AAV9-Mediated Intrastriatal Delivery of GNAO1 Reduces Hyperlocomotion in Gnao1 Heterozygous R209H Mutant Mice.

Mutations in the GNAO1 gene, which encodes the abundant brain G-protein Gα o, result in neurologic disorders characterized by developmental delay, epilepsy, and movement abnormalities. There are over 50 mutant alleles associated with GNAO1 disorders; the R209H mutation results in dystonia, choreoathetosis, and developmental delay without seizures. Mice heterozygous for the human mutant allele (Gnao1 +/R209H) exhibit hyperactivity in open field tests but no seizures. We developed self-complementary adeno-associated virus serotype 9 (scAAV9) vectors expressing two splice variants of human GNAO1 Gα o isoforms 1 (GoA, GNAO1.1) and 2 (GoB, GNAO1.2). Bilateral intrastriatal injections of either scAAV9-GNAO1.1 or scAAV9-GNAO1.2 significantly reversed mutation-associated hyperactivity in open field tests. GNAO1 overexpression did not increase seizure susceptibility, a potential side effect of GNAO1 vector treatment. This represents the first report of successful preclinical gene therapy for GNAO1 encephalopathy applied in vivo. Further studies are needed to uncover the molecular mechanism that results in behavior improvements after scAAV9-mediated Gα o expression and to refine the vector design. SIGNIFICANCE STATEMENT: GNAO1 mutations cause a spectrum of developmental, epilepsy, and movement disorders. Here we show that intrastriatal delivery of scAAV9-GNAO1 to express the wild-type Gα o protein reduces the hyperactivity of the Gnao1 +/R209H mouse model, which carries one of the most common movement disorder-associated mutations. This is the first report of a gene therapy for GNAO1 encephalopathy applied in vivo on a patient-allele model.

Epidermal growth factor receptor (EGFR) signaling is a key mediator of hormone-induced leukocyte infiltration in the pubertal female mammary gland.

It is well documented that macrophages and eosinophils play important roles in normal murine pubertal mammary gland development. Although it is accepted that estrogen (E) and progesterone (P) are key players in mammary gland development, the roles these hormones might play in regulating the actions of leukocytes in that process is an understudied area. We show here that P and E, respectively, induce unique, but overlapping, sets of proinflammatory and angiogenic cytokines and chemokines, in the pubertal female BALB/c mammary gland, as well as induce infiltration of macrophages and eosinophils to the mammary periepithelium. This extends earlier studies showing P induction of proinflammatory products in pubertal and adult mammary epithelial organoids and P-induced in vivo infiltration of leukocytes to the adult mammary periepithelium. Importantly, epidermal growth factor receptor-signaling, which is likely mediated by amphiregulin (Areg), a downstream mediator of E and P, is both necessary and sufficient for both E- and P-induced recruitment of macrophages and eosinophils to the pubertal mammary periepithelium. We further show that receptor activator of nuclear factor κB ligand (RANKL), although not sufficient of itself to cause macrophage and eosinophil recruitment, contributes to an optimal response to P. The potency of Areg is highlighted by the fact that it is sufficient to induce macrophage and eosinophil recruitment at levels equivalent to that induced by either E or P. Our finding of a dominant role for Areg in hormonally induced leukocyte recruitment to the pubertal mammary gland parallels its dominance in regulating ductal outgrowth and its role in P-induced proliferation in the pubertal gland.

Amphiregulin mediates progesterone-induced mammary ductal development during puberty.

INTRODUCTION: Puberty is a period of increased susceptibility to factors that cause increased breast cancer risk in adulthood. Mammary end buds (EBs) that develop during puberty are believed to be the targets of breast cancer initiation. Whereas the role of estrogen (E) has been extensively studied in pubertal mammary gland development, the role of progesterone (P) during puberty is less defined. METHODS: Pubertal and prepubertal ovariectomized mice were treated with vehicle control (C), E, P, or E+P. Mammary glands from these mice were analyzed for changes in morphology, proliferation, and expression of the downstream targets amphiregulin (AREG) and receptor activator of NF-κB ligand (RANKL). RESULTS: P, acting specifically through the progesterone receptor, induced increases in mammary gland proliferation and EB formation that were associated with increased AREG expression in ducts and EBs. E, acting specifically through the estrogen receptor, produced similar responses also mediated by AREG. Blocking AREG action by treatment with an EGFR inhibitor completely abrogated the effect of P on EB formation and proliferation and significantly reduced proliferation within ducts. P also increased expression of RANKL, primarily in ducts. Treatment with RANK-Fc, an inhibitor of RANKL, reduced P-dependent proliferation in ducts and to a lesser extent in EB, but did not cause EB regression. CONCLUSIONS: These results demonstrate a novel P-specific effect through AREG to cause EB formation and proliferation in the developing mammary gland both before and during puberty. Thus, hormones and/or factors in addition to E that upregulate AREG can promote mammary gland development and have the potential to affect breast cancer risk associated with pubertal mammary gland development.

