Heterozygous KO Research Articles

AAV9/SLC6A1 gene therapy rescues abnormal EEG patterns and cognitive behavioral deficiencies in Slc6a1-/- mice.

The SLC6A1 gene encodes the gamma-aminobutyric acid (GABA) transporter GAT-1, the deficiency of which is associated with infantile encephalopathy with intellectual disability. We designed two AAV9 vectors, with either the JeT or MeP promoter, and conducted preclinical gene therapy studies using heterozygous and homozygous Slc6a1 KO mice at different developmental ages and various routes of administration. Neonatal intracerebroventricular administration of either vector resulted in significantly normalized EEG patterns in Slc6a1-/- or Slc6a1+/- mice, as well as improvement in several behavioral phenotypes of Slc6a1-/- mice. However, some mortality and adverse effects were observed in neonatal-treated mice. Intrathecal administration of either vector at postnatal day (PND) 5 normalized EEG patterns in Slc6a1+/- mice, but in Slc6a1-/- mice the treatment only rescued nest building without impact on EEG. Both vectors were well-tolerated in all mice treated at PND5 or later (including WT mice), up to 1 year post-injection. Overall, our data demonstrate compelling efficacy when mice are treated at an early development age. We also identified that outside of the neonatal treatment window, the severe homozygous KO model is more refractory to treatment, whereas our treatments in the heterozygous mice, which genotypically match human patients, have resulted in stronger benefits.

Consequences of GMPPB deficiency for neuromuscular development and maintenance.

Guanosine diphosphate-mannose pyrophosphorylase B (GMPPB) catalyzes the conversion of mannose-1-phosphate and GTP to GDP-mannose, which is required as a mannose donor for the biosynthesis of glycan structures necessary for proper cellular functions. Mutations in GMPPB have been associated with various neuromuscular disorders such as muscular dystrophy and myasthenic syndromes. Here, we report that GMPPB protein abundance increases during brain and skeletal muscle development, which is accompanied by an increase in overall protein mannosylation. To model the human disorder in mice, we generated heterozygous GMPPB KO mice using CIRSPR/Cas9. While we were able to obtain homozygous KO mice from heterozygous matings at the blastocyst stage, homozygous KO embryos were absent beyond embryonic day E8.5, suggesting that the homozygous loss of GMPPB results in early embryonic lethality. Since patients with GMPPB loss-of-function manifest with neuromuscular disorders, we investigated the role of GMPPB in vitro. Thereby, we found that the siRNA-mediated knockdown of Gmppb in either primary myoblasts or the myoblast cell line C2C12 impaired myoblast differentiation and resulted in myotube degeneration. siRNA-mediated knockdown of Gmppb also impaired the neuron-like differentiation of N2A cells. Taken together, our data highlight the essential role of GMPPB during development and differentiation, especially in myogenic and neuronal cell types.

Elovl4b knockout zebrafish as a model for ocular very-long-chain PUFA deficiency

Very-long-chain PUFAs (VLC-PUFAs) are a group of lipids with chain lengths >24 carbons, and the ELOVL4 (elongation of very-long-chain FA-4) enzyme is responsible for vertebrate VLC-PUFA biosynthesis. Studies on the role of VLC-PUFAs in vision have been hindered because of the need for adequate animal models to capture the global loss of VLC-PUFAs. Since homozygous Elovl4 ablation is lethal in neonatal mice because of catastrophic drying from the loss of their protective skin barrier, we established a zebrafish (Danio rerio) model of Elovl4 ablation. We generated Elovl4b KO zebrafish by creating a 56-bp deletion mutation in exon 2 of the Elovl4b gene using CRISPR-Cas9. We used GC-MS and LC-MS/MS to analyze the VLC-PUFA and lipid profiles from wild-type and Elovl4b KO fish eyes. We also performed histology and visual-behavioral tests. We found that heterozygous and homozygous Elovl4b KO zebrafish eyes had altered lipid profiles and a significantly lower C30 to C36 VLC-PUFA abundance than wild-type fish. Moreover, Elovl4b+/− and Elovl4b−/− KO larvae had significantly lower motor activity in response to light-dark cycles than their age-matched controls. Elovl4b−/− adult fish showed no obvious differences in gross retinal morphology and lamination compared with wild type, except for the presence of lipid droplets within the retinal pigment epithelial cell layer of Elovl4b−/− fish. Our data indicate that the loss of Elovl4b in zebrafish changes ocular lipid profiles and leads to visual abnormalities and subtle retinal changes. These findings highlight the use of zebrafish as a model for VLC-PUFA depletion and ELOVL4-related dysfunction.

