Actin Promoter Research Articles

Activated myofibroblasts promote cardiac hypertrophy and systolic dysfunction independently of cardiac fibrosis in experimental autoimmune myocarditis

Abstract Background/Introduction Heart-specific inflammation – myocarditis is a common cause dilated cardiomyopathy which is characterized by pathological tissue remodeling, ventricular stiffening, cardiomyopathy and heart failure. In experimental autoimmune myocarditis (EAM) susceptible mice immunized with alpha myosin heavy chain (αMyHC) and complete Freund's adjuvant (CFA) develop acute myocarditis driven by autoreactive CD4+ T cells that is followed by progressive fibrosis, cardiomyopathy and systolic dysfunction. Purpose The aim of the study was to investigate the role of cardiac fibroblasts and myofibroblasts in myocarditis and post-inflammatory dilated cardiomyopathy in mouse model of EAM. Methods EAM was induced in BALB/c mice by immunization with αMyHC/CFA. We used reporter mice expressing EGFP under collagen type I promoter (Coll-EGFP) and RFP under a control of α-smooth muscle actin (αSMA) promoter (αSMA-RFP) and transgenic αSMA-TK mice with ganciclovir-inducible ablation of proliferating myofibroblasts. Cardiac cells were quantified using flow cytometry. Cardiac fibroblasts (CD45-CD31-EGFP+) were sorted from healthy and myocarditis-positive (day 21) mice using BD FACSAria™ II Cell Sorter and analyzed for the whole genome transcriptomics by RNA sequencing. Echocardiography was performed on Vevo 2100 Imaging System. Cardiac fibrosis was assessed by Trichrome Massons's staining and hydroxyproline assay, whereas cardiac hypertrophy by analysing cross-sectional cardiomyocyte area. Profibrotic gene expression was assessed by qRT-PCR. Results The total number of cardiac fibroblasts (CD45-CD31-EGFP+) and the subset of myofibroblasts (CD45-CD31-EGFP+RFP+) remained unchanged at inflammatory (d21) and fibrotic stages (d40). Analysis of differentially expressed genes (min. 2x fold change, p value <0.05) pointed out activation of immune processes (mainly chemokine production), response to stress, cytoskeletal and extracellular matrix re-organization in cardiac fibroblasts in response to myocarditis. αSMA-TK mice treated with ganciclovir (from day 21) showed comparable percent of fibrotic area, but significantly reduced heart weight, decreased cardiomyocyte hypertrophy and improved ejection fraction and cardiac output at day 40 comparing to PBS-treated mice. Ganciclovir-treated mice showed also attenuated cardiac Acta2 and Srf but markedly enhanced Mmp2 expression. Conclusions In EAM model cardiac fibroblasts actively participate in proinflammatory and profibrotic responses, while activated myofibroblasts contribute to dilated cardiomyopathy development independently of cardiac fibrosis. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Science Centre (Poland)

Choroideremia Gene Therapy.

Choroideremia Gene Therapy.

Knockdown of Dehydrodolichyl Diphosphate Synthase in the Drosophila Retina Leads to a Unique Pattern of Retinal Degeneration.

Dehydrodolichyl diphosphate synthase (DHDDS) is a ubiquitously expressed enzyme that catalyzes cis-prenyl chain elongation to produce the poly-prenyl backbone of dolichol. It appears in all tissues including the nervous system and it is a highly conserved enzyme that can be found in all animal species. Individuals who have biallelic missense mutations in the DHDDS gene are presented with non-syndromic retinitis pigmentosa with unknown underlying mechanism. We have used the Drosophila model to compromise DHDDS ortholog gene (CG10778) in order to look for cellular and molecular mechanisms that, when defective, might be responsible for this retinal disease. The Gal4/UAS system was used to suppress the expression of CG10778 via RNAi-mediated-knockdown in various tissues. The resulting phenotypes were assessed using q-RT-PCR, transmission-electron-microscopy (TEM), electroretinogram, antibody staining and Western blot analysis. Targeted knockdown of CG10778-mRNA in the early embryo using the actin promoter or in the developing wings using the nub promoter resulted in lethality, or wings loss, respectively. Targeted expression of CG10778-RNAi using the glass multiple reporter (GMR)-Gal4 driver (GMR-DHDDS-RNAi) in the larva eye disc and pupal retina resulted in a complex phenotype: (a) TEM retinal sections revealed a unique pattern of retinal-degeneration, where photoreceptors R2 and R5 exhibited a nearly normal structure of their signaling-compartment (rhabdomere), but only at the region of the nucleus, while all other photoreceptors showed retinal degeneration at all regions. (b) Western blot analysis revealed a drastic reduction in rhodopsin levels in GMR-DHDDS-RNAi-flies and TEM sections showed an abnormal accumulation of endoplasmic reticulum (ER). To conclude, compromising DHDDS in the developing retina, while allowing formation of the retina, resulted in a unique pattern of retinal degeneration, characterized by a dramatic reduction in rhodopsin protein level and an abnormal accumulation of ER membranes in the photoreceptors cells, thus indicating that DHDDS is essential for normal retinal formation.

