M. David Stewart

Restoration of testis function in hypogonadotropic hypogonadal mice harboring a misfolded GnRHR mutant by pharmacoperone drug therapy

Significance Many diseases result from genetic mutations that cause protein misfolding. Medical treatments often address the symptoms, but do not correct the underlying etiology. This study illustrates proof of principle that a disease caused by a misfolded cell surface receptor can be corrected with a pharmacoperone, a unique class of target-specific drugs that assist protein folding. Mutations in receptors, ion channels, and enzymes are frequently recognized by the cellular quality control system as misfolded and retained in the endoplasmic reticulum (ER) or otherwise misrouted. Retention results in loss of function at the normal site of biological activity and disease. Pharmacoperones are target-specific small molecules that diffuse into cells and serve as folding templates that enable mutant proteins to pass the criteria of the quality control system and route to their physiologic site of action. Pharmacoperones of the gonadotropin releasing hormone receptor (GnRHR) have efficacy in cell culture systems, and their cellular and biochemical mechanisms of action are known. Here, we show the efficacy of a pharmacoperone drug in a small animal model, a knock-in mouse, expressing a mutant GnRHR. This recessive mutation (GnRHR E90K) causes hypogonadotropic hypogonadism (failed puberty associated with low or apulsatile luteinizing hormone) in both humans and in the mouse model described. We find that pulsatile pharmacoperone therapy restores E90K from ER retention to the plasma membrane, concurrently with responsiveness to the endogenous natural ligand, gonadotropin releasing hormone, and an agonist that is specific for the mutant. Spermatogenesis, proteins associated with steroid transport and steroidogenesis, and androgen levels were restored in mutant male mice following pharmacoperone therapy. These results show the efficacy of pharmacoperone therapy in vivo by using physiological, molecular, genetic, endocrine and biochemical markers and optimization of pulsatile administration. We expect that this newly appreciated approach of protein rescue will benefit other disorders sharing pathologies based on misrouting of misfolded protein mutants.

Uterine Gland Formation in Mice Is a Continuous Process, Requiring the Ovary after Puberty, But Not after Parturition

Uterine gland formation occurs postnatally in an ovary- and steroid-independent manner in many species, including humans. Uterine glands secrete substances that are essential for embryo survival. Disruption of gland development during the postnatal period prevents gland formation, resulting in infertility. Interestingly, stabilization of beta-catenin (CTNNB1) in the uterine stroma causes a delay in gland formation rather than a complete absence of uterine glands. Thus, to determine if a critical postnatal window for gland development exists in mice, we tested the effects of extending the endocrine environment of pregnancy on uterine gland formation by treating neonatal mice with estradiol, progesterone, or oil for 5 days. One uterine horn was removed before puberty, and the other was collected at maturity. Some mice were also ovariectomized before puberty. The hormone-treated mice exhibited a delay in uterine gland formation. Hormone-treatment increased the abundance of uterine CTNNB1 and estrogen receptor alpha (ESR1) before puberty, indicating possible mechanisms for delayed gland formation. Despite having fewer glands, progesterone-treated mice were fertile, suggesting that a threshold number of glands is required for pregnancy. Mice that were ovariectomized before puberty did not undergo further uterine growth or gland development. Finally, to establish the role of the ovary in postpartum uterine gland regeneration, mice were either ovariectomized or given a sham surgery after parturition, and uteri were evaluated 1 wk later. We found that the ovary is not required for uterine growth or gland development following parturition. Thus, uterine gland development occurs continuously in mice and requires the ovary after puberty, but not after parturition.

Sertoli Cell Behaviors in Developing Testis Cords and Postnatal Seminiferous Tubules of the Mouse

Sertoli cells are the primary structural component of the fetal testis cords and postnatal seminiferous tubules. Live imaging technologies facilitate the visualization of cell morphologies and behaviors through developmental processes. A transgenic mouse line was generated using a fragment of the rat Gata4 gene to direct the expression of a dual-color fluorescent protein reporter in fetal and adult Sertoli cells. The reporter encoded a red fluorescent protein, monomeric Cherry (mCherry), fused to histone 2B and enhanced green fluorescent protein (EGFP) fused to a glycosylphosphatidylinositol sequence, with a self-cleaving 2A polypeptide separating the two fusion proteins. After translation, the red and green fluorescent proteins translocated to the nucleus and plasma membrane, respectively, of Sertoli cells. Transgene expression in testes was first detected by fluorescent microscopy around Embryonic Day 12.0. Sertoli cell division and migration were visualized during testis cord formation in organ culture. Initially, the Sertoli cells had mesenchyme-like morphologies and behaviors, but later, the cells migrated to the periphery of the testis cords to become epithelialized. In postnatal seminiferous tubules, Sertoli nuclei were evenly spaced when viewed from the external surface of tubules, and Sertoli cytoplasm and membranes were associated with germ cells basally in a rosette pattern. This mouse line was bred to previously described transgenic mouse lines expressing EGFP in Sertoli cytoplasm or a nuclear cyan fluorescent protein (Cerulean) and mCherry in plasma membranes of germ cells. This revealed the physical relationship between Sertoli and germ cells in developing testis cords and provided a novel perspective on Sertoli cell development.