GFPT1 deficiency is a novel cause of a dilated cardiomyopathy and is required for myocardial adaptation to chronic pressure-overload stress-induced heart failure

Abstract Background GFPT1 is the rate-limiting enzyme of a pathway called the hexosamine biosynthesis pathway (HBP) that turns fructose 6-phosphate derived from glucose into another monosaccharide called N-acetylglucosamine (GlcNAc). GlcNAc is an important substrate for modifying diverse cellular proteins on their serine and threonine residues termed O-GlcNAcylation often competing with phosphorylation and altering their activity or stability (Bond & Hanover; 2015). Increased GFPT1 activity is known to stimulate the cellular integrated stress response improving protein quality control and cell survival (Denzel et al 2014). In the heart, GFPT1 expression and O-GlcNAcylation increases in the early stages of chronic cardiac stress but the physiological role of GFPT1 in regulating heart function including its role during chronic stress remains unclear. Identifying molecular pathways that promote adaptive cardiac remodelling is a promising approach to discover novel therapies for heart failure. Methods Cardiomyocyte-specific GFPT1 knockout mice were generated to study the effects of GFPT1 deletion in hearts. Heart function was examined by echocardiography. HBP activity was quantified using Western blotting of O-GlcNAc-modified proteins. Abdominal aortic banding (AAB) was used to induce chronic pressure-overload heart failure. Results Homozygous GFPT1 KO mice spontaneously develop a dilated cardiomyopathy (DCM) with a 30% mortality rate (ejection fraction 32% compared with 62% for floxed controls and end diastolic volume 125 microL versus 60 microL; n=5-6 per group; p <0.05). Hearts showed reduced levels of total protein O-GlcNAcylation suggesting impaired hexosamine biosynthesis pathway activity. Heart function could be rescued by supplementing their diets with oral glucosamine bypassing GFPT1 to provide metabolites required for hexosamine biosynthesis and normalising O-GlcNAcylation (ejection fraction improved to 40% in KO with supplementation versus KO without; n=4-6 per group; p <0.05). Heterozygous KO mice do not develop a DCM but when subject to pressure-overload cardiac stress, develop worse heart failure with a 50% mortality rate over 9 weeks of pressure-overload (ejection fraction 43% in het KO versus 53% in floxed control; n= 3-6 per group; p = 0.0025). Conclusion Deficiency of GFPT1 is a novel cause of a spontaneous dilated cardiomyopathy. GFPT1 is required for physiological growth of the heart that may in part involve the O-GlcNAcylation of proteins. Partial GFPT1 deficiency is associated with adverse cardiac remodelling following pressure-overload stress. Increasing GFPT1 activity may be a key therapeutic target in developing novel heart failure treatments.

Conditional deletion of LRRC8A in the brain reduces stroke damage independently of swelling-activated glutamate release

The ubiquitous volume-regulated anion channels (VRACs) facilitate cell volume control and contribute to many other physiological processes. Treatment with non-specific VRAC blockers or brain-specific deletion of the essential VRAC subunit LRRC8A is highly protective in rodent models of stroke. Here, we tested the widely accepted idea that the harmful effects of VRACs are mediated by release of the excitatory neurotransmitter glutamate. We produced conditional LRRC8A knockout either exclusively in astrocytes or in the majority of brain cells. Genetically modified mice were subjected to an experimental stroke (middle cerebral artery occlusion). The astrocytic LRRC8A knockout yielded no protection. Conversely, the brain-wide LRRC8A deletion strongly reduced cerebral infarction in both heterozygous (Het) and full KO mice. Yet, despite identical protection, Het mice had full swelling-activated glutamate release, whereas KO animals showed its virtual absence. These findings suggest that LRRC8A contributes to ischemic brain injury via a mechanism other than VRAC-mediated glutamate release.

Complementary Square-Wave Voltammetry and LC-MS/MS Analysis to Elucidate Induced Damaged and Mutated Mitochondrial and Nuclear DNA from in Vivo Knockdown of the BRCA1 Gene in the Mouse Skeletal Muscle.

Breast cancer 1 gene (BRCA1) DNA mutations impact skeletal muscle functions. Inducible skeletal muscle specific Brca1 homozygote knockout (Brca1KOsmi, KO) mice accumulate mitochondrial DNA (mtDNA) mutations resulting in loss of muscle quality.1 Complementary electrochemical andmass spectrometry analyses were utilized to rapidly assess mtDNA or nuclear DNA (nDNA) extracted directly from mouse skeletal muscles. Oxidative peak currents (Ip) from DNA immobilized layer by layer (LbL) were monitored using square-wave voltammetry (SWV) via Ru(bpy)32+ electrocatalysis. Ip significantly decreased (p < 0.05) for KO mtDNA compared to heterozygous KO (Het) or wild type (WT), indicative of decreases in the guanine content. nDNA Ip significantly increased in KO compared to WT (p < 0.05), suggesting an accumulation of damaged nDNA. Guanine or oxidatively damaged guanine content was monitored via appropriate m/z mass transitions using liquid chromatography-tandem mass spectroscopy (LC-MS/MS). Guanine in both KO mtDNA and nDNA was significantly lower, while oxidatively damaged guanine in KO nDNA was significantly elevated versus WT. These data demonstrate a loss of guanine content consistent with mtDNA mutation accumulation. Oxidative damage in KO nDNA suggests that repair processes associated with Brca1 are impacted. Overall, electrochemical and LC-MS/MS analysis can provide chemical-level answers to biological model phenotypic responses as a rapid and cost-effective analysis alternative to established assays.