An efficient vector-based CRISPR/Cas9 system in an Oreochromis mossambicus cell line using endogenous promoters

CRISPR/Cas9 gene editing is effective in manipulating genetic loci in mammalian cell cultures and whole fish but efficient platforms applicable to fish cell lines are currently limited. Our initial attempts to employ this technology in fish cell lines using heterologous promoters or a ribonucleoprotein approach failed to indicate genomic alteration at targeted sites in a tilapia brain cell line (OmB). For potential use in a DNA vector approach, endogenous tilapia beta Actin (OmBAct), EF1 alpha (OmEF1a), and U6 (TU6) promoters were isolated. The strongest candidate promoter determined by EGFP reporter assay, OmEF1a, was used to drive constitutive Cas9 expression in a modified OmB cell line (Cas9-OmB1). Cas9-OmB1 cell transfection with vectors expressing gRNAs driven by the TU6 promoter achieved mutational efficiencies as high as 81% following hygromycin selection. Mutations were not detected using human and zebrafish U6 promoters demonstrating the phylogenetic proximity of U6 promoters as critical when used for gRNA expression. Sequence alteration to TU6 improved mutation rate and cloning efficiency. In conclusion, we report new tools for ectopic expression and a highly efficient, economical system for manipulation of genomic loci and evaluation of their causal relationship with adaptive cellular phenotypes by CRISPR/Cas9 gene editing in fish cells.

Generation of Transgenic Medaka Oryzias curvinotus (Nichols & Pope, 1927) Carrying a Cyan Fluorescent Protein Gene Driven by Alpha Actin Promoter

The study aimed to produce fluorescent protein transgenic medaka Oryzias curvinotus (Nichols & Pope, 1927) as a novel strain of ornamental fish. These fish were produced by transferring a plasmid consisting of a fluorescent reporter gene and a strong promoter into one-cell stage embryos. For this purpose, myosin light chain 2, but not other promoters, was mainly used. The study also evaluated the stability of the transgenic medaka germline acquiring vivid fluorescent phenotypes via the transgenesis of the cyan fluorescent protein (CFP) gene under the control of O. curvinotus skeletal alpha-actin (OCacta) promoter. The pOCacta-CFP plasmid, containing a OCacta promoter and CFP reporter gene, was transferred into the one-cell stage of O. curvinotus embryos by a microinjection technique. As a result, 36 of 1386 microinjected O. curvinotus embryos exhibited CFP signals in their trunks. The expressed CFP signals in O. curvinotus embryos and adults were detected under a microscope using a green fluorescent protein (GFP) filter (450–490 nm wavelength), and blue LED light (400–450 nm wavelength). Five O. curvinotus founders showing clear CFP signals were selected and crossed with non-transgenic counterparts to produce subsequent generations. Among strains, the frequency of germline transmission from founder to F1 was highly variable. Only two of the five founders successfully pass the transgene to the F1 generation. At present, the progeny of subsequent generations is being produced and tested for the expression of CFP signals, and therefore, stable lines are ongoing.

TGF-\u03b2-induced \u03b1-SMA expression is mediated by C/EBP\u03b2 acetylation in human alveolar epithelial cells