Defective myogenesis in the absence of the muscle-specific lysine methyltransferase SMYD1.

The SMYD (SET and MYND domain) family of lysine methyltransferases harbor a unique structure in which the methyltransferase (SET) domain is intervened by a zinc finger protein-protein interaction MYND domain. SMYD proteins methylate both histone and non-histone substrates and participate in diverse biological processes including transcriptional regulation, DNA repair, proliferation and apoptosis. Smyd1 is unique among the five family members in that it is specifically expressed in striated muscles. Smyd1 is critical for development of the right ventricle in mice. In zebrafish, Smyd1 is necessary for sarcomerogenesis in fast-twitch muscles. Smyd1 is expressed in the skeletal muscle lineage throughout myogenesis and in mature myofibers, shuttling from nucleus to cytosol during myoblast differentiation. Because of this expression pattern, we hypothesized that Smyd1 plays multiple roles at different stages of myogenesis. To determine the role of Smyd1 in mammalian myogenesis, we conditionally eliminated Smyd1 from the skeletal muscle lineage at the myoblast stage using Myf5(cre). Deletion of Smyd1 impaired myoblast differentiation, resulted in fewer myofibers and decreased expression of muscle-specific genes. Muscular defects were temporally restricted to the second wave of myogenesis. Thus, in addition to the previously described functions for Smyd1 in heart development and skeletal muscle sarcomerogenesis, these results point to a novel role for Smyd1 in myoblast differentiation.

Mice Harboring Gnrhr E90K, a Mutation that Causes Protein Misfolding and Hypogonadotropic Hypogonadism in Humans, Exhibit Testis Size Reduction and Ovulation Failure

GnRH, produced in the hypothalamus, acts on pituitary gonadotropes to stimulate release of the gonadotropins LH and FSH. Reduced responsiveness of gonadotropes to GnRH is a primary cause of hypogonadotropic hypogonadism (HH), a disease characterized by gonadal dysfunction and low blood levels of gonadotropins. Loss-of-function mutations in the gene encoding the receptor for GnRH (GNRHR) are a common cause of HH. Sequencing of the GNRHR gene in patients with HH revealed mainly point mutations producing single amino acid substitutions that cause misfolding and misrouting of this G protein-coupled receptor. To generate a mouse model that mimics the human disease, we introduced a single amino acid substitution (E90K) into the mouse Gnrhr gene, which is identical to a known human recessive mutation. In humans, E90K causes severe HH by preventing formation of the E90-K121 salt bridge, which is essential for correct folding. In cell cultures, E90K causes misfolding that leads to almost complete retention by the protein quality control system and subsequent degradation. Here we report that the primary phenotype of mice homozygous for E90K is female infertility due to ovulation failure. Mutant males are fertile despite reduced gonadotropin levels and smaller testes. These results suggest decreased GnRH receptor signaling in the mutant animal, compared with wild type. Our findings suggest that a threshold level of GnRH receptor activity is required for ovulation.

Smyd1 facilitates heart development by antagonizing oxidative and ER stress responses

Smyd1/Bop is an evolutionary conserved histone methyltransferase previously shown by conventional knockout to be critical for embryonic heart development. To further explore the mechanism(s) in a cell autonomous context, we conditionally ablated Smyd1 in the first and second heart fields of mice using a knock-in (KI) Nkx2.5-cre driver. Robust deletion of floxed-Smyd1 in cardiomyocytes and the outflow tract (OFT) resulted in embryonic lethality at E9.5, truncation of the OFT and right ventricle, and additional defects consistent with impaired expansion and proliferation of the second heart field (SHF). Using a transgenic (Tg) Nkx2.5-cre driver previously shown to not delete in the SHF and OFT, early embryonic lethality was bypassed and both ventricular chambers were formed; however, reduced cardiomyocyte proliferation and other heart defects resulted in later embryonic death at E11.5-12.5. Proliferative impairment prior to both early and mid-gestational lethality was accompanied by dysregulation of transcripts critical for endoplasmic reticulum (ER) stress. Mid-gestational death was also associated with impairment of oxidative stress defense—a phenotype highly similar to the previously characterized knockout of the Smyd1-interacting transcription factor, skNAC. We describe a potential feedback mechanism in which the stress response factor Tribbles3/TRB3, when directly methylated by Smyd1, acts as a co-repressor of Smyd1-mediated transcription. Our findings suggest that Smyd1 is required for maintaining cardiomyocyte proliferation at minimally two different embryonic heart developmental stages, and its loss leads to linked stress responses that signal ensuing lethality.

