Mutant Mice Research Articles

Upregulation of NFE2L1 reduces ROS levels and α-synuclein aggregation caused by GBA1 knockdown

Biallelic mutations in the GBA1 gene result in Gaucher disease (GD), and both patients with GD and carriers of a single GBA1 mutation have an increased susceptibility to Parkinson's disease (PD), but the underlying mechanisms of this association are not yet clear. In previous studies, we established Gba1 F213I point mutation mice and found that homozygous Gba1 F213I mutant mice died shortly after birth, while heterozygous mice could survive normally. In this study, we investigated the transcriptomic changes in the brain tissue of Gba1 F213I heterozygous mice, identifying 138 differentially expressed genes. Among them, Nfe2l1 was the most significantly downregulated gene. Inhibition or knockdown of GBA1 in BE(2)-M17 cells resulted in decreased expression levels of NFE2L1. Knockdown of GBA1 or NFE2L1 could lead to an elevation in intracellular aggregation of α-synuclein (α-syn) and reactive oxygen species (ROS) levels, while upregulation of NFE2L1 effectively mitigated those cellular manifestations induced by GBA1 knockdown. In summary, our in vitro results showed that upregulation of NFE2L1 may provide a therapeutic benefit for cellular phenotypes resulting from GBA1 knockdown, providing new insights for future research on GD and GBA1-associated PD.

An efficient inducible model for the control of gene expression in renin cells.

Fate mapping and genetic manipulation of renin cells have relied on either noninducible Cre lines that can introduce the developmental effects of gene deletion or bacterial artificial chromosome transgene-based inducible models that may be prone to spurious and/or ectopic gene expression. To circumvent these problems, we generated an inducible mouse model in which CreERT2 is under the control of the endogenous Akr1b7 gene, an independent marker of renin cells that is expressed in a few extrarenal tissues. We confirmed the proper expression of Cre using Akr1b7CreERT2/+;R26RmTmG/+ mice in which Akr1b7+/renin+ cells become green fluorescent protein (GFP)+ upon tamoxifen administration. In embryos and neonates, GFP was found in juxtaglomerular cells, along the arterioles, and in the mesangium, and in adults, GFP was present mainly in juxtaglomerular cells. In mice treated with captopril and a low-salt diet to induce recruitment of renin cells, GFP extended along the afferent arterioles and in the mesangium. We generated Akr1b7CreERT2/+;Ren1cFl/-;R26RmTmG/+ mice to conditionally delete renin in adult mice and found a marked reduction in kidney renin mRNA and protein and mean arterial pressure in mutant animals. When subjected to a homeostatic threat, mutant mice were unable to recruit renin+ cells. Most importantly, these mice developed concentric vascular hypertrophy ruling out potential developmental effects on the vasculature due to the lack of renin. We conclude that Akr1b7CreERT2 mice constitute an excellent model for the fate mapping of renin cells and for the spatial and temporal control of gene expression in renin cells.NEW & NOTEWORTHY Fate mapping and genetic manipulation are important tools to study the identity of renin cells. Here, we report on a novel Cre mouse model, Akr1b7CreERT2, for the spatial and temporal regulation of gene expression in renin cells. Cre is properly expressed in renin cells during development and in the adult under basal conditions and under physiological stress. Moreover, renin can be efficiently deleted in the adult, leading to the development of concentric vascular hypertrophy.

FOXC1 and FOXC2 Ablation Causes Abnormal Valvular Endothelial Cell Junctions and Lymphatic Vessel Formation in Myxomatous Mitral Valve Degeneration.