Background Although the morbidity and mortality rates associated with idiopathic pulmonary fibrosis (IPF) are high, there is still lack of powerful and precise therapeutic options for IPF.ObjectThrough in vitro model, this study sought to determine whether binding of acetylated CCAAT/enhancer binding protein β (C/EBPβ) to alpha-smooth muscle actin (α-SMA) promoter could affect the activity of the latter as well as assess if it is essential for epithelial-to-mesenchymal transition (EMT) and extracellular matrix deposition in IPF.MethodsThe expression of EMT and C/EBPβ in A549 cells treated with transforming growth factor-beta (TGF-β) as pulmonary fibrotic model was detected by western blotting and qPCR. Collagen-I expression using ELISA was performed. The luciferase activity was used to examine the activity of C/EBPβ. Knockdown of C/EBPβ was performed by siRNA. We also investigated the effect of deacetylation of C/EBPβ on EMT using sirtuin 1 (SIRT1). The binding ability of C/EBPβ with α-SMA promoter was affirmed via chromatin immunoprecipitation (ChIP) and electrophoresis mobility shift assay (EMSA). The relationship between α-SMA and acetylated C/EBPβ was determined with co-immunoprecipitation (Co-IP). SiRNA-mediated knockdown of C/EBPβ in A549 cells attenuated TGF-β1-induced myofibroblast differentiation and ECM deposition. The extent of association between acetylated C/EBPβ and α-SMA promoter was dynamically monitored.ResultsIt was confirmed that deacetylation of C/EBPβ in A549 cells successfully ameliorated TGF-β1-induced EMT, as shown by reduction in α-SMA expression and excessive collagen-I accumulation. ConclusionThe EMT and fibrotic effect of TGF-β1 is dependent on acetylated C/EBPβ-mediated regulation of α-SMA gene activity. Thus, C/EBPβ acetylation may play a central role in pulmonary fibrosis.

FoxP1 is a transcriptional repressor associated with cancer cachexia that induces skeletal muscle wasting and weakness.

BackgroundSkeletal muscle wasting is a devastating consequence of cancer that affects up to 80% of cancer patients and associates with reduced survival. Herein, we investigated the biological significance of Forkhead box P1 (FoxP1), a transcriptional repressor that we demonstrate is up‐regulated in skeletal muscle in multiple models of cancer cachexia and in cachectic cancer patients.MethodsInducible, skeletal muscle‐specific FoxP1 over‐expressing (FoxP1iSkmTg/Tg) mice were generated through crossing conditional Foxp1a transgenic mice with HSA‐MCM mice that express tamoxifen‐inducible Cre recombinase under control of the skeletal muscle actin promoter. To determine the requirement of FoxP1 for cancer‐induced skeletal muscle wasting, FoxP1‐shRNA was packaged and targeted to muscles using AAV9 delivery prior to inoculation of mice with Colon‐26 Adenocarcinoma (C26) cells.ResultsUp‐regulation of FoxP1 in adult skeletal muscle was sufficient to induce features of cachexia, including 15% reduction in body mass (P < 0.05), and a 16–27% reduction in skeletal muscle mass (P < 0.05) that was characterized by a 20% reduction in muscle fibre cross‐sectional area of type IIX/B muscle fibres (P = 0.020). Skeletal muscles from FoxP1iSkmTg/Tg mice also showed significant damage and myopathy characterized by the presence of centrally nucleated myofibres, extracellular matrix expansion, and were 19–26% weaker than controls (P < 0.05). Transcriptomic analysis revealed FoxP1 as a potent transcriptional repressor of skeletal muscle gene expression, with enrichment of pathways related to skeletal muscle structure and function, growth signalling, and cell quality control. Because FoxP1 functions, at least in part, as a transcriptional repressor through its interaction with histone deacetylase proteins, we treated FoxP1iSkmTg/Tg mice with Trichostatin A, and found that this completely prevented the loss of muscle mass (p = 0.007) and fibre atrophy (P < 0.001) in the tibialis anterior. In the context of cancer, FoxP1 knockdown blocked the cancer‐induced repression of myocyte enhancer factor 2 (MEF2)‐target genes critical to muscle differentiation and repair, improved muscle ultrastructure, and attenuated muscle fibre atrophy by 50% (P < 0.05).ConclusionsIn summary, we identify FoxP1 as a novel repressor of skeletal muscle gene expression that is increased in cancer cachexia, whose up‐regulation is sufficient to induce skeletal muscle wasting and weakness, and required for the normal wasting response to cancer.

(Pro)renin Receptor Knockdown Attenuates Liver Fibrosis Through Inactivation of ERK/TGF-β1/SMAD3 Pathway.

