are revealed, and an ontogenesis-dependent expression of Septin7 is demonstrated. KD mice displayed a characteristic hunchback phenotype with skeletal deformities, reduction in in vivo and in vitro force generation, and disorganized mitochondrial networks. Furthermore, knockout of Septin7 in C2C12 cells resulted in complete loss of cell division while KD cells provided evidence that Septin7 is essential for proper myotube differentiation. These and the transient increase in Septin7 expression following muscle injury suggest that it may be involved in muscle regeneration and development.

Septin7 is indispensable for proper skeletal muscle architecture and function

Today septins are considered as the fourth component of the cytoskeleton, with the Septin7 isoform playing a critical role in the formation of higher-order structures. While its importance has already been confirmed in several intracellular processes of different organs, very little is known about its role in skeletal muscle. Here, using Septin7 conditional knockdown (KD) mouse model, the C2C12 cell line, and enzymatically isolated adult muscle fibers, the organization and localization of septin filaments are revealed, and an ontogenesis-dependent expression of Septin7 is demonstrated. KD mice displayed a characteristic hunchback phenotype with skeletal deformities, reduction in in vivo and in vitro force generation, and disorganized mitochondrial networks. Furthermore, knockout of Septin7 in C2C12 cells resulted in complete loss of cell division while KD cells provided evidence that Septin7 is essential for proper myotube differentiation. These and the transient increase in Septin7 expression following muscle injury suggest that it may be involved in muscle regeneration and development.

Ketamine triggers rapid antidepressant effects by modulating synaptic plasticity in a new depressive-like mouse model based on astrocyte glutamate transporter GLT-1 knockdown in infralimbic cortex

ObjectiveRecently, we reported on a new MDD-like mouse model based on a regionally selective knockdown of astroglial glutamate transporters, GLAST/GLT-1, in infralimbic cortex (IL) which evokes widespread changes in mouse brain associated with the typical alterations found in MDD patients. To further characterize this new MDD-like mouse model, here we examine some transcriptional elements of glutamatergic/GABAergic neurotransmission and neuroplasticity in forebrain regions in the GLT-1 knockdown mice. Furthermore, we assess the acute ketamine effects on these transcriptional processes. Material and methodsWe used a small interfering RNA (siRNA) pool targeting GLT-1 mRNA to disrupt the GLT-1 transcription in mouse IL. Histological assays were performed to examine postsynaptic density protein-95 (PSD95), neuritin (NRN), glutamine acid descarboxilase-65 (GAD65), and GLT-1 mRNA expression in IL and hippocampus. ResultsKnockdown of GLT-1 in mouse IL leads to decreased expression of PSD95 and NRN neuroplasticity mRNAs in IL and hippocampus, which was reversed by an acute dose of ketamine antidepressant. Likewise, a single dose of ketamine also increased the mRNA levels of GAD65 and GLT-1 in IL of GLT-1 knockdown mice, reaching the basal values of control mice. ConclusionsThe glutamatergic neuronal hyperactivity and deficits in the GABA system resulting from siRNA-induced astroglial glutamate transporter knockdown in IL can compromise the integrity/plasticity of neurocircuits affected in MDD. Suitable depressive-like animal models to address the neurobiological changes in MDD are an unmet need and the development of the GLAST/GLT-1 knockdown mouse model may represent a better option to understand the rapid-acting antidepressant effects of ketamine.

F-Actin Dysplasia Involved in Organ of Corti Deformity in Gjb2 Knockdown Mouse Model.