Mitral valve (MV) disease including myxomatous degeneration is the most common form of valvular heart disease with an age-dependent frequency. Genetic evidence indicates that mutations of the human transcription factor FOXC1 are associated with MV defects, including MV regurgitation. In this study, we sought to determine whether murine Foxc1 and its closely related factor, Foxc2, are required in valvular endothelial cells (VECs) for the maintenance of MV leaflets, including VEC junctions and the stratified trilaminar ECM (extracellular matrix). Adult mice carrying tamoxifen-inducible, vascular endothelial cell (EC), and lymphatic EC-specific, compound Foxc1;Foxc2 mutations (ie, EC-Foxc-DKO and lymphatic EC-Foxc-DKO mice, respectively) were used to study the function of Foxc1 and Foxc2 in the maintenance of MVs. The EC and lymphatic EC mutations of Foxc1/c2 were induced at 7 to 8 weeks of age by tamoxifen treatment, and abnormalities in the MVs of these mutant mice were assessed via whole-mount immunostaining, immunohistochemistry/RNAscope, Movat pentachrome/Masson Trichrome staining, and Evans blue injection. EC deletions of Foxc1 and Foxc2 in mice resulted in abnormally extended and thicker MVs by causing defects in the regulation of ECM organization with increased proteoglycan and decreased collagen. Notably, reticular adherens junctions were found in VECs of control MV leaflets, and these reticular structures were severely disrupted in EC-Foxc-DKO mice. PROX1 (prospero homeobox protein 1), a key regulator in a subset of VECs on the fibrosa side of MVs, was downregulated in EC-Foxc1/c2 mutant VECs. Furthermore, we determined the precise location of lymphatic vessels in murine MVs, and these lymphatic vessels were aberrantly expanded and dysfunctional in EC-Foxc1/c2 mutant MVs. Lymphatic EC deletion of Foxc1/c2 also resulted in similar structural/ECM abnormalities as seen in EC-Foxc1/c2 mutant MVs. Our results indicate that Foxc1 and Foxc2 are required for maintaining the integrity of the MV, including VEC junctions, ECM organization, and lymphatic vessel formation/function to prevent myxomatous MV degeneration.

Abstract P434: Piezo2 Channels in JG cells do not Regulate Renin Expression or Renin Release to the Circulation

Kidneys are central to blood pressure regulation through the renin-angiotensin system. Renin-producing juxtaglomerular (JG) cells act as renal baroreceptors by controlling renin release according to the changes in perfusion pressure. Recent studies suggest that Piezo2 calcium channels may regulate the baroreceptor mechanism of renin release. However, our single-cell RNAseq and RNAScope analysis on adult kidneys showed a very low expression for Piezo2 in the JG cells. Since it is unclear if Piezo2 in the JG cells regulates renin expression and release, we defined the in vivo role of Piezo2 in the current study using Piezo2 conditional mutant mice. For this we generated Piezo2 fl/fl ;Ren1 d Cre, Piezo2 fl/del ;Ren1 d Cre and Piezo2 fl/fl ;Akr1b7.Cre-Er T2 mice. Though we detected an efficient deletion of Piezo2 by RNAScope in the JG cells of the mutant kidneys, we did not observe a significant difference in the renin signals between the control and the mutant groups by renin immunostaining. q-RT-PCR of the kidney cortices to measure renin mRNA levels and renin-specific ELISA to quantify plasma renin levels also indicated no significant differences (NS) between the control and the mutant groups ( Piezo2 fl/fl ;Ren1 d Cre - Relative renin mRNA levels: Control (n=11) 1.05±0.113 Mutants (n=6) 0.90±0.20, NS; Plasma renin levels (pg/ml): Control (n=11) 17472±3712 Mutants (n=6) 14934±3547, NS; Piezo2 fl/del ;Ren1 d Cre - Relative renin mRNA levels: Control (n=3) 0.98±0.13 Mutants (n=4) 1.04±0.19, NS; Plasma renin levels (pg/ml): Control (n=3) 20221±4303 Mutants (n=4) 15014±1901, NS). Furthermore, subjecting the mice to hypotension and volume depletion with captopril and a low salt diet for one week did not affect the renin expression due to Piezo2 deletion in the renin cells. Finally, our results from the aortic coarctation surgery model showed that Piezo2 deletion in the adult JG cells of Piezo2 fl/fl ;Akr1b7.Cre-Er T2 mice did not affect the changes in renin expression between the left and right kidneys in response to perfusion pressure (Relative renin mRNA levels: Control (n=11) LK-1.102 ±0.173 RK-0.316±0.046, p=0.002 Mutants (n=9) LK-1.191±0.266 RK-0.524±0.212, p=0.05). Collectively, our in vivo data shows that renin expression is unaffected during normal conditions and when homeostasis is threatened by hypotension, sodium depletion, or changes in blood pressure, indicating that Piezo2 channels are not necessary for renin synthesis and release to circulation in JG cells.