A reporter system for enriching CRISPR/Cas9 knockout cells in technically challenging settings like patient models

CRISPR/Cas9 represents a valuable tool to determine protein function, but technical hurdles limit its use in challenging settings such as cells unable to grow in vitro like primary leukemia cells and xenografts derived thereof (PDX). To enrich CRISPR/Cas9-edited cells, we improved a dual-reporter system and cloned the genomic target sequences of the gene of interest (GOI) upstream of an out-of-frame fluorochrome which was expressed only upon successful gene editing. To reduce rounds of in vivo passaging required for PDX leukemia growth, targets of 17 GOI were cloned in a row, flanked by an improved linker, and PDX cells were lentivirally transduced for stable expression. The reporter enriched scarce, successfully gene-edited PDX cells as high as 80%. Using the reporter, we show that KO of the SRC-family kinase LYN increased the response of PDX cells of B precursor cell ALL towards Vincristine, even upon heterozygous KO, indicating haploinsufficiency. In summary, our reporter system enables enriching KO cells in technically challenging settings and extends the use of gene editing to highly patient-related model systems.

Jazf1 acts as a regulator of insulin-producing β-cell differentiation in induced pluripotent stem cells and glucose homeostasis in mice.

Genetic susceptibility of type 2 diabetes and Juxtaposed with another zinc finger protein 1 (Jazf1) has been reported; however, the precise role of Jazf1 in metabolic processes remains elusive. In this study, using Jazf1-knockout (KO)-induced pluripotent stem cells (iPSC), pancreatic beta cell line MIN6 cells, and Jazf-1 heterozygous KO (Jazf1+/- ) mice, the effect of Jazf1 on gradual differentiation was investigated. We checked the alterations of the genes related with β-cell specification, maturation, and insulin release against glucose treatment by the gain and loss of the Jazf1 gene in the MIN6 cells. Because undifferentiated Jazf1-KO iPSC were not significantly different from wild-type (WT) iPSC, the size and endoderm marker expression after embryoid body (EB) and teratoma formation were investigated. Compared to EB and teratomas formed with WT iPSC, the EB and teratomas from with Jazf1-KO iPSC were smaller, and in teratomas, the expression of proliferation markers was reduced. Moreover, the expression of the gene sets for β-cell differentiation and the levels of insulin and C-peptide secreted by insulin precursor cells were notably reduced in β-cells differentiated from Jazf1-KO iPSC compared with those differentiated from WT iPSC. A comparison of Jazf1+/- and WT mice showed that Jazf1+/- mice had lower levels of serum insulin, pancreatic insulin expression, and decreased pancreatic β-cell size, which resulted in defects in the glucose homeostasis. These findings suggest that Jazf1 plays a pivotal role in the differentiation of β-cells and glucose homeostasis.

Mutations of MAP1B encoding a microtubule-associated phosphoprotein cause sensorineural hearing loss.

The pathophysiology underlying spiral ganglion cell defect–induced deafness remains elusive. Using the whole exome sequencing approach, in combination with functional assays and a mouse disease model, we identified the potentially novel deafness-causative MAP1B gene encoding a highly conserved microtubule-associated protein. Three novel heterozygous MAP1B mutations (c.4198A>G, p.1400S>G; c.2768T>C, p.923I>T; c.5512T>C, p.1838F>L) were cosegregated with autosomal dominant inheritance of nonsyndromic sensorineural hearing loss in 3 unrelated Chinese families. Here, we show that MAP1B is highly expressed in the spiral ganglion neurons in the mouse cochlea. Using otic sensory neuron–like cells, generated by pluripotent stem cells from patients carrying the MAP1B mutation and control subject, we demonstrated that the p.1400S>G mutation caused the reduced levels and deficient phosphorylation of MAP1B, which are involved in the microtubule stability and dynamics. Strikingly, otic sensory neuron–like cells exhibited disturbed dynamics of microtubules, axonal elongation, and defects in electrophysiological properties. Dysfunctions of these derived otic sensory neuron–like cells were rescued by genetically correcting MAP1B mutation using CRISPR/Cas9 technology. Involvement of MAP1B in hearing was confirmed by audiometric evaluation of Map1b heterozygous KO mice. These mutant mice displayed late-onset progressive sensorineural hearing loss that was more pronounced in the high frequencies. The spiral ganglion neurons isolated from Map1b mutant mice exhibited the deficient phosphorylation and disturbed dynamics of microtubules. Map1b deficiency yielded defects in the morphology and electrophysiology of spiral ganglion neurons, but it did not affect the morphologies of cochlea in mice. Therefore, our data demonstrate that dysfunctions of spiral ganglion neurons induced by MAP1B deficiency caused hearing loss.