Mutations in the GJB2 gene encoding connexin26 (Cx26) protein are one of the most common causes of hereditary deafness. Previous studies have found that different Cx26-null mouse models have severe hearing loss and deformity of the organ of Corti (OC) as well as a reduction in microtubules in pillar cells (PCs). To explore the underlying mechanism of OC deformity caused by Cx26 downregulation further, we established Cx26 knockdown (KD) mouse models at postnatal days (P)0 and P8. The actin filaments contained in the pillar cells of mice in the P0 KD group were reduced by 54.85% and vinculin was increased by 22%, while the outer hair cells (OHCs) showed normal F-actin content. In the P8 KD group, PCs and OHCs of mice also showed almost normal F-actin content. The G-actin/F-actin ratio increased by 38% in the P0 KD group. No significant change was found in the mRNA or protein expression level of G-actin or the cadherin–catenin core complex in the P0 KD group at P6. Moreover, immunofluorescence showed that the intensity of LRRK2 was reduced by 97% in the P0 KD group at P6. Our results indicate that Cx26 is involved in the maturation of the cytoskeleton during the development of the OC at the early postnatal stage. The polymerization of G-actin into F-actin is prevented in Cx26 KD mice.

Clinical characterization of a novel RAB39B nonstop mutation in a family with ASD and severe ID causing RAB39B downregulation and study of a Rab39b knock down mouse model.

Autism spectrum disorder (ASD) and intellectual disability (ID) often exist together in patients. The RAB39B gene has been reported to be mutated in ID patients with additional clinical features ranging from ASD, macrocephaly, seizures and/or early-onset parkinsonism. Here, we describe a novel RAB39B nonstop mutation [Xq28; c.640 T > C; p.(*214Glnext*21)] in a family with ASD, severe ID and poor motor coordination, and we assessed the pathogenicity of the mutation. A heterologous cell system and a Rab39b knockdown (KD) murine model, which mimic the nonstop mutation, were used to validate the deleterious effect of the RAB39B mutation. The mutation led to RAB39B protein instability, resulting in its increased degradation and consequent downregulation. Using a Rab39b KD mouse model, we demonstrated that the downregulation of RAB39B led to increased GluA2 lacking Ca2+-permeable AMPAR composition at the hippocampal neuronal surface and increased dendritic spine density that remained in an immature filopodia-like state. These phenotypes affected behavioural performance in a disease-specific manner. Rab39b KD mice revealed impaired social behaviour but intact social recognition. They also showed normal anxiety-like, exploratory and motivational behaviours but impaired working and associative memories. In conclusion, we found a novel RAB39B nonstop variant that segregated in a family with a clinical phenotype including ID, ASD and poor motor coordination. The pathogenicity of mutations causing the downregulation of RAB39B proteins, impacting AMPAR trafficking and dendritic spine morphogenesis, reinforced the idea that AMPAR modulation and dendritic spine assets could be considered hallmarks of neurodevelopmental disorders.

Deletion of Trpm4 Alters the Function of the Nav1.5 Channel in Murine Cardiac Myocytes.

Transient receptor potential melastatin member 4 (TRPM4) encodes a Ca2+-activated, non-selective cation channel that is functionally expressed in several tissues, including the heart. Pathogenic mutants in TRPM4 have been reported in patients with inherited cardiac diseases, including conduction blockage and Brugada syndrome. Heterologous expression of mutant channels in cell lines indicates that these mutations can lead to an increase or decrease in TRPM4 expression and function at the cell surface. While the expression and clinical variant studies further stress the importance of TRPM4 in cardiac function, the cardiac electrophysiological phenotypes in Trpm4 knockdown mouse models remain incompletely characterized. To study the functional consequences of Trpm4 deletion on cardiac electrical activity in mice, we performed perforated-patch clamp and immunoblotting studies on isolated atrial and ventricular cardiac myocytes and surfaces, as well as on pseudo- and intracardiac ECGs, either in vivo or in Langendorff-perfused explanted mouse hearts. We observed that TRPM4 is expressed in atrial and ventricular cardiac myocytes and that deletion of Trpm4 unexpectedly reduces the peak Na+ currents in myocytes. Hearts from Trpm4−/− mice presented increased sensitivity towards mexiletine, a Na+ channel blocker, and slower intraventricular conduction, consistent with the reduction of the peak Na+ current observed in the isolated cardiac myocytes. This study suggests that TRPM4 expression impacts the Na+ current in murine cardiac myocytes and points towards a novel function of TRPM4 regulating the Nav1.5 function in murine cardiac myocytes.