Background & AimsActivation of the (pro)renin receptor (PRR) up-regulates the expression of profibrotic genes in the kidney and heart. We aimed to investigate the role of PRR in hepatic fibrogenesis.MethodsHuman hepatic PRR levels were measured in patients with or without liver fibrosis. PRR expression was analyzed in primary mouse hepatic stellate cells (HSCs). Experimental fibrosis was studied in thioacetamide (TAA)-treated or methionine choline-deficient (MCD) diet-fed C57BL/6 mice. Lentivirus-mediated PRR short hairpin RNA was used to knockdown hepatic PRR expression. Lentiviral vectors expressing PRR short hairpin RNA or complementary DNA from the α-smooth muscle actin promoter were used for myofibroblast-specific gene knockdown or overexpression.ResultsPRR is up-regulated in human and mouse fibrotic livers, and in activated HSCs. Hepatic PRR knockdown reduced liver fibrosis by suppressing the activation of HSCs and expression of profibrotic genes in TAA or MCD diet–injured mice without significant changes in hepatic inflammation. Renin and prorenin increased the expression of PRR and production of TGF-β1 in human activated HSC Lieming Xu-2 cells, and knockdown of PRR inactivated Lieming Xu-2 cells with decreased production of transforming growth factor (TGF)-β1 and Mothers against decapentaplegic homolog 3 (Smad3) phosphorylation. Myofibroblast-specific PRR knockdown also attenuated liver fibrosis in TAA or MCD diet–injured mice. Mice with both myofibroblast-specific and whole-liver PRR knockdown showed down-regulation of the hepatic extracellular signal-regulated kinase (ERK)/TGF-β1/Smad3 pathway. Myofibroblast-specific PRR overexpression worsened TAA-induced liver fibrosis by up-regulating the ERK/TGF-β1/Smad3 pathway.ConclusionsPRR contributes to liver fibrosis and HSC activation, and its down-regulation attenuates liver fibrosis through inactivation of the ERK/TGF-β1/Smad3 pathway. Therefore, PRR is a promising therapeutic target for liver fibrosis.

JSRV Intragenic Enhancer Element Increases Expression from a Heterologous Promoter and Promotes High Level AAV-mediated Transgene Expression in the Lung and Liver of Mice.

Jaagsiekte sheep retrovirus (JSRV) induces tumors in the distal airways of sheep and goats. A putative intragenic enhancer, termed JE, localized to the 3′ end of the JSRV env gene, has been previously described. Herein we provide further evidence that the JE functions as a transcriptional enhancer, as it was able to enhance gene expression when placed in either forward or reverse orientation when combined with a heterologous chicken beta actin promoter. We then generated novel composite promoters designed to improve transgene expression from adeno-associated virus (AAV) gene therapy vectors. A hybrid promoter consisting of the shortest JE sequence examined (JE71), the U3 region of the JSRV long terminal repeat (LTR), and the chicken beta actin promoter, demonstrated robust expression in vitro and in vivo, when in the context of AAV vectors. AAV-mediated transgene expression in vivo from the hybrid promoter was marginally lower than that observed for AAV vectors encoding the strong CAG promoter, but greatly reduced in the heart, making this promoter/enhancer combination attractive for non-cardiac applications, particularly respiratory tract or liver directed therapies. Replacement of the murine leukemia virus intron present in the original vector construct with a modified SV40 intron reduced the promoter/enhancer/intron cassette size to 719 bp, leaving an additional ~4 kb of coding capacity when packaged within an AAV vector. Taken together, we have developed a novel, compact promoter that is capable of directing high level transgene expression from AAV vectors in both the liver and lung with diminished transgene expression in the heart.

Myocarditis triggers inflammatory response in cardiac fibroblasts and profibrotic activation of myeloid and endothelial cells in mouse model of experimental autoimmune myocarditis

Abstract Background/Introduction Myocarditis, a heart-specific inflammation, is a common cause of pathological tissue remodeling and cardiac fibrosis resulting in stiffening of ventricles, functional impairment and heart failure. Immunization of susceptible mice with alpha myosin heavy chain (αMyHC) and complete Freund's adjuvant (CFA) induces CD4+ T cell-mediated experimental autoimmune myocarditis (EAM). In EAM model, resolution of acute cardiac inflammation is followed by a progressive dilated cardiomyopathy and systolic dysfunction. Purpose The aim of our study was to identify the role of resident cardiac fibroblasts, cardiac endothelial as well as inflammatory myeloid cells during the course of EAM. Methods EAM was induced by immunization with αMyHC/CFA in reporter BALB/c mice expressing EGFP under collagen type I promoter (Coll-EGFP) and RFP under a control of α-smooth muscle actin (αSMA) promoter (αSMA-RFP). Using flow cytometry analysis, cardiac cells were phenotyped and quantified at inflammatory (d19–21) and fibrotic (d40) stage of EAM. Sorted EGFP-positive cardiac fibroblasts obtained from healthy and myocarditis-positive mice (day 21 of EAM) were comparatively analyzed for the whole genome transcriptomics using the Next Generation Sequencing with read length 2x150bp and 20–30 million reads per sample. Results A massive infiltration of inflammatory CD45+CD11b+ myeloid cells (mainly CD11b+CD36+ macrophages, CD11b+CD36–Ly6GhiLy6chi neutrophils, CD11b+CD36–Ly6G–Ly6c– monocytes, CD11b+CD36–Ly6G–Ly6chi inflammatory monocytes) was observed at day 21 of EAM. Myeloid cells as well as endothelial cells showed increased production of type I collagen at day 21, which was further reduced at day 40 of EAM. At day 21, collagen-producing endothelial cells showed particularly elevated levels of adhesion molecules ICAM and VCAM. On the other hand, the total number of EGFP-positive cardiac fibroblasts remained unchanged during the course of EAM, as well as the percentage of cardiac fibroblasts positive for αSMA (myofibroblasts). Gene ontology analysis of transcripts differentially regulated in cardiac fibroblasts during acute myocarditis pointed mainly to activation of immune processes, response to stress, cytoskeletal and extracellular matrix organization. Specifically, in EAM at day 21 cardiac fibroblasts increased transcription of chemokines (Ccl6, Ccl9, Cxcl2, Cxcl3, Cxcl5, Cxcl9, Cxcl13), collagens (Col6a4, Col6a5, Col9a1, Col9a3, Col11a2, Col12a1, Col24a1, Col28a1), and genes involved in ECM biology (Bmp7, Kng2, Lgals3, Cthrc1, Cela1, Spn). Conclusions In EAM model, inflammatory myeloid and cardiac endothelial cells seem to contribute to excessive collagen type I production, whereas cardiac fibroblasts actively participate in inflammatory and profibrotic responses. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): The National Science Centre (Poland)