Homozygous G650del nexilin variant causes cardiomyopathy in mice.

Nexilin (NEXN) was recently identified as a component of the junctional membrane complex required for development and maintenance of cardiac T-tubules. Loss of Nexn in mice leads to a rapidly progressive dilated cardiomyopathy (DCM) and premature death. A 3 bp deletion (1948–1950del) leading to loss of the glycine in position 650 (G650del) is classified as a variant of uncertain significance in humans and may function as an intermediate risk allele. To determine the effect of the G650del variant on cardiac structure and function, we generated a G645del-knockin (G645del is equivalent to human G650del) mouse model. Homozygous G645del mice express about 30% of the Nexn expressed by WT controls and exhibited a progressive DCM characterized by reduced T-tubule formation, with disorganization of the transverse-axial tubular system. On the other hand, heterozygous Nexn global KO mice and genetically engineered mice encoding a truncated Nexn missing the first N-terminal actin-binding domain exhibited normal cardiac function, despite expressing only 50% and 20% of the Nexn, respectively, expressed by WT controls, suggesting that not only quantity but also quality of Nexn is necessary for a proper function. These findings demonstrated that Nexn G645 is crucial for Nexn’s function in tubular system organization and normal cardiac function.

Peptidylarginine Deiminase 4 Promotes the Renal Infiltration of Neutrophils and Exacerbates the TLR7 Agonist-Induced Lupus Mice

Peptidylarginine deiminase 4 (PAD4), encoded by PADI4, plays critical roles in the immune system; however, its contribution to the pathogenesis of lupus nephritis remains controversial. The pathological roles of PAD4 were investigated in lupus model mice. An imiquimod (IMQ)-induced lupus model was analyzed in wild-type (WT) and Padi4-knockout (KO) mice. Proteinuria, serum anti-double stranded DNA (anti-dsDNA) antibody, and renal infiltrated cells were evaluated. Neutrophil migration and adhesion were assessed using adoptive transfer and adhesion assay. PAD4-regulated pathways were identified by RNA-sequencing of Padi4 KO neutrophils. Padi4 KO mice exhibited significant improvements in proteinuria progression compared with WT mice, whereas, serum anti-dsDNA antibody and immune complex deposition in the glomeruli showed no difference between both mice strains. Padi4 KO mice showed decreased neutrophil infiltration in the kidneys. Adoptively transferred Padi4 KO neutrophils showed decreased migration to the kidneys of IMQ-treated WT mice, and adhesion to ICAM-1 was impaired in Padi4 KO neutrophils. Padi4 KO neutrophils exhibited reduced upregulation of p38 mitogen-activated protein kinase (MAPK) pathways. Toll-like receptor 7 (TLR7)-primed Padi4 KO neutrophils demonstrated reduced phosphorylation of p38 MAPK and lower expression of JNK-associated leucine zipper protein (JLP), a p38 MAPK scaffold protein. Neutrophils from heterozygous Jlp KO mice showed impaired adhesion to ICAM-1 and decreased migration to the kidneys of IMQ-treated WT mice. These results indicated a pivotal role of PAD4-p38 MAPK pathway in renal neutrophil infiltration in TLR7 agonist-induced lupus nephritis, and the importance of neutrophil-mediated kidney inflammation. Inhibition of the PAD4-p38 MAPK pathway may help in formulating a novel therapeutic strategy against lupus nephritis.

Reduced Expression of the PP2A Methylesterase, PME-1, or the PP2A Methyltransferase, LCMT-1, Alters Sensitivity to Beta-Amyloid-Induced Cognitive and Electrophysiological Impairments in Mice.