Fibroblast Growth Factor (FGF) 13

Fibroblast Growth Factor (FGF) 13, also referred to as FGF homologous factor (FHF) 2, is a member of the FGF11 subfamily that is characterized as having sequence similarities to classical FGF receptor (FGFR)-binding FGFs, but functionally do not bind FGFRs. In this primer mini-review, we summarize current knowledge regarding FGF13 expression, mutant analyses, and gene and protein structure. Similar to other FHFs, FGF13 has been considered a non-secreted protein that lacks an amino signal and is prominently expressed in developing and mature neurons of the central and peripheral nervous systems, as well as the heart. The expression of FGF13 is not limited to early embryonic stages and has been shown to persist in adult tissues. As well, FGF13 is known to localize subcellularly, both within the cytoplasm and the nucleus. FGF13 is extremely adaptable, as it interacts with MAPK scaffolding protein islet brain 2 (IB2), stabilizes microtubules, or binds to voltage-gated sodium channels. Fgf13 mutant mouse lines display various neurological pathologies. Through sequence mapping, FGF13 is considered a candidate causative gene that is mutated in multiple human X-linked neurological diseases.

Abstract 63: Acetylation of Mitochondrial Cyclophilin D Increases vascular Oxidative Stress, Induces Glycolitic Switch, Promotes Endothelial Dysfunction and Hypertension

Background: Nearly half of adults have hypertension, a major risk factor for cardiovascular disease. Mitochondrial hyperacetylation is linked to hypertension, but the role of acetylation of specific proteins is not clear. We hypothesized that acetylation of mitochondrial Cyclophilin D (CypD) at K166 contributes to endothelial dysfunction and hypertension. Methods: To test this hypothesis, we studied CypD acetylation in patients with essential hypertension, defined a pathogenic role of CypD acetylation in deacetylation mimetic CypD-K166R mutant mice and endothelial specific general control of amino acid synthesis 5 like 1 (GCN5L1) deficient mice using an angiotensin II model of hypertension. Results: Arterioles from hypertensive patients had 280% higher CypD acetylation coupled with reduced Sirt3 and increased GCN5L1 levels. GCN5L1 regulates mitochondrial protein acetylation and promotes CypD acetylation which is counteracted by mitochondrial deacetylase Sirt3. In human aortic endothelial cells GCN5L1 depletion prevents superoxide overproduction. Deacetylation mimetic CypD-K166R mice were protected from vascular oxidative stress, endothelial dysfunction and angiotensin II-induced hypertension. Angiotensin II-induced hypertension increased mitochondrial GCN5L1 and reduced Sirt3 levels resulting in 250% increase in GCN5L1/Sirt3 ratio leading to imbalance between GCN5L1-mediated acetylation and Sirt3-mediated deacetylation and promoting CypD acetylation. Treatment with mitochondria-targeted scavenger of cytotoxic isolevuglandins, mito2HOBA, normalized GCN5L1/Sirt3 ratio, reduced CypD acetylation, and attenuated hypertension. The role of mitochondrial acetyltransferase GCN5L1 in endothelial function was tested in endothelial specific GCN5L1 knockout mice. Depletion of endothelial GCN5L1 prevented angiotensin II-induced mitochondrial oxidative stress, reduced maladaptive switch of vascular metabolism to glycolysis, prevented inactivation of endothelial nitric oxide, preserved endothelial-dependent relaxation, and attenuated hypertension. Conclusions: These data support the pathogenic role of CypD acetylation in endothelial dysfunction and hypertension. We suggest that targeting cytotoxic mitochondrial isolevuglandins and GCN5L1 reduces CypD acetylation which may be beneficial in cardiovascular disease.

