Caralina Marín de Evsikova

Aging in inbred strains of mice: study design and interim report on median lifespans and circulating IGF1 levels

To better characterize aging in mice, the Jackson Aging Center carried out a lifespan study of 31 genetically‐diverse inbred mouse strains housed in a specific pathogen‐free facility. Clinical assessments were carried out every 6 months, measuring multiple age‐related phenotypes including neuromuscular, kidney and heart function, body composition, bone density, hematology, hormonal levels, and immune system parameters. In a concurrent cross‐sectional study of the same 31 strains at 6, 12, and 20 months, more invasive measurements were carried out followed by necropsy to assess apoptosis, DNA repair, chromosome fragility, and histopathology. In this report, which is the initial paper of a series, the study design, median lifespans, and circulating insulin‐like growth factor 1 (IGF1) levels at 6, 12, and 18 months are described for the first cohort of 32 females and 32 males of each strain. Survival curves varied dramatically among strains with the median lifespans ranging from 251 to 964 days. Plasma IGF1 levels, which also varied considerably at each time point, showed an inverse correlation with a median lifespan at 6 months (R = −0.33, P = 0.01). This correlation became stronger if the short‐lived strains with a median lifespan < 600 days were removed from the analysis (R = −0.53, P < 0.01). These results support the hypothesis that the IGF1 pathway plays a key role in regulating longevity in mice and indicates that common genetic mechanisms may exist for regulating IGF1 levels and lifespan.

PPARG by dietary fat interaction influences bone mass in mice and humans

Adult BMD, an important risk factor for fracture, is the result of genetic and environmental interactions. A quantitative trait locus (QTL) for the phenotype of volumetric BMD (vBMD), named Bmd8, was found on mid‐distal chromosome (Chr) 6 in mice. This region is homologous to human Chr 3p25. The B6.C3H‐6T (6T) congenic mouse was previously created to study this QTL. Using block haplotyping of the 6T congenic region, expression analysis in the mouse, and examination of nonsynonymous SNPs, peroxisome proliferator activated receptor γ (Pparg) was determined to be the most likely candidate gene for the Bmd8 QTL of the 630 genes located in the congenic region. Furthermore, in the C3H/HeJ (C3H) strain, which is the donor strain for the 6T congenic, several polymorphisms were found in the Pparg gene. On challenge with a high‐fat diet, we found that the 6T mouse has a lower areal BMD (aBMD) and volume fraction of trabecular bone (BV/TV%) of the distal femur compared with B6 mice. Interactions between SNPs in the PPARG gene and dietary fat for the phenotype of BMD were examined in the Framingham Offspring Cohort. This analysis showed that there was a similar interaction of the PPARG gene and diet (fat intake) on aBMD in both men and women. These findings suggest that dietary fat has a significant influence on BMD that is dependent on the alleles present for the PPARG gene.

NPHP4 is necessary for normal photoreceptor ribbon synapse maintenance and outer segment formation, and for sperm development

Nephronophthisis (NPHP) is an autosomal recessive kidney disease that is often associated with vision and/or brain defects. To date, 11 genes are known to cause NPHP. The gene products, while structurally unrelated, all localize to cilia or centrosomes. Although mouse models of NPHP are available for 9 of the 11 genes, none has been described for nephronophthisis 4 (Nphp4). Here we report a novel, chemically induced mutant, nmf192, that bears a nonsense mutation in exon 4 of Nphp4. Homozygous mutant Nphp4(nmf192/nmf192) mice do not exhibit renal defects, phenotypes observed in human patients bearing mutations in NPHP4, but they do develop severe photoreceptor degeneration and extinguished rod and cone ERG responses by 9 weeks of age. Photoreceptor outer segments (OS) fail to develop properly, and some OS markers mislocalize to the inner segments and outer nuclear layer in the Nphp4(nmf192/nmf192) mutant retina. Despite NPHP4 localization to the transition zone in the connecting cilia (CC), the CC appear to be normal in structure and ciliary transport function is partially retained. Likewise, synaptic ribbons develop normally but then rapidly degenerate by P14. Finally, Nphp4(nmf192/nmf192) male mutants are sterile and show reduced sperm motility and epididymal sperm counts. Although Nphp4(nmf192/nmf192) mice fail to recapitulate the kidney phenotype of NPHP, they will provide a valuable tool to further elucidate how NPHP4 functions in the retina and male reproductive organs.