Beta-amyloid (Aβ) is thought to play a critical role in Alzheimer's disease (AD), and application of soluble oligomeric forms of Aβ produces AD-like impairments in cognition and synaptic plasticity in experimental systems. We found previously that transgenic overexpression of the PP2A methylesterase, PME-1, or the PP2A methyltransferase, LCMT-1, altered the sensitivity of mice to Aβ-induced impairments, suggesting that PME-1 inhibition may be an effective approach for preventing or treating these impairments. To explore this possibility, we examined the behavioral and electrophysiological effects of acutely applied synthetic Aβ oligomers in male and female mice heterozygous for either a PME-1 KO or an LCMT-1 gene-trap mutation. We found that heterozygous PME-1 KO mice were resistant to Aβ-induced impairments in cognition and synaptic plasticity, whereas LCMT-1 gene-trap mice showed increased sensitivity to Aβ-induced impairments. The heterozygous PME-1 KO mice produced normal levels of endogenous Aβ and exhibited normal electrophysiological responses to picomolar concentrations of Aβ, suggesting that reduced PME-1 expression in these animals protects against Aβ-induced impairments without impacting normal physiological Aβ functions. Together, these data provide additional support for roles for PME-1 and LCMT-1 in regulating sensitivity to Aβ-induced impairments, and suggest that inhibition of PME-1 may constitute a viable therapeutic approach for selectively protecting against the pathologic actions of Aβ in AD.SIGNIFICANCE STATEMENT Elevated levels of β-amyloid (Aβ) in the brain are thought to contribute to the cognitive impairments observed in Alzheimer's disease patients. Here we show that genetically reducing endogenous levels of the PP2A methylesterase, PME-1, prevents the cognitive and electrophysiological impairments caused by acute exposure to pathologic concentrations of Aβ without impairing normal physiological Aβ function or endogenous Aβ production. Conversely, reducing endogenous levels of the PP2A methyltransferase, LCMT-1, increases sensitivity to Aβ-induced impairments. These data offer additional insights into the molecular factors that control sensitivity to Aβ-induced impairments, and suggest that inhibiting PME-1 may constitute a viable therapeutic avenue for preventing Aβ-related impairments in Alzheimer's disease.

Abstract P3-03-07: TNFR-signaling in breast cancer development

Abstract Introduction: Breast cancer (BC) is the 2nd leading cause of cancer death among women in the US. Known risk factors for the development of BC include thoracic radiotherapy during puberty and obesity with the latter turning into a nation-wide health crisis. The underlying mechanisms that lead to increased BC incidences in these two scenarios are incompletely understood. However, exposure to ionizing radiation (IR) or obesity generate pro-inflammatory conditions that have been linked to mammary carcinogenesis. As inflammation involves signaling through TNF receptors (TNFR), we have therefore employed crosses between mice that spontaneously develop BC (MMTV-Wnt1) and TNFR KO mice to study the impact of TNFR downstream signaling on BC development. Material and Methods: MMTV-Wnt1 were crossed with TNFR1 or TNFR2 KO mice to explore the BC incidence during the first year post-partum in both genders. Mammary gland area and ductal outgrowth were evaluated by whole mount histological staining with 6-week-old female (end of puberty) and 14-week-old mice (adult). Tumor cells were harvested from tumor-developed MMTV-Wnt1 and MMTV-Wnt1 x TNFR2 KO female mice, the population of tumor initiating cells (EpCAMlow/CD49fhi) was analyzed by FACS. Vasculature formation (CD31), cell proliferation (Ki67) and immune system (CD4, CD8, F4/80) were evaluated within tumors by immunohistochemistry staining. Tumor cells were digested out from primary tumors and cultured in spheres condition. In-vitro sphere formation assay was performed w/o IR or TNFα treatment. All quantitative results were treated by ANOVA and Bonferroni analysis, upon verification of normal data distribution with α value at 0.05. Results: TNFR2KO drastically accelerates BC in female animals but not in males. In contrary, TNFR1 KOhad no effect on female BC development but completely prevented BC in males. TNFR2 KO led to elongation of mammary gland duct system, expansion of mammary epithelial stem cell pool and increase of BC initiating cells, as well as more vasculatures and proliferating cells in developed tumors. T-cellsand macrophage content in established tumors don’t correlate with differences in BC incidence between the crosses. MMTV-Wnt1 x TNFR2 KO mammospheres were more resistant to IR compared to Wnt1 x TNFR1 KO and MMTV-Wnt1. Pro-inflammatory TNFα ligand increased the sphere formation in a dose-dependent manner within Wnt1 x TNFR1 KO and MMTV-Wnt1 spheres, but not in Wnt1 x TNFR2 KO ones. Conclusion: Heterozygous TNFR2 KO in female MMTV-Wnt1 Tg mice increases the BC incidence, along with more resistance to IR, suggesting the downstream of TNFR2 may potentially suppress breast carcinogenesis. Also TNFR2 KO leads to more mammary gland branching and expansion of epithelial stem cells during development, which imply the potential use of TNFR2 agonist in thoracic radiotherapy and obese scenarios to reduce the BC incidence. Citation Format: Ling He, Kruttika Bhat, Paul Medina, Claudia Alli, Mohammad Saki, Fei Cheng, Frank Pajonk. TNFR-signaling in breast cancer development [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-03-07.