CHCHD2 mutant mice display mitochondrial protein accumulation and disrupted energy metabolism.

Mutations in the mitochondrial cristae protein CHCHD2 lead to a late-onset autosomal dominant form of Parkinson's disease (PD) which closely resembles idiopathic PD, providing the opportunity to gain new insights into the mechanisms of mitochondrial dysfunction contributing to PD. To begin to address this, we used CRISPR genome-editing to generate CHCHD2 T61I point mutant mice. CHCHD2 T61I mice had normal viability, and had only subtle motor deficits with no signs of premature dopaminergic (DA) neuron degeneration. Nonetheless, CHCHD2 T61I mice exhibited robust molecular changes in the brain including increased CHCHD2 insolubility, accumulation of CHCHD2 protein preferentially in the substantia nigra (SN), and elevated levels of α-synuclein. Metabolic analyses revealed an increase in glucose metabolism through glycolysis relative to the TCA cycle with increased respiratory exchange ratio, and immune-electron microscopy revelated disrupted mitochondria in DA neurons. Moreover, spatial genomics revealed decreased expression of mitochondrial complex I and III respiratory chain proteins, while proteomics revealed increased respiratory chain and other mitochondrial protein-protein interactions. As such, the CHCHD2 T61I point-mutation mice exhibit robust mitochondrial disruption and a consequent metabolic shift towards glycolysis. These findings thus establish CHCHD2 T61I mice as a new model for mitochondrial-based PD, and implicate disrupted respiratory chain function as a likely causative driver.

Gene inactivation of lysyl oxidase in smooth muscle cells reduces atherosclerosis burden and plaque calcification in hyperlipidemic mice

Background and aimsLysyl oxidase (LOX) catalyzes the crosslinking of collagen and elastin to maintain tensile strength and structural integrity of the vasculature. Excessive LOX activity increases vascular stiffness and the severity of occlusive diseases. Herein, we investigated the mechanisms by which LOX controls atherogenesis and osteogenic differentiation of vascular smooth muscle cells (SMC) in hyperlipidemic mice. MethodsGene inactivation of Lox in SMC was achieved in conditional knockout mice after tamoxifen injections. Atherosclerosis burden and vascular calcification were assessed in hyperlipidemic conditional [Loxf/fMyh11-CreERT2ApoE−/−] and sibling control mice [Loxwt/wtMyh11-CreERT2ApoE−/−]. Mechanistic studies were performed with primary aortic SMC from Lox mutant and wild type mice. ResultsInactivation of Lox in SMCs decreased > 70 % its RNA expression and protein level in the aortic wall and significantly reduced LOX activity without compromising vascular structure and function. Moreover, LOX deficiency protected mice against atherosclerotic burden (13 ± 2 versus 23 ± 1 %, p < 0.01) and plaque calcification (5 ± 0.4 versus 11.8 ± 3 %, p < 0.05) compared to sibling controls. Interestingly, gene inactivation of Lox in SMCs preserved the contractile phenotype of vascular SMC under hyperlipidemic conditions as demonstrated by single-cell RNA sequencing and immunofluorescence. Mechanistically, the absence of LOX in SMC prevented excessive collagen crosslinking and the subsequent activation of the pro-osteogenic FAK/β-catenin signaling axis. ConclusionsLox inactivation in SMC protects mice against atherosclerosis and plaque calcification by reducing SMC modulation and FAK/β-catenin signaling.