CARM1 Regulates AMPK Signaling in Skeletal Muscle.

SummaryCoactivator-associated arginine methyltransferase 1 (CARM1) is an emerging mediator of skeletal muscle plasticity. We employed genetic, physiologic, and pharmacologic approaches to determine whether CARM1 regulates the master neuromuscular phenotypic modifier AMP-activated protein kinase (AMPK). CARM1 skeletal muscle-specific knockout (mKO) mice displayed reduced muscle mass and dysregulated autophagic and atrophic processes downstream of AMPK. We observed altered interactions between CARM1 and AMPK and its network, including forkhead box protein O1, during muscle disuse. CARM1 methylated AMPK during the early stages of muscle inactivity, whereas CARM1 mKO mitigated progression of denervation-induced atrophy and was accompanied by attenuated phosphorylation of AMPK targets such as unc-51 like autophagy-activating kinase 1Ser555. Lower acetyl-coenzyme A corboxylaseSer79 phosphorylation, as well as reduced peroxisome proliferator-activated receptor-γ coactivator-1α, was also observed in mKO animals following acute administration of the direct AMPK activator MK-8722. Our study suggests that targeting CARM1-AMPK interplay may have broad impacts on neuromuscular health and disease.

Adeno-Associated Virus Capsid-Promoter Interactions in the Brain Translate from Rat to the Nonhuman Primate.

Recently, we established an adeno-associated virus (AAV9) capsid-promoter interaction that directly determined cell-specific gene expression across two synthetic promoters, Cbh and CBA, in the rat striatum. These studies not only expand this capsid-promoter interaction to include another promoter in the rat striatum but also establish AAV capsid-promoter interactions in the nonhuman primate brain. When AAV serotype 9 (AAV9) vectors were injected into the rat striatum, the minimal synthetic promoter JetI drove green fluorescent protein (GFP) gene expression predominantly in oligodendrocytes. However, similar to our previous findings, the insertion of six alanines into VP1/VP2 of the AAV9 capsid (AAV9AU) significantly shifted JetI-driven GFP gene expression to neurons. In addition, previous retrograde tracing studies in the nonhuman primate brain also revealed the existence of a capsid-promoter interaction. When rAAV2-Retro vectors were infused into the frontal eye field (FEF) of rhesus macaques, local gene expression was prominent using either the hybrid chicken beta actin (CAG) or human synapsin (hSyn) promoters. However, only the CAG promoter, not the hSyn promoter, led to gene expression in the ipsilateral claustrum and contralateral FEF. Conversely, infusion of rAAV2-retro-hSyn vectors, but not rAAV2-retro-CAG, into the macaque superior colliculus led to differential and selective retrograde gene expression in cerebellotectal afferent cells. Clearly, this differential promoter/capsid expression profile could not be attributed to promoter inactivation from retrograde transport of the rAAV2-Retro vector. In summary, we document the potential for AAV capsid/promoter interactions to impact cell-specific gene expression across species, experimental manipulations, and engineered capsids, independent of capsid permissivity.

Adult-Onset Myopathy with Constitutive Activation of Akt following the Loss of hnRNP-U.