Abstract WP321: The Role of Neuronal Sirt1 in Ischemic Stroke: Regulation of the Metabolic Imbalance

Introduction: Sirt1 is a NAD+ dependent deacetylase that is known as an important metabolic regulator for type 2 diabetes melitus. To determine the role of Sirt1 in ischemic stroke-induced metabolic imbalance, we generated the neuronal specific Sirt1 knock-in (nSirt1 KI) and knock-out (Sirt1 KO) mice and examined its effect and mechanism. Methods: To determine the role of Sirt1 in ischemic stroke-induced impaired metabolic actions including insulin and glucose abnormality, we tested the pharmacological effects of Metformin and Sirtinol as Sirt1 activator and inhibitor. To determine the genetic role of neuronal Sirt1 in the brain, we generated the nSirt1 KI mice and Sirt1 KO. We used the Ischemia/Reperfusion (I/R: 1h middle cerebral artery occlusion (MCAO)/23h reperfusion) injury model to induce the abnormal metabolic changes. Infarction volume was measured by TTC staining and we determined neurological deficit score (NDS) at 23h after 1h MCAO. The level of glucose and insulin were measured by a drop of tail blood using test-strip and ELISA Kit before, during (every 10 minutes interval), and after MCAO. Results: Metformin reduced infarction volume and Sirtinol worsened the infarction volume via Sirt1 signaling pathway. Heterozygous Sirt1 KO (Sirt1 +/-) mice showed decreased relative cerebral blood flow (rCBF) and worsen infarction volume in ischemic areas, whereas nSirt1 KI mice exhibited increased rCBF, improved NDS, regulating abnormal blood glucose and insulin, and reduced infarction volume by 42% compare to that of littermate control in I/R injury model (n=7, p&lt;0.05). Based on western blotting analysis of brain lysates in nSirt1 KI mice, the expression of Sirt1 was significantly increased. Glucose regulation by overexpression of Sirt1 and reduced infarction volume in nSirt1 KI mice were reversed by Sirtinol. Conclusion: These findings indicate that the important neuroprotective effect of neuronal cells is mediated by Sirt1 overexpression and suggest that upregulation of neuronal Sirt1 may be an important therapeutic target for ischemic stroke via regulating metabolic imbalance.

P313ADGRL2 is an essential surface molecule for cardiac lineage specification and heart development

Abstract Background Specific surface markers that enable monitoring of cell subsets would be valuable for establishing the conditions under which pluripotent stem cells (PSCs) differentiate into cardiac progenitor cells (CPCs) and cardiomyocytes (CMCs). Methods and results To verify whether a specific marker is expressed during heart development, we assessed its expression using the CLARITY technique. After immersion in a solution with a refractive index matching that of the CLARITY hybrid, the mouse embryo became transparent. After immunostaining the cleared embryo sample, Adgrl2 was exclusively observed in cardiac cells expressing α-SA at embryonic day E9.5 and E10.5. Our clarified 3D images and movies show that four chambers of the heart are fully developed at E10.5 but not at E9.5. At E9.5, Adgrl2 is observed at the ventricle and atrium, while Adgrl2 is present in all chambers of the heart at E10.5. Next, we performed LacZ (β-Gal) staining in heterozygous Adgrl2 KO embryos to evaluate Adgrl2 expression. As a result, LacZ staining showed that Adgrl2 was predominantly expressed in the heart during the embryonic developmental stage. Adgrl2 knockout in mice was embryonically lethal because of severe heart, but not vascular, defects. To examine the use of Adgrl2 as a bona fide CPC marker during heart development, we tracked Adgrl2 expression during early embryonic development. The heart of Adgrl2−/− embryos at E10.5 exhibited occlusion of the RV, and the expression levels of Gata4 and Nkx2.5 were not as high as those in wild-type and Adgrl2+/− embryos. Interestingly, the heart of Adgrl2−/− embryos, unlike those of wild-type and Adgrl2+/− embryos between E13.5 and E15.5 had a single ventricle revealing a ventricular septal defect. The specific expression pattern of Adgrl2 in PSC-derived cardiac lineage cells as well as in embryonic heart, adult mice, and human heart tissues. Conclusion We demonstrate that Adgrl2 plays a pivotal and functional role across all strata of the cardiomyogenic lineage, as early as the precursor stage of heart development. These findings shed light on heart development and regeneration. Acknowledgement/Funding Grants from “Strategic Center of Cell and Bio Therapy” (grant number: HI17C2085) and “Korea Research-Driven Hospital” (HI14C1277)