Amphiregulin mediates estrogen, progesterone, and EGFR signaling in the normal rat mammary gland and in hormone-dependent rat mammary cancers.

Both estrogen (E) and progesterone (P) are implicated in the etiology of human breast cancer. Defining their mechanisms of action, particularly in vivo, is relevant to the prevention and therapy of breast cancer. We investigated the molecular and cellular mechanisms of E and/or P-induced in vivo proliferation, in the normal rat mammary gland and in hormone-dependent rat mammary cancers which share many characteristics with the normal human breast and hormone-dependent breast cancers. We show that E+P treatment induced significantly greater proliferation in both the normal gland and mammary cancers compared to E alone. In both the normal gland and tumors, E+P-induced proliferation was mediated through the increased production of amphiregulin (Areg), an epidermal growth factor receptor (EGFR) ligand, and the activation of intracellular signaling pathways (Erk, Akt, JNK) downstream of EGFR that regulate proliferation. In vitro experiments using rat primary mammary organoids or T47D breast cancer cells confirmed that Areg and the synthetic progestin, R5020, synergize to promote cell proliferation through EGFR signaling. Iressa, an EGFR inhibitor, effectively blocked this proliferation. These results indicate that mediators of cross talk between E, P, and EGFR pathways may be considered as relevant molecular targets for the therapy of hormone-dependent breast cancers, especially in premenopausal women.

Progesterone receptor A-regulated gene expression in mammary organoid cultures.

Progesterone, through the progesterone receptor (PR), promotes development of the normal mammary gland and is implicated in the etiology of breast cancer. We identified PRA-regulated genes by microarray analysis of cultured epithelial organoids derived from pubertal and adult mouse mammary glands, developmental stages with differing progesterone responsiveness. Microarray analysis showed significant progestin (R5020)-regulation of 162 genes in pubertal organoids and 104 genes in adult organoids, with 68 genes regulated at both developmental stages. Greater induction of receptor activator of NFkappaB ligand and calcitonin expression was observed in adult organoids, suggesting possible roles in the differential progesterone responsiveness of the adult and pubertal mammary glands. Analysis of the R5020-responsive transcriptome revealed several enriched biological processes including cell adhesion, immune response, and survival. R5020 both induced Agtr1 and potentiated angiotensin II-stimulated proliferation, highlighting the functional significance of the latter process. Striking up-regulation of genes involved in innate immunity processes included the leukocyte chemoattractants serum amyloid A1, 2 and 3 (Saa1, 2, 3). In vivo analysis revealed that progesterone treatment increased SAA1 protein expression and leukocyte density in mammary gland regions undergoing epithelial expansion. These studies reveal novel targets of PRA in mammary epithelial cells and novel linkages of progesterone action during mammary gland development.

Topography of the prostaglandin endoperoxide H2 synthase-2 in membranes.

The topology of association of the monotopic protein cyclooxygenase-2 (COX-2) with membranes has been examined using EPR spectroscopy of spin-labeled recombinant human COX-2. Twenty-four mutants, each containing a single free cysteine substituted for an amino acid in the COX-2 membrane binding domain were expressed using the baculovirus system and purified, then conjugated with a nitroxide spin label and reconstituted into liposomes. Determining the relative accessibility of the nitroxide-tagged amino acid side chains for the solubilized COX-2 mutants, or COX-2 reconstituted into liposomes to nonpolar (oxygen) and polar (NiEDDA or CrOx) paramagnetic reagents allowed us to map the topology of COX-2 interaction with the lipid bilayer. When spin-labeled COX-2 was reconstituted into liposomes, EPR power saturation curves showed that side chains for all but two of the 24 mutants tested had limited accessibility to both polar and nonpolar paramagnetic relaxation agents, indicating that COX-2 associates primarily with the interfacial membrane region near the glycerol backbone and phospholipid head groups. Two amino acids, Phe(66) and Leu(67), were readily accessible to the non-polar relaxation agent oxygen, and thus likely inserted into the hydrophobic core of the lipid bilayer. However these residues are co-linear with amino acids in the interfacial region, so their extension into the hydrophobic core must be relatively shallow. EPR and structural data suggest that membrane interaction of COX-2 is also aided by partitioning of 4 aromatic amino acids, Phe(59), Phe(66), Tyr(76), and Phe(84) to the interfacial region, and by the electrostatic interactions of two basic amino acids, Arg(62) and Lys(64), with the phospholipid head groups.