Adaptation to exercise-induced stress is not dependent on cardiomyocyte α1A-adrenergic receptors

The ‘fight or flight’ response to physiological stress involves sympathetic nervous system activation, catecholamine release and adrenergic receptor stimulation. In the heart, this induces positive inotropy, previously attributed to the β1-adrenergic receptor subtype. However, the role of the α1A-adrenergic receptor, which has been suggested to be protective in cardiac pathology, has not been investigated in the setting of physiological stress. To explore this, we developed a tamoxifen-inducible, cardiomyocyte-specific α1A-adrenergic receptor knock-down mouse model, challenged mice to four weeks of endurance swim training and assessed cardiac outcomes. With 4-OH tamoxifen treatment, expression of the α1A-adrenergic receptor was knocked down by 80–89%, without any compensatory changes in the expression of other adrenergic receptors, or changes to baseline cardiac structure and function. Swim training caused eccentric hypertrophy, regardless of genotype, demonstrated by an increase in heart weight/tibia length ratio (30% and 22% in vehicle- and tamoxifen-treated animals, respectively) and an increase in left ventricular end diastolic volume (30% and 24% in vehicle- and tamoxifen-treated animals, respectively) without any change in the wall thickness/chamber radius ratio. Consistent with physiological hypertrophy, there was no increase in fetal gene program (Myh7, Nppa, Nppb or Acta1) expression. In response to exercise-induced volume overload, stroke volume (39% and 30% in vehicle- and tamoxifen-treated animals, respectively), cardiac output/tibia length ratio (41% in vehicle-treated animals) and stroke work (61% and 33% in vehicle- and tamoxifen-treated animals, respectively) increased, regardless of genotype. These findings demonstrate that cardiomyocyte α1A-adrenergic receptors are not necessary for cardiac adaptation to endurance exercise stress and their acute ablation is not deleterious.

Ursodesoxycholic acid alleviates liver fibrosis via proregeneration by activation of the ID1-WNT2/HGF signaling pathway.

BackgroundThe human liver possesses a remarkable capacity for self‐repair. However, liver fibrosis remains a serious medical concern, potentially progressing to end‐stage liver cirrhosis and even death. Liver fibrosis is characterized by excess accumulation of extracellular matrix in response to chronic injury. Liver regenerative ability, a strong indicator of liver health, is important in resisting fibrosis. In this study, we provide evidence that ursodesoxycholic acid (UDCA) can alleviate liver fibrosis by promoting liver regeneration via activation of the ID1‐WNT2/hepatocyte growth factor (HGF) pathway.MethodsBile duct ligation (BDL) and partial hepatectomy (PH) mouse models were used to verify the effects of UDCA on liver fibrosis, regeneration, and the ID1‐WNT2/HGF pathway. An Id1 knockdown mouse model was also used to assess the role of Id1 in UDCA alleviation of liver fibrosis.ResultsOur results demonstrate that UDCA can alleviate liver fibrosis in the BDL mice and promote liver regeneration via the ID1‐WNT2/HGF pathway in PH mice. In addition, Id1 knockdown abolished the protection afforded by UDCA in BDL mice.ConclusionsWe conclude that UDCA protects against liver fibrosis by proregeneration via activation of the ID1‐WNT2/HGF pathway.

Relative Adipose Tissue Failure in Alström Syndrome Drives Obesity-Induced Insulin Resistance.