SummarySkeletal muscle has the remarkable ability to modulate its mass in response to changes in nutritional input, functional utilization, systemic disease, and age. This is achieved by the coordination of transcriptional and post-transcriptional networks and the signaling cascades balancing anabolic and catabolic processes with energy and nutrient availability. The extent to which alternative splicing regulates these signaling networks is uncertain. Here we investigate the role of the RNA-binding protein hnRNP-U on the expression and splicing of genes and the signaling processes regulating skeletal muscle hypertrophic growth. Muscle-specific Hnrnpu knockout (mKO) mice develop an adult-onset myopathy characterized by the selective atrophy of glycolytic muscle, the constitutive activation of Akt, increases in cellular and metabolic stress gene expression, and changes in the expression and splicing of metabolic and signal transduction genes. These findings link Hnrnpu with the balance between anabolic signaling, cellular and metabolic stress, and physiological growth.

Inducible deletion of skeletal muscle AMPKα reveals that AMPK is required for nucleotide balance but dispensable for muscle glucose uptake and fat oxidation during exercise

ObjectiveEvidence for AMP-activated protein kinase (AMPK)-mediated regulation of skeletal muscle metabolism during exercise is mainly based on transgenic mouse models with chronic (lifelong) disruption of AMPK function. Findings based on such models are potentially biased by secondary effects related to a chronic lack of AMPK function. To study the direct effect(s) of AMPK on muscle metabolism during exercise, we generated a new mouse model with inducible muscle-specific deletion of AMPKα catalytic subunits in adult mice. MethodsTamoxifen-inducible and muscle-specific AMPKα1/α2 double KO mice (AMPKα imdKO) were generated by using the Cre/loxP system, with the Cre under the control of the human skeletal muscle actin (HSA) promoter. ResultsDuring treadmill running at the same relative exercise intensity, AMPKα imdKO mice showed greater depletion of muscle ATP, which was associated with accumulation of the deamination product IMP. Muscle-specific deletion of AMPKα in adult mice promptly reduced maximal running speed and muscle glycogen content and was associated with reduced expression of UGP2, a key component of the glycogen synthesis pathway. Muscle mitochondrial respiration, whole-body substrate utilization, and muscle glucose uptake and fatty acid (FA) oxidation during muscle contractile activity remained unaffected by muscle-specific deletion of AMPKα subunits in adult mice. ConclusionsInducible deletion of AMPKα subunits in adult mice reveals that AMPK is required for maintaining muscle ATP levels and nucleotide balance during exercise but is dispensable for regulating muscle glucose uptake, FA oxidation, and substrate utilization during exercise.

Peroxisome Proliferator Activator Receptor Gamma Coactivator-1α Overexpression in Amyotrophic Lateral Sclerosis: A Tale of Two Transgenics.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder manifesting with upper and lower neuron loss, leading to impairments in voluntary muscle function and atrophy. Mitochondrial dysfunction in metabolism and morphology have been implicated in the pathogenesis of ALS, including atypical oxidative metabolism, reduced mitochondrial respiration in muscle, and protein aggregates in the mitochondrial outer membrane. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) plays an essential role in the regulation of mitochondrial biogenesis, the process by which existing mitochondria grow and divide. PGC-1α has been previously reported to be downregulated in the spinal cord of individuals with ALS. Towards targeting PGC-1α as a therapeutic mechanism, we have previously reported improved motor function and survival in the SOD1G93A mouse model of ALS by neuron-specific over-expression of PGC-1α under a neuron-specific enolase (NSE) promoter. As pharmacological intervention targeting PGC-1α would result in whole-body upregulation of this transcriptional co-activator, in the current study we investigated whether global expression of PGC-1α is beneficial in a SOD1G93A mouse model, by generating transgenic mice with PGC-1α transgene expression driven by an actin promoter. Actin-PGC-1α expression levels were assayed and confirmed in spinal cord, brain, muscle, liver, kidney, and spleen. To determine the therapeutic effects of global expression of PGC-1α, wild-type, actin-PGC-1α, SOD1G93A, and actin-PGC-1α/SOD1G93A animals were monitored for weight loss, motor performance by accelerating rotarod test, and survival. Overexpression of actin-PGC-1α did not confer significant improvement in these assessed outcomes. A potential explanation for this difference is that the actin promoter may not induce levels of PGC-1α relevant to disease pathophysiology in the cells that are specifically relevant to the pathogenesis of ALS. This evidence strongly supports future therapeutic approaches that target PGC-1α primarily in neurons.