201-LB: The Neuronal Sirt1 Regulate the Metabolic Imbalance in Ischemic Stroke

Background: Sirt1 is a NAD+ dependent deacetylase that is known as an important metobolic regulator for type 2 diabetes mellitus. To determine the role of Sirt1 in ischemic stroke induced metabolic imbalance, we generated the neuronal specific Sirt1 knock-in (nSirt1 KI) and knock-out (Sirt1 KO) mice and examined its effect and mechanism. Methods: To determine the role of Sirt1 in ischemic stroke induced impaired metabolic actions including insulin and glucose abnormality, we tested the pharmacological effects of metformin and sirtinol. To determine the genetic role of neuronal Sirt1 in the brain, we generated the nSirt1 KI mice. We used the Ischemia/Reperfusion (I/R: 1h MCAO/23h reperfusion) injury model to induce the abnormal metabolic changes. Infarction volume was measured by TTC staining and we determined neurological deficit score (NDS) at 23h after 1h MCAO. The level of glucose was measured by a drop of tail blood using test-strip before, during (every 10 minutes interval), and after MCAO. Results: Metformin reduced infarction volume and sirtinol worsened the infarction volume via Sirt1 signaling pathway. Heterozygous Sirt1 KO (Sirt1+/-) mice showed decreased relative cerebral blood flow and worsen infarction volume in ischemic areas, whereas nSirt1 KI mice exhibited increased rCBF, improved NDS, regulating abnormal blood glucose, and reduced infarction volume by 42% compare to that of littermate control in I/R injury model (n=7, p&amp;lt;0.05). Based on western blotting analysis of brain tissue lysates in nSirt1 KI mice, the expression of Sirt1 was significantly increased. Glucose regulation by overexpression of Sirt1 and reduced infarction volume in nSirt1 KI mice was reversed by sirtinol. Conclusion: These findings indicate that the important neuroprotective effect of neuronal cells is mediated by Sirt1 overexpression and suggest that upregulation of neuronal Sirt1 may be an important therapeutic target for ischemic stroke via regulating metabolic imbalance. Disclosure G. Li: None. H. Kim: None. Funding Jeongseon County

Vps4b heterozygous mice do not develop tooth defects that replicate human dentin dysplasia I

BackgroundVacuolar protein sorting-associated protein 4B (VPS4B) is a member of the ATP enzyme AAA protein family, and is mainly involved in protein degradation and cell membrane fusion. Recently, a dominant mutation in this gene was identified in human dentin dysplasia type I (DD-I). Herein, we report the generation of Vps4b knockout (Vps4b KO) mice; however, the homozygous Vps4b KO mutation was embryonic lethal at the early stages of embryo development, and we therefore report the results of heterozygous mutant mice.ResultsMice heterozygous for Vps4b did not develop tooth defects replicating human DD-I. Immunohistochemistry showed that gene KO was successful, as there was decreased expression of Vps4b in heterozygous mice; hematoxylin and eosin (H&E) staining also showed that the width of the pre-dentin zone was increased in heterozygous mice, although the arrangement of the odontoblasts was not significantly different from wild-type (WT) mice. However, H&E staining showed no obvious abnormalities in the bones of heterozygous mice. Moreover, stereomicroscopic and X-ray radiography results indicated no abnormal manifestations in teeth or bones. Furthermore, statistical analysis of the volume and density of dentin and enamel, as well as skeletal analysis, including the volume and separation of trabecular bone analyzed by micro-CT, all showed no differences between Vps4b heterozygotes and WT mice. In addition, there also were no significant differences in bone or cartilage mineralization as evaluated by Alcian blue–Alizarin red staining.ConclusionsThe heterozygous Vps4b KO mice do not develop tooth defects that replicate human DD-I and this is likely to be due to differences in tooth development between the two species. Consequently, further studies are needed to determine whether mice are an appropriate animal model for human tooth diseases.

12: Evidence for the crucial role of estrogen signaling with preterm labor and perinatal neuroinflammation