Pathology reduction and motor behavior improvement associated with ultrasound-mediated delivery of arctiin to the motor cortex in a mutant SOD1 mouse model of amyotrophic lateral sclerosis

BackgroundAmyotrophic lateral sclerosis (ALS) is marked by the deterioration of both cortical and spinal cord motor neurons. Despite the underlying causes of the disease remain elusive, there has been a growing attention on the well-being of cortical motor neurons in recent times. Focused ultrasound combined with microbubbles (FUS/MB) for opening the blood–brain barrier (BBB) provides a means for drug delivery to specific brain regions, holding significant promise for the treatment of neurological disorders. ObjectivesWe aim to explore the outcomes of FUS/MB-mediated delivery of arctiin (Arc), a natural compound with anti-inflammatory activities, to the cerebral motor cortex area by using a transgenic ALS mouse model. MethodsThe ALS mouse model with the SOD1G93A mutation was used and subjected to daily Arc administration with FUS/MB treatment twice a week. After six-week treatments, the motor performance was assessed by grip strength, wire hanging, and climbing-pole tests. Mouse brains, spinal cords and gastrocnemius muscle were harvested for histological staining. ResultsCompared with the mice given Arc administration only, the combined treatments of FUS/MB with Arc induced further mitigation of the motor function decline, accompanied by improved health of the gastrocnemius muscle. Furthermore, notable neuroprotective effect was evidenced by the amelioration of motor neuron failure in the cortex and lumbar spinal cord. ConclusionThese preliminary results indicated that the combined treatment of FUS/MB and arctiin exerted a potentially beneficial effect on neuromuscular function in the ALS disease.

Genotyping single point mutations in rd1 and rd8 mice using melting curve analysis of qPCR fragments

PCR is tolerant to single nucleotide mismatches. Therefore, genotyping of point mutations by PCR requires special conditions for the amplification of allele-specific PCR fragments. MS-PCR (mutagenically separated PCR) is an improved version of ARMS (amplification refractory mutation system) in which additional nucleotide mismatches near the mutation site are used to separate the wt fragments from the mutant fragments in a single-tube PCR. In the originally described procedure, the resulting fragments are resolved on agarose gels according to differences in size introduced by different lengths of the allele-specific primers. In order to evaluate the PCR fragments by melting curve analysis, we enlarged the difference in the melting temperatures of the fragments of the two alleles by increasing the GC content of the longer allele-specific primer resulting in a higher melting temperature of the corresponding fragment. Using the murine retinal degeneration mutations rd1 and rd8 as an example, we show that such primers result in an easy to handle genotyping procedure: qPCR followed by melting curve analysis. In summary, MS-PCR is a simple and easy-to-use method for detecting single nucleotide variants.

Focused Ultrasound Impels the Delivery and Penetration of Model Therapeutics into Cerebral Cavernous Malformations.

Cerebral cavernous malformations (CCMs) are vascular neoplasms in the brain that can cause debilitating symptoms. Current treatments pose significant risks to some patients, motivating the development of new nonsurgical options. We recently discovered that focused ultrasound-mediated blood-brain barrier opening (FUS) arrests CCM formation and growth. Here, we build on this discovery and assess the ability of FUS to deliver model therapeutics into CCMs. Quantitative T1 mapping MRI sequences were used with 1 kDa (MultiHance; MH) and 17 kDa (GadoSpin D; GDS) contrast agents to assess the FUS-mediated delivery and penetration of model small molecule drugs and biologics, respectively, into CCMs of Krit1 mutant mice. FUS elevated the rate of MH delivery to both the lesion core (4.6-fold) and perilesional space (6.7-fold). Total MH delivery more than doubled in the lesion core and tripled in the perilesional space when FUS was applied immediately prior to MH injection. For the model biologic drug (i.e. GDS), FUS was of greater relative benefit, resulting in 21.7-fold and 3.8-fold delivery increases to the intralesional and perilesional spaces, respectively. FUS is capable of impelling the delivery and penetration of therapeutics into the complex and disorganized CCM microenvironment. Benefits to small molecule drug delivery are more evident in the perilesional space, while benefits to biologic delivery are more evident in CCM cores. These findings, when combined with ability of FUS alone to control CCMs, highlight the potential of FUS to serve as a powerful non-invasive therapeutic platform for CCM.

TFEB/3 Govern Repair Schwann Cell Generation and Function Following Peripheral Nerve Injury.