Dysregulation of Inositol Synthesis Affects Obesity and Causes Developmental Birth Defects

Myo‐inositol is a six‐carbon sugar alcohol that is a precursor of phosphatidylinositol (PI), a cell membrane phospholipid. Inositol is synthesized from glucose‐6‐phosphate by the product of the Drosophila melanogaster Inos gene, myo‐inositol‐3‐phosphate synthase (MIPSp). Abnormalities in myo‐inositol metabolism have been implicated in diseases and complications such as type 2 diabetes, polycystic ovary syndrome, and bipolar disorder. Obesity can be induced by a high sucrose diet in D. melanogaster third instar larvae. The hypothesis of this study was that obesity induced in D. melanogaster by high sucrose diet can be alleviated by inositol, either dietary or endogenously synthesized. A float buoyancy assay was used to assess obesity of larvae of three strains: a wild type strain (CS), a homozygous P‐element insertion strain with UASGAL4 5’ of the Inos gene (inosP‐UAS), and a heterozygous inosP‐UAS strain with GAL4 production driven by an actin promoter (inosP‐UAS ; Act5C‐Gal4). This last strain has up‐regulated inositol synthesis. Larval Inos transcript and MIPS protein expression levels in the three strains were confirmed by RT‐qPCR and Western blots. The inosP‐UAS larvae with GAL4 production driven by the actin promoter (inosP‐UAS ; Act5C‐Gal4) have the highest levels of Inos transcript and MIPS protein relative to the homozygous inosP‐UAS insertion larvae which have an intermediate level of expression, and the wild‐type CS with the lowest level of expression. In the float‐buoyancy assay, larvae with increased buoyancy are scored as ‘obese’. Provision of dietary inositol reduced the percentage of ‘obese’ wild‐type larvae from 74.75±1.94 to 63.95 ±1.70. Moreover, up‐regulation of inositol synthesis via inosP‐UAS ; Act5C‐Gal4 dramatically reduced the percentage of ‘obese’ larvae to 13.28±4.42. During these studies, it became apparent that dysregulation of inositol synthesis resulted in developmental abnormalities and birth defects. Attempts to grow inosP‐UAS ; Act5C‐Gal4 larvae to maturity (adult flies) resulted in a surprising outcome – nearly all flies died as pharate adults. Some structural alterations of wings, halteres, and legs were immediately apparent. The few flies that did eclose died within a few days. Scanning electron and light microscope imaging revealed that they all had malformed or missing proboscises. These data give us a better understanding of the importance of the regulation of inositol in obesity and development.Support or Funding InformationThis project is supported in part by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R25GM071638 and by the Frank Schatzlein Endowed Student Award.

Abstract QS32: Characterizing Cellular Expansion In Response To Long Bone Distraction Osteogenesis

Purpose: Distraction Osteogenesis (DO) is a commonly used procedure to correct for skeletal abnormalities. However, the cellular contribution responsible for inducing bone formation during DO is not well understood. We hypothesize that the clonal expansion observed during distraction is due to skeletal stem cells. Methods: Actin;R26VT2/GK3 (rainbow) is a multicolor, ubiquitous, genetic driver that marks cells under the actin promoter. Upon tamoxifen, induction cells are randomly and genetically marked with one of ten color combinations. Subsequent daughter cells are marked with the same color as their parent population enabling for lineage tracing. 10-week old male rainbow mice underwent distraction surgery that consisted of a five-day latency period followed by a twice-daily distraction rate of 0.15 mm for 10 days, ending with a consolidation period of 28 days. At post-operative day (POD 43) the distracted tibias of the rainbow mice (n=3) were harvested and imaged using confocal microscopy. Furthermore, the functional assessment of fracture and distracted skeletal stem cells (SSCs) was determined using proliferation (n=5) and osteogenic assays (n=5). Wild-type 10-week-old male mice were divided into two groups: fracture and distraction. At POD 10, fracture and distracted tibias were harvested for fluorescent activated cell sorting (FACS) of SSCs. Results: Rainbow labeling of an uninjured tibia shows successful genetic marking of the diaphysis. During distraction, a clonal expansion of cells is observed preceded by successful bone formation. SSCs sorted from distraction calluses at POD 10 form robust colonies and larger bone nodules in comparison to fracture calluses in vitro. In addition, quantification analysis indicates distraction SSCs form more colonies (***p <0.001) and have increased incidence of in vitro bone formation (****p<0.0001). Conclusions: We demonstrate successful rainbow labeling of the mouse tibia and show clonal expansion of skeletal stem cells in response to distraction. Future studies will delineate the role of SSCs in response to distraction.

Transient Transfection of the Zoonotic Parasite Babesia microti.