Obesity is a major risk factor for insulin resistance (IR) and its attendant complications. The pathogenic mechanisms linking them remain poorly understood, partly due to a lack of intermediary monogenic human phenotypes. Here, we report on a monogenic form of IR-prone obesity, Alström syndrome (ALMS). Twenty-three subjects with monogenic or polygenic obesity underwent hyperinsulinemic-euglycemic clamping with concomitant adipose tissue (AT) microdialysis and an in-depth analysis of subcutaneous AT histology. We have shown a relative AT failure in a monogenic obese cohort, a finding supported by observations in a novel conditional mouse model (Almsflin/flin) and ALMS1-silenced human primary adipocytes, whereas selective reactivation of ALMS1 gene in AT of an ALMS conditional knockdown mouse model (Almsflin/flin; Adipo-Cre+/−) restores systemic insulin sensitivity and glucose tolerance. Hence, we show for the first time the relative AT failure in human obese cohorts to be a major determinant of accelerated IR without evidence of lipodystrophy. These new insights into adipocyte-driven IR may assist development of AT-targeted therapeutic strategies for diabetes.

Placenta-Derived Osteoprotegerin Is Required for Glucose Homeostasis in Gestational Diabetes Mellitus.

Osteoprotegerin (OPG) is involved in various biological processes, including bone remodeling, vascular calcification and pancreatic β-cell function. Although some clinical studies have shown an increase in serum OPG level during pregnancy, the role of OPG in gestational diabetes mellitus (GDM) is largely unknown. Therefore, we explored the effect of OPG in metabolic homeostasis during pregnancy. We initially evaluated serum OPG levels using ELISA and western blotting techniques on samples from GDM patients. We also assessed OPG expression levels in maternal mice. We then used blastocysts transduced with lentiviruses capable of trophoblast-specific transgene expression to establish placenta-specific OPG knockdown or overexpression mouse models for functional and mechanistic investigation after embryo transplantation. We found that OPG expression was positively associated with GDM in clinical samples, and OPG levels were significantly increased in GDM patient sera and term placenta. Serum OPG was significantly increased in maternal compared to non-pregnant mice, and expression levels of OPG were the highest in placenta compared with other organs, including bone, liver and pancreas. OPG was also significantly increased in pregnant mice fed a high-fat diet (HFD). Placenta-specific OPG knockdown induced glucose intolerance, decreased β-cell proliferation and decreased serum insulin levels, whereas placenta-specific OPG overexpression promoted glucose tolerance and enhanced β-cell proliferation, which increased serum insulin production and decreased fetal weight in HFD-feeding pregnant mice. Placenta-derived OPG (pl-OPG) regulated glucose homeostasis during pregnancy via enhancement of β-cell proliferation, which suggests a potential therapeutic application of OPG for GDM.

CAMSAP3 facilitates basal body polarity and the formation of the central pair of microtubules in motile cilia

Synchronized beating of cilia on multiciliated cells (MCCs) generates a directional flow of mucus across epithelia. This motility requires a "9 + 2" microtubule (MT) configuration in axonemes and the unidirectional array of basal bodies of cilia on the MCCs. However, it is not fully understood what components are needed for central MT-pair assembly as they are not continuous with basal bodies in contrast to the nine outer MT doublets. In this study, we discovered that a homozygous knockdown mouse model for MT minus-end regulator calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), Camsap3tm1a/tm1a , exhibited multiple phenotypes, some of which are typical of primary ciliary dyskinesia (PCD), a condition caused by motile cilia defects. Anatomical examination of Camsap3tm1a/tm1a mice revealed severe nasal airway blockage and abnormal ciliary morphologies in nasal MCCs. MCCs from different tissues exhibited defective synchronized beating and ineffective generation of directional flow likely underlying the PCD-like phenotypes. In normal mice, CAMSAP3 localized to the base of axonemes and at the basal bodies in MCCs. However, in Camsap3tm1a/tm1a , MCCs lacked CAMSAP3 at the ciliary base. Importantly, the central MT pairs were missing in the majority of cilia, and the polarity of the basal bodies was disorganized. These phenotypes were further confirmed in MCCs of Xenopus embryos when CAMSAP3 expression was knocked down by morpholino injection. Taken together, we identified CAMSAP3 as being important for the formation of central MT pairs, proper orientation of basal bodies, and synchronized beating of motile cilia.