TFEB and TFE3 (TFEB/3), key regulators of lysosomal biogenesis and autophagy, play diverse roles depending on cell type. This study highlights a hitherto unrecognized role of TFEB/3 crucial for peripheral nerve repair. Specifically, they promote the generation of progenitor-like repair Schwann cells after axonal injury. In Schwann cell-specific TFEB/3 double knock-out mice of either sex, the TFEB/3 loss disrupts the transcriptomic reprogramming that is essential for the formation of repair Schwann cells. Consequently, mutant mice fail to populate the injured nerve with repair Schwann cells and exhibit defects in axon regrowth, target reinnervation, and functional recovery. TFEB/3 deficiency inhibits the expression of injury-responsive repair Schwann cell genes, despite the continued expression of c-jun, a previously identified regulator of repair Schwann cell function. TFEB/3 binding motifs are enriched in the enhancer regions of injury-responsive genes, suggesting their role in repair gene activation. Autophagy-dependent myelin breakdown is not impaired despite TFEB/3 deficiency. These findings underscore a unique role of TFEB/3 in adult Schwann cells that is required for proper peripheral nerve regeneration.

HnRNP R promotes O-GlcNAcylation of eIF4G and facilitates axonal protein synthesis

Motoneurons critically depend on precise spatial and temporal control of translation for axon growth and the establishment and maintenance of neuromuscular connections. While defects in local translation have been implicated in the pathogenesis of motoneuron disorders, little is known about the mechanisms regulating axonal protein synthesis. Here, we report that motoneurons derived from Hnrnpr knockout mice show reduced axon growth accompanied by lowered synthesis of cytoskeletal and synaptic components in axons. Mutant mice display denervated neuromuscular junctions and impaired motor behavior. In axons, hnRNP R is a component of translation initiation complexes and, through interaction with O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (Ogt), modulates O-GlcNAcylation of eIF4G. Restoring axonal O-GlcNAc levels rescued local protein synthesis and axon growth defects of hnRNP R knockout motoneurons. Together, these findings demonstrate a function of hnRNP R in controlling the local production of key factors required for axon growth and formation of neuromuscular innervations.

Cant1 Affects Cartilage Proteoglycan Properties: Aggrecan and Decorin Characterization in a Mouse Model of Desbuquois Dysplasia Type 1.

Desbuquois dysplasia type 1 (DBQD1) is a recessive chondrodysplasia caused by mutations in the CANT1 gene, encoding for the Golgi Calcium-Activated Nucleotidase 1 (CANT1). The enzyme hydrolyzes UDP, the by-product of glycosyltransferase reactions, but it might play other roles in different cell types. Using a Cant1 knock-out mouse, we demonstrated that CANT1 is crucial for glycosaminoglycan (GAG) synthesis; however, its impact on the biochemical properties of cartilage proteoglycans remains unknown. Thus, in this work, we characterized decorin and aggrecan from primary chondrocyte cultures and cartilage biopsies of mutant mice at post-natal day 4 by Western blots and further investigated their distribution in the cartilage extracellular matrix (ECM) by immunohistochemistry. We demonstrated that the GAG synthesis defect caused by CANT1 impairment led to the synthesis and secretion of proteoglycans with shorter GAG chains compared with wild-type animals. However, this alteration did not result in the synthesis and secretion of decorin and aggrecan in the unglycanated form. Interestingly, the defect was not cartilage-specific since also skin decorin showed a reduced hydrodynamic size. Finally, immunohistochemical studies in epiphyseal sections of mutant mice demonstrated that the proteoglycan structural defect moderately affected decorin distribution in the ECM.

Microtubules Sequester Acetylated YAP in the Cytoplasm and Inhibit Heart Regeneration.