Mouse myofibers lacking the SMYD1 methyltransferase are susceptible to atrophy, internalization of nuclei and myofibrillar disarray

ABSTRACT The Smyd1 gene encodes a lysine methyltransferase specifically expressed in striated muscle. Because Smyd1-null mouse embryos die from heart malformation prior to formation of skeletal muscle, we developed a Smyd1 conditional-knockout allele to determine the consequence of SMYD1 loss in mammalian skeletal muscle. Ablation of SMYD1 specifically in skeletal myocytes after myofiber differentiation using Myf6cre produced a non-degenerative myopathy. Mutant mice exhibited weakness, myofiber hypotrophy, prevalence of oxidative myofibers, reduction in triad numbers, regional myofibrillar disorganization/breakdown and a high percentage of myofibers with centralized nuclei. Notably, we found broad upregulation of muscle development genes in the absence of regenerating or degenerating myofibers. These data suggest that the afflicted fibers are in a continual state of repair in an attempt to restore damaged myofibrils. Disease severity was greater for males than females. Despite equivalent expression in all fiber types, loss of SMYD1 primarily affected fast-twitch muscle, illustrating fiber-type-specific functions for SMYD1. This work illustrates a crucial role for SMYD1 in skeletal muscle physiology and myofibril integrity. Summary: Elimination of the lysine methyltransferase SMYD1 from mouse skeletal muscle caused myopathy with excessive internal nuclei, atrophy, myofibrillar disorganization and broad upregulation of muscle gene expression.

Mesp1 Marked Cardiac Progenitor Cells Repair Infarcted Mouse Hearts

Mesp1 directs multipotential cardiovascular cell fates, even though it’s transiently induced prior to the appearance of the cardiac progenitor program. Tracing Mesp1-expressing cells and their progeny allows isolation and characterization of the earliest cardiovascular progenitor cells. Studying the biology of Mesp1-CPCs in cell culture and ischemic disease models is an important initial step toward using them for heart disease treatment. Because of Mesp1’s transitory nature, Mesp1-CPC lineages were traced by following EYFP expression in murine Mesp1Cre/+; Rosa26EYFP/+ ES cells. We captured EYFP+ cells that strongly expressed cardiac mesoderm markers and cardiac transcription factors, but not pluripotent or nascent mesoderm markers. BMP2/4 treatment led to the expansion of EYFP+ cells, while Wnt3a and Activin were marginally effective. BMP2/4 exposure readily led EYFP+ cells to endothelial and smooth muscle cells, but inhibition of the canonical Wnt signaling was required to enter the cardiomyocyte fate. Injected mouse pre-contractile Mesp1-EYFP+ CPCs improved the survivability of injured mice and restored the functional performance of infarcted hearts for at least 3 months. Mesp1-EYFP+ cells are bona fide CPCs and they integrated well in infarcted hearts and emerged de novo into terminally differentiated cardiac myocytes, smooth muscle and vascular endothelial cells.

Cardiomyocyte-specific conditional knockout of the histone chaperone HIRA in mice results in hypertrophy, sarcolemmal damage and focal replacement fibrosis

ABSTRACT HIRA is the histone chaperone responsible for replication-independent incorporation of histone variant H3.3 within gene bodies and regulatory regions of actively transcribed genes, and within the bivalent promoter regions of developmentally regulated genes. The HIRA gene lies within the 22q11.2 deletion syndrome critical region; individuals with this syndrome have multiple congenital heart defects. Because terminally differentiated cardiomyocytes have exited the cell cycle, histone variants should be utilized for the bulk of chromatin remodeling. Thus, HIRA is likely to play an important role in epigenetically defining the cardiac gene expression program. In this study, we determined the consequence of HIRA deficiency in cardiomyocytes in vivo by studying the phenotype of cardiomyocyte-specific Hira conditional-knockout mice. Loss of HIRA did not perturb heart development, but instead resulted in cardiomyocyte hypertrophy and susceptibility to sarcolemmal damage. Cardiomyocyte degeneration gave way to focal replacement fibrosis and impaired cardiac function. Gene expression was widely altered in Hira conditional-knockout hearts. Significantly affected pathways included responses to cellular stress, DNA repair and transcription. Consistent with heart failure, fetal cardiac genes were re-expressed in the Hira conditional knockout. Our results suggest that transcriptional regulation by HIRA is crucial for cardiomyocyte homeostasis. Summary: Deletion of HIRA, a gene encoding a histone chaperone that lies within the critical region of 22q11.2 deletion syndrome, alters cardiac gene expression and leads to cardiomyopathy.

Confirmation of recombination site functionality in gene targeting vectors using recombinase-expressing bacteria

Recognition sequences for the site-specific DNA recombinases Cre and FLP are commonly incorporated into gene targeting vectors for the purposes of removing selection markers or generating conditional alleles. Gene targeting vectors typically contain a positive selection marker, such as the neomycin resistance gene, flanked by loxP sites. Thus, the selection marker can be removed by breeding to a mouse strain which expresses Cre recombinase in its germ line. Conditional knockout vectors typically have one or more exons flanked by loxP sites and the positive selection marker flanked by FRT sites. Thus, the selection marker is removed with FLP recombinase and the knockout allele is generated in tissues expressing Cre recombinase. Because the generation of mice by gene targeting in embryonic stem (ES) cells is an expensive and time-consuming process, it is important to confirm that the recombination sites in your targeting vector are functional prior to electroporation of ES cells. This chapter describes a simple method for testing the functionality of loxP and FRT sites in vivo using Cre- or FLP-expressing bacteria.