The development of genetic manipulation techniques has been reported in many protozoan parasites over the past few years. However, these techniques have not been established for Babesia microti. Here, we report the first successful transient transfection of B. microti. The plasmids containing the firefly luciferase reporter gene were transfected into B. microti by an AMAXA 4D Nucleofection system. Twenty-four-hour synchronization, the 5′-actin promoter, program FA100, and 50 μg of plasmid DNA constituted the best conditions for the transient transfection of B. microti. This finding is the first step towards a stable transfection method for B. microti, which may contribute to a better understanding of the biology of the parasite.

Improved cell-specificity of adeno-associated viral vectors for medullary thyroid carcinoma using calcitonin gene regulatory elements.

Targeted gene therapy using recombinant adeno-associated virus (rAAV) vectors is a potential therapeutic strategy for treating cancer, and tissue-specific promoters may help with tissue targeting. Medullary thyroid carcinoma (MTC) is a disease of the calcitonin secreting thyroid C cells, and calcitonin is highly expressed in MTC tumors compared to other cells. To target MTC cells, we evaluated an rAAV serotype 2 vector (rAAV2-pM+104-GFP) containing a modified calcitonin/calcitonin gene related peptide promoter (pM+104) and a green fluorescent protein (GFP) reporter gene. In vitro transduction experiments comparing the MTC TT cell line with non-MTC cell lines demonstrated that rAAV2-pM+104-GFP infection yielded significantly (p < 0.05) higher GFP expression in TT cells than in non-MTC cell lines (HEK293 and HeLa), and significantly higher expression than in TT cells infected with the positive control rAAV2-pCBA-GFP vector. The rAAV2-pCBA-GFP control vector included a well-characterized, ubiquitously expresses control promoter, the chicken beta actin promoter with a cytomegalovirus enhancer (pCBA). In vivo experiments using a TT cell xenograft tumor mouse model showed that tumors directly injected with 2 x 1010 vg of rAAV2-pM+104-GFP vector resulted in GFP expression detected in 21.7% of cells, 48 hours after the injection. Furthermore, GFP expression was significantly higher for rAAV-pM+104-GFP treatments with a longer vector treatment duration and higher vector dose, with up to 52.6% (q < 0.05) GFP cells detected 72 hours after injecting 1x 1011 vg/tumor. These data show that we have developed an rAAV vector with improved selectivity for MTC.

P300 and cAMP response element-binding protein-binding protein in skeletal muscle homeostasis, contractile function, and survival.

BackgroundReversible ε‐amino acetylation of lysine residues regulates transcription as well as metabolic flux; however, roles for specific lysine acetyltransferases in skeletal muscle physiology and function are unknown. In this study, we investigated the role of the related acetyltransferases p300 and cAMP response element‐binding protein‐binding protein (CBP) in skeletal muscle transcriptional homeostasis and physiology in adult mice.MethodsMice with skeletal muscle‐specific and inducible knockout of p300 and CBP (PCKO) were generated by crossing mice with a tamoxifen‐inducible Cre recombinase expressed under the human α‐skeletal actin promoter with mice having LoxP sites flanking exon 9 of the Ep300 and Crebbp genes. Knockout of PCKO was induced at 13–15 weeks of age via oral gavage of tamoxifen for 5 days to both PCKO and littermate control [wildtype (WT)] mice. Body composition, food intake, and muscle function were assessed on day 0 (D0) through 5 (D5). Microarray and tandem mass tag mass spectrometry analyses were performed to assess global RNA and protein levels in skeletal muscle of PCKO and WT mice.ResultsAt D5 after initiating tamoxifen treatment, there was a reduction in body weight (−15%), food intake (−78%), stride length (−46%), and grip strength (−45%) in PCKO compared with WT mice. Additionally, ex vivo contractile function [tetanic tension (kPa)] was severely impaired in PCKO vs. WT mice at D3 (~70–80% lower) and D5 (~80–95% lower) and resulted in lethality within 1 week—a phenotype that is reversed by the presence of a single allele of either p300 or CBP. The impaired muscle function in PCKO mice was paralleled by substantial transcriptional alterations (3310 genes; false discovery rate < 0.1), especially in gene networks central to muscle contraction and structural integrity. This transcriptional uncoupling was accompanied by changes in protein expression patterns indicative of impaired muscle function, albeit to a smaller magnitude (446 proteins; fold‐change > 1.25; false discovery rate < 0.1).ConclusionsThese data reveal that p300 and CBP are required for the control and maintenance of contractile function and transcriptional homeostasis in skeletal muscle and, ultimately, organism survival. By extension, modulating p300/CBP function may hold promise for the treatment of disorders characterized by impaired contractile function in humans.