The Hippo pathway effector YAP (Yes-associated protein) plays an essential role in cardiomyocyte proliferation and heart regeneration. In response to physiological changes, YAP moves in and out of the nucleus. The pathophysiological mechanisms regulating YAP subcellular localization after myocardial infarction remain poorly defined. We identified YAP acetylation at site K265 by in vitro acetylation followed by mass spectrometry analysis. We used adeno-associated virus to express YAP-containing mutations that either abolished acetylation (YAP-K265R) or mimicked acetylation (YAP-K265Q) and studied how acetylation regulates YAP subcellular localization in mouse hearts. We generated a cell line with YAP-K265R mutation and investigated the protein-protein interactors by YAP immunoprecipitation followed by mass spectrometry, then validated the YAP interaction in neonatal rat ventricular myocytes. We examined colocalization of YAP and TUBA4A (tubulin α 4A) by superresolution imaging. Furthermore, we developed YAP-K265R and αMHC-MerCreMer (MCM); Yap-loxP/K265R mutant mice to examine the pathophysiological role of YAP acetylation in cardiomyocytes during cardiac regeneration. We found that YAP is acetylated at K265 by CBP (CREB-binding protein)/P300 (E1A-binding protein P300) and is deacetylated by nicotinamide phosphoribosyltransferase/nicotinamide adenine dinucleotide/sirtuins axis in cardiomyocytes. After myocardial infarction, YAP acetylation is increased, which promotes YAP cytoplasmic localization. Compared with controls, mice that were genetically engineered to express a K265R mutation that prevents YAP K256 acetylation showed improved cardiac regenerative ability and increased YAP nuclear localization. Mechanistically, YAP acetylation facilitates its interaction with TUBA4A, a component of the microtubule network that sequesters acetylated YAP in the cytoplasm. After myocardial infarction, the microtubule network increased in cardiomyocytes, resulting in the accumulation of YAP in the cytoplasm. After myocardial infarction, decreased sirtuin activity enriches YAP acetylation at K265. The growing TUBA4A network sequesters acetylated YAP within the cytoplasm, which is detrimental to cardiac regeneration.

Myeloperoxidase-deficient mice exposed to Zymosan exhibit severe neutrophilia and anemia with enhanced granulopoiesis and reduced erythropoiesis, accompanied by pulmonary inflammation

We previously reported that myeloperoxidase-deficient (MPO-/-) mice develop more severe neutrophil-rich lung inflammation than wild-type mice following intranasal Zymosan administration. Interestingly, we found that these mutant mice with severe lung inflammation also displayed pronounced neutrophilia and anemia, characterized by increased granulopoiesis and decreased erythropoiesis in the bone marrow, compared to wild-type mice. This condition was associated with higher concentrations of granulocyte-colony stimulating factor (G-CSF) in both the lungs and serum, a factor known to enhance granulopoiesis. Neutrophils accumulating in the lungs of MPO-/- mice produced greater amounts of G-CSF than those in wild-type mice, indicating that they are a significant source of G-CSF. In vitro experiments using signal transduction inhibitors and Western blot analysis revealed that MPO-/- neutrophils express higher levels of G-CSF mRNA in response to Zymosan, attributed to the upregulation of the IκB kinase/nuclear factor (NF)-κB pathway and the extracellular-signal-regulated kinase/NF-κB pathway. These findings highlight MPO as a critical regulator of granulopoiesis and erythropoiesis in inflamed tissues.

Late development of OCD-like phenotypes in Dlgap1 knockout mice.

Despite variants in the Dlgap1 gene having the two lowest p-value in a genome-wide association study of obsessive compulsive disorder (OCD), previous studies reported the absence of OCD-like phenotypes in Dlgap1 knockout (KO) mice. Since these studies observed behavioral phenotypes only for a short period, development of OCD-like phenotypes in these mice at older ages was still plausible. To examine the presence or absence of development of OCD-like phenotypes in Dlgap1 KO mice and their responsiveness to fluvoxamine. Newly produced Dlgap1 KO mice were observed for a year. Modified SHIRPA primary screen in 2-month-old homozygous mutant mice showed only weak signs of anxiety, stress conditions and aggression. At older ages, however, these mutant mice exhibited excessive self-grooming characterized by increased scratching which led to skin lesions. A significant sex difference was observed in this scratching behavior. The penetrance of skin lesions reached 50% at 6-7 months of age and 90% at 12 months of age. In the open-field test performed just after the appearance of these lesions, homozygous mutant mice spent significantly less time in the center, an anxiety-like behavior, than did their wild-type and heterozygous littermates, none and less than 10% of which showed skin lesions at 1 year, respectively. The skin lesions and excessive self-grooming were significantly alleviated by two-week treatment with fluvoxamine. Usefulness of Dlgap1 KO mice as a tool for investigating the pathogenesis of OCD-like phenotypes and its translational relevance was suggested.