Female offspring are less susceptible to prematurity-related neuroinflammatory injury than males. We hypothesize such differences are attributed to sex-specific hormones, specifically estrogen. Using a murine prematurity model, we evaluated the role of estrogen signaling in the process of perinatal brain injury following exposure to intrauterine inflammation (IUI) by: 1) Characterizing the principle components of signaling (Esr1 and Esr2) and synthesis (Cyp19) and 2) Evaluating the phenotype with Esr1 deletion. To simulate IUI, intrauterine lipopolysaccharide (LPS) was used to invoke preterm labor and fetal neuroinflammation and compared to controls. In the set of CD1 dams, fetal brains were collected at 6 hours and analyzed by qRT-PCR, western blot and immunohistochemistry. Additionally, females heterozygous for Esr1 KO allele were bred to heterozygous Esr1 KO males. In this set, dams were monitored for preterm labor and embryo viability. Esr1 and Esr2 transcripts significantly increased with LPS exposure while protein levels declined (Figure 1A-B). Though inverse, the level of Cyp19 did not differ by mRNA or protein analysis. Both Esr1 and Esr2 co-localized to the nuclei of developing neural cells and decreased with LPS (Figure 1C). Dams heterozygous for the Esr1 KO allele did not tolerate LPS with 100% mortality in all exposed offspring (stillborn at gestational days 18-19 of the 21 expected days). These offspring were also significantly smaller, by pup weight as well as head and body size, compared to controls (Figure 2). The reduction of Esr1 and Esr2 by western blot suggests that these proteins may be degraded with neuroinflammation. It is possible that transcriptional upregulation of Esr1 and Esr2 occurs to compensate for the loss of these proteins. Alternatively, the translation of Esr1 and Esr2 mRNAs is disrupted with LPS and upregulation is due to a feedback mechanism. Based on the receptors' nuclear location, it is possible that their transcriptional activity and regulation are altered with neuroinflammation. Correspondingly, lack of fetal viability resulting from Esr1 insufficiency with LPS injury supports the importance of estrogen signaling with preterm labor. Future research will evaluate whether these findings represent an exaggerated maternal response or a susceptible fetal genotype.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Partial loss of psychiatric risk gene Mir137 in mice causes repetitive behavior and impairs sociability and learning via increased Pde10a.

Genetic analyses have linked MicroRNA-137 (MIR137) to neuropsychiatric disorders, including schizophrenia and autism spectrum disorder (ASD). MiR-137 plays important roles in neurogenesis and neuronal maturation, but the impact of miR-137 loss of function in vivo remains unclear. Here we show the complete loss of miR-137 in the mouse germline (gKO) or nervous system (cKO) leads to postnatal lethality, while heterozygous gKO and cKO mice remain viable. Partial loss of miR-137 in heterozygous cKO mice results in dysregulated synaptic plasticity, repetitive behavior, and impaired learning and social behavior. Transcriptomic and proteomic analyses revealed that the miR-137 mRNA target, Phosphodiesterase 10a (Pde10a), is elevated in heterozygous KO mice. Treatment with the PDE10A inhibitor papaverine or knockdown of Pde10a ameliorates the deficits observed in the heterozygous cKO mice. Collectively, our results suggest that MIR137 plays essential roles in postnatal neurodevelopment, and that dysregulation of miR-137 potentially contributes to neuropsychiatric disorders in humans.

Abstract 100: PRDM16 is a Novel Regulator of Cardiac Hypertrophy, Remodeling and Mitochondrial Dynamics

Elucidation of the mechanisms that cause cardiac hypertrophy is essential for developing novel therapeutic strategies to prevent or reverse these prevalent conditions. Prdm16/Mel1 (PRDM16) belongs to the Prdm family of transcription factors involved in cell fate determination and development. PRDM16 deletion, found in humans with 1p36 deletion syndrome, has been associated with two main cardiomyopathies: left ventricle non-compaction (LVNC) and dilated cardiomyopathy. Nonetheless, its role in the heart and the underlying mechanisms by which these mutations contribute to the development of cardiomyopathy are unknown. We sought to directly assess the role of Prdm16 in the developing and adult heart by generating both a germline (cPRDM16) and a tamoxifen-inducible (ciPRDM16) cardiomyocyte-specific Prdm16 knockout mice. cPRDM16KO mice died shortly after birth, showing grossly abnormal ventricles that were reduced in size and often exhibiting a cleft at their apex, consistent with LVCN. Both heterozygous mice (cPRDM16KOhet) and ciPRDM16KO are viable and displayed markedly increased heart size, cardiomyocyte cross sectional area, collagen deposition and expression of genes involved in cardiac hypertrophy and ECM production. We observed an increased in TGF-β receptors mRNA expression as well as activation of Smad signaling in the hearts of cPRDM16KO neonates, cPRDM16 heterozygous, and ciPRDM16 KO mice. Interestingly, we observed a systematic reduction in Drp1 phosphorylation on Ser616 (activation) in the hearts of cPRDM16KO and cPRDM16het mice. The reduction in Drp1 phosphorylation was associated with more tubular mitochondria in cPRDM16KO mice. Interestingly, by 25 weeks, cPRDM16het mice accumulated more mitochondrial proteins, despite exhibiting no change in PGC1α. Together, these data reveal previously unknown roles for Prdm16 in protection against cardiac hypertrophy and fibrosis through the suppression of TGF-β/Smad signaling in the heart. They also reveal roles for PRDM16 in the modulation of mitochondrial dynamics and clearance in the heart, a process that likely involves the fission protein Drp1.