SELENOI is important for maintenance of phosphatidylethanolamine and glycosylphosphatidylinositol anchor protein during T cell activation and immune response

Abstract Selenoproteins are important for both innate and adaptive immune responses. As one of the selenoprotein members, SELENOI is characterized by its ethanolamine phosphotransferase activity in the Kennedy pathway of phosphatidylethanolamine (PE) synthesis and PE is also the donor of phosphoethanolamine moiety in glycosylphosphatidylinositol anchor protein (GPI-AP). To explore how SELENOI is involved in the immune response, we generated three mouse models with different SELENOI expression levels, including defined selenium diets, doxycycline inducible SELENOI knockdown (KD) and T cell specific SELENOI knockout (KO) mice models. We analyzed both the quantity of PE species on activated CD3 T cell membrane by LCMS and GPI-AP expression on activated T cell surface by FACS in all these three models. The SELENOI KO T cells exhibit a lower level of PE species on T cell membrane compared to WT T cells, and reduced expression of GPI-anchored proteins including CD52, CD73, CD48, CD109 and CD44 on cell surface upon activation as well. We also detected the same pattern of reduced PE species and GPI-anchor proteins on defined selenium diets and doxycycline-inducible SELENOI KD mouse models. Furthermore, the SELENOI KO T cells exhibited a reduced proliferation rate upon CD3/CD28 co-stimulation. Vaccination with LCMV peptides in SELENOI deficient mice results in less CD8+ and CD4+ LCMV specific T cells and reduced IFN-γ production, indicating an impaired immune response when SELENOI is absent. These results indicate that SELENOI is crucial for both PE and GPI-AP synthesis during T cell activation and proliferation due to the importance of PE during the activation process.

FKBPL, a novel player in cardiac ischaemia and fibrosis

FKBPL is a key anti-angiogenic protein. In an FKBPL knockdown mouse model, impaired cardiac and vascular function were previously observed. In this study, we investigated the role of FKBPL in ischaemic heart disease and fibrosis, which are the leading causes of mortality in people with cardiovascular disease, using relevant in vivo and in vitro experimental models.

Tsg101 positively regulates P62-Keap1-Nrf2 pathway to protect hearts against oxidative damage

Currently, most antioxidants do not show any favorable clinical outcomes in reducing myocardial ischemia-reperfusion (I/R) injury, suggesting an urgent need for exploring a new regulator of redox homeostasis in I/R hearts. Here, using heart-specific transgenic (TG) and knockdown (KD) mouse models, tumor susceptibility gene 101 (Tsg101) is defined as a novel cardiac-protector against I/R-triggered oxidative stress. RNA sequencing and bioinformatics data surprisingly reveal that most upregulated genes in Tsg101-TG hearts are transcribed by Nrf2. Accordingly, pharmacological inhibition of Nrf2 offsets Tsg101-elicited cardio-protection. Mechanistically, Tsg101 interacts with SQSTM1/p62 through its PRR domain, and promotes p62 aggregation, leading to recruitment of Keap1 for degradation by autophagosomes and release of Nrf2 to the nucleus. Furthermore, knockout of p62 abrogates Tsg101-induced cardio-protective effects during I/R. Hence, our findings uncover a previously unrecognized role of Tsg101 in the regulation of p62/Keap1/Nrf2 signaling cascades and provide a new strategy for the treatment of ischemic heart disease.

A critical appraisal of the tafazzin knockdown mouse model of Barth syndrome: what have we learned about pathogenesis and potential treatments?