G protein–coupled receptor kinase 5 regulates thrombin signaling in platelets

BackgroundOur prior genome-wide association study of thrombin-induced platelet aggregation identified a G protein–coupled receptor kinase 5 (GRK5) noncoding variant (rs10886430-G) that is strongly associated with increased platelet reactivity to thrombin. This variant predisposes to increased risk of stroke, pulmonary embolism, and venous thromboembolism. ObjectivesTo determine role of platelet specific GRK5 in platelet responses to agonists and injury. MethodsPlatelets from GRK5 mutant mice have been shown to have increased thrombin sensitivity, indicating that GRK5 may be a negative regulator of platelet activation. However, this has not been studied in a platelet-specific manner. We therefore used platelet-specific GRK5 mutant mice and models of thrombosis and pulmonary embolism. ResultsWe now demonstrate that mice lacking GRK5 specifically in platelets had a mild increase in thrombin responses in vitro and a shortened time to arterial thrombosis in vivo. In addition, platelet GRK5 mutant mice had increased thrombin but not collagen-induced thrombus burden in a mouse model of pulmonary embolism. ConclusionThese data indicate that platelet GRK5 has a significant role in limiting platelet responses to thrombin.

Carrying both the heterozygous Myh6-R453C and Tnnt2-R92W mutations aggravate the hypertrophic cardiomyopathy phenotype in mice

Hypertrophic cardiomyopathy (HCM) is an inherited disease of the heart muscle that is dominated by variations in eight genes encoding sarcomere proteins. Although there are clinical or basic research reports that carrying double mutations can lead to more severe HCM phenotypes, there are also research reports that after reanalyzing the reported mutations, the severity of clinical symptoms in patients with double mutations did not significantly increase compared to patients with only one mutation. To determine whether double pathogenic mutations can aggravate the phenotype of hypertrophic cardiomyopathy in mice, we constructed mice carrying single pathogenic heterozygous mutation Myh6-R453C or Tnnt2-R92W and mice carrying both pathogenic heterozygous mutations. Our results showed that mice with double heterozygous mutations exhibited significant hypertrophic cardiomyopathy phenotypes at 4 weeks of age, and the degree of hypertrophy was significantly higher than that of single heterozygous mutant mice of the same age. Our study suggests that carrying the two pathogenic heterozygous mutations simultaneously can aggravate the phenotype of HCM in mice, which provides experimental evidence for the genotype-phenotype relationship of double pathogenic mutations and provides reference significance for clinical risk stratification of HCM patients.

Npc1 deficiency impairs microglia function via TREM2-mTOR signaling in Niemann-Pick disease type C

Niemann-Pick disease Type C (NPC) is a neurodegenerative disease mainly caused by the mutation in NPC1 gene, leading to massive accumulation of unesterified cholesterol in the late endosome/lysosome of cells. Impaired phenotype of microglia is a hallmark in Npc1 mutant mice (Npc1−/− mice). However, the mechanism of Npc1 in regulating microglial function is still unclear. Here, we showed that the reactive microglia in the neonatal Npc1−/− mice indicated by the increased lysosome protein CD68 and phagocytic activity were associated with disrupted TREM2-mTOR signaling in microglia. Furthermore, in Npc1-deficient BV2 cells, genetic deletion of Trem2 partially restored microglial function, probably via restored mTOR signaling. Taken together, our findings indicated that loss of Npc1 in microglia caused changes of their morphologies and the impairment of lysosomal function, which were linked to the TREM2-mTOR signaling pathway.