Pediatric heart failure remains poorly understood, distinct in many aspects from adult heart failure. Limited data point to roles of altered mitochondrial functioning and, in particular, changes in mitochondrial lipids, especially cardiolipin. Barth syndrome is a mitochondrial disorder caused by tafazzin mutations that lead to abnormal cardiolipin profiles. Patients are afflicted by cardiomyopathy, skeletal myopathy, neutropenia, and growth delay. A mouse model of Barth syndrome was developed a decade ago, which relies on a doxycycline-inducible short hairpin RNA to knock down expression of tafazzin mRNA (TAZKD). Our objective was to review published data from the TAZKD mouse to determine its contributions to our pathogenetic understanding of, and potential treatment strategies for, Barth syndrome. In regard to the clinical syndrome, the reported physiological, biochemical, and ultrastructural abnormalities of the mouse model mirror those in Barth patients. Using this model, the peroxisome proliferator-activated receptor pan-agonist bezafibrate has been suggested as potential therapy because it ameliorated the cardiomyopathy in TAZKD mice, while increasing mitochondrial biogenesis. A clinical trial is now underway to test bezafibrate in Barth syndrome patients. Thus the TAZKD mouse model of Barth syndrome has led to important insights into disease pathogenesis and therapeutic targets, which can potentially translate to pediatric heart failure.

Novel roles of ER stress in repressing neural activity and seizures through Mdm2- and p53-dependent protein translation.

Seizures can induce endoplasmic reticulum (ER) stress, and sustained ER stress contributes to neuronal death after epileptic seizures. Despite the recent debate on whether inhibiting ER stress can reduce neuronal death after seizures, whether and how ER stress impacts neural activity and seizures remain unclear. In this study, we discovered that the acute ER stress response functions to repress neural activity through a protein translation-dependent mechanism. We found that inducing ER stress promotes the expression and distribution of murine double minute-2 (Mdm2) in the nucleus, leading to ubiquitination and down-regulation of the tumor suppressor p53. Reduction of p53 subsequently maintains protein translation, before the onset of translational repression seen during the latter phase of the ER stress response. Disruption of Mdm2 in an Mdm2 conditional knockdown (cKD) mouse model impairs ER stress-induced p53 down-regulation, protein translation, and reduction of neural activity and seizure severity. Importantly, these defects in Mdm2 cKD mice were restored by both pharmacological and genetic inhibition of p53 to mimic the inactivation of p53 seen during ER stress. Altogether, our study uncovered a novel mechanism by which neurons respond to acute ER stress. Further, this mechanism plays a beneficial role in reducing neural activity and seizure severity. These findings caution against inhibition of ER stress as a neuroprotective strategy for seizures, epilepsies, and other pathological conditions associated with excessive neural activity.

The protective effect of betacellulin against acute pancreatitis is ERBB4 dependent.

The EGFR ligand betacellulin (BTC) has been previously shown to protect mice against experimentally induced acute pancreatitis (AP). BTC binds both autonomous ERBB receptors EGFR and ERBB4. In this study, we evaluated the mechanism underlying the protection from AP-associated inflammation in detail. AP was induced with cerulein or L-arginine and investigated in a pancreas-specific ERBB4 knockout and in an EGFR knockdown mouse model (EgfrWa5/+). Pancreatitis was evaluated by scoring inflammation, necrosis, and edema, while microarrays were performed to analyze alterations in the transcriptome between mice with AP and animals which were protected against AP. The intracellular domain (ICD) of ERBB4 was analyzed in different cell compartments. While the pancreas of BTC transgenic mice in the background of EgfrWa5/+ is still protected against AP, the BTC-mediated protection is no longer present in the absence of ERBB4. We further demonstrate that BTC activates the ICD of ERBB4, and increases the expression of the extracellular matrix (ECM) proteins periostin and matrix gla protein as well as the ECM modulators matrix metalloproteinases 2 and 3, but only in the presence of ERBB4. Notably, the increased expression of these proteins is not accompanied by anincreased ECM amount. These findings suggest that BTC derivates, as a drug, or the ERBB4 receptor, as a druggable target protein, could play an important role in modulating the course of AP and even prevent AP in humans.