Gene expression during the oocyte-to-embryo transition in mammals

The seminal question in modern developmental biology is the origins of new life arising from the unification of sperm and egg. The roots of this question begin from 19th to 20th century embryologists studying fertilization and embryogenesis. Although the revolution of molecular biology has yielded significant insight into the complexity of this process, the overall orchestration of genes, molecules, and cells is still not fully formed. Early mammalian development, specifically the oocyte‐to‐embryo transition, is essentially under “maternal command” from factors deposited in the cytoplasm during oocyte growth, independent of de novo transcription from the nascent embryo. Many of the advances in understanding this developmental period occurred in tandem with application of new methods and techniques from molecular biology, from protein electrophoresis to sequencing and assemblies of whole genomes. From this bed of knowledge, it appears that precise control of mRNA translation is a key regulator coordinating the molecular and cellular events occurring during oocyte‐to‐embryo transition. Notably, oocyte transcriptomes share, yet retain some uniqueness, common genetic motifs among all chordates. The common genetic motifs typically define fundamental processes critical for cellular maintenance, whereas the unique genetic features may be a source of variation and a substrate for sexual selection, genetic drift, or gene flow. One purpose for this complex interplay among genes, proteins, and cells may allow for evolution to transform and act upon the underlying processes, at molecular, structural and organismal levels, to increase diversity, which is the ultimate goal of sexual reproduction. Mol. Reprod. Dev. 76: 805–818, 2009.

Photoreceptor Degeneration, Azoospermia, Leukoencephalopathy, and Abnormal RPE Cell Function in Mice Expressing an Early Stop Mutation in CLCN2

PURPOSE To determine the molecular basis and the pathologic consequences of a chemically induced mutation in a mouse model of photoreceptor degeneration, nmf240. METHODS Mice from a G3 N-ethyl-N-nitrosourea mutagenesis program were screened by indirect ophthalmoscopy for abnormal fundi. A chromosomal position for the recessive nmf240 mutation was determined by a genome-wide linkage analysis by use of simple sequence length polymorphic markers in an F2 intercross. The critical region was refined, and candidate genes were screened by direct sequencing. The nmf240 phenotype was characterized by histologic analysis of the retina, brain, and male reproductive organs and by electroretinogram (ERG)-based studies of the retina and retinal pigment epithelium (RPE). RESULTS Clinically, homozygous nmf240 mutants exhibit a grainy retina that progresses to panretinal patches of depigmentation. The mutation was localized to a region on chromosome 16 containing Clcn2, a gene associated with retinal degeneration. Sequencing identified a missense C-T mutation at nucleotide 1063 in Clcn2 that converts a glutamine to a stop codon. Mice homozygous for the Clcn2(nmf240) mutation experience a severe loss of photoreceptor cells at 14 days of age that is preceded by an elongation of RPE apical microvilli. Homozygous mutants also experience leukoencephalopathy in multiple brain areas and male sterility. Despite a normal retinal histology in nmf240 heterozygotes, the ERG light peak, generated by the RPE, is reduced. CONCLUSIONS The nmf240 phenotype closely resembles that reported for Clcn2 knockout mice. The observation that heterozygous nmf240 mice present with a reduced ERG light peak component suggests that CLCN2 is necessary for the generation of this response component.

Transcriptome analysis reveals an unexpected role of a collagen tyrosine kinase receptor gene, Ddr2, as a regulator of ovarian function

Mice homozygous for the smallie (slie) mutation lack a collagen receptor, discoidin domain receptor 2 (DDR2), and are dwarfed and infertile due to peripheral dysregulation of the endocrine system of unknown etiology. We used a systems biology approach to identify biological networks affected by Ddr2(slie/slie) mutation in ovaries using microarray analysis and validate findings using molecular, cellular, and functional biological assays. Transcriptome analysis indicated several altered gene categories in Ddr2(slie/slie) mutants, including gonadal development, ovulation, antiapoptosis, and steroid hormones. Subsequent biological experiments confirmed the transcriptome analysis predictions. For instance, a significant increase of TUNEL-positive follicles was found in Ddr2(slie/slie) mutants vs. wild type, which confirm the transcriptome prediction for decreased chromatin maintenance and antiapoptosis. Decreases in gene expression were confirmed by RT-PCR and/or qPCR; luteinizing hormone receptor and prostaglandin type E and F receptors in Ddr2(slie/slie) mutants, compared with wild type, confirm hormonal signaling pathways involved in ovulation. Furthermore, deficiencies in immunohistochemistry for DDR2 and luteinizing hormone receptor in the somatic cells, but not the oocytes, of Ddr2(slie/slie) mutant ovaries suggest against an intrinsic defect in germ cells. Indeed, Ddr2(slie/slie) mutants ovulated significantly fewer oocytes; their oocytes were competent to complete meiosis and fertilization in vitro. Taken together, our convergent data signify DDR2 as a novel critical player in ovarian function, which acts upon classical endocrine pathways in somatic, rather than germline, cells.

Effects of sub-lethal teratogen exposure during larval development on egg laying and egg quality in adult Caenorhabditis elegans

Background: Acute high dose exposure to teratogenic chemicals alters the proper development of an embryo leading to infertility, impaired fecundity, and few viable offspring. However, chronic exposure to sub-toxic doses of teratogens during early development may also have long-term impacts on egg quality and embryo viability. Methods: To test the hypothesis that low dose exposure during early development can impact long-term reproductive health, Caenorhabditis elegans larvae were exposed to 10 teratogens during larval development, and subsequently were examined for the pattern of egg-laying and egg quality (hatched larvae and embryo viability) as gravid adults. After the exposure, adult gravid worms were transferred to untreated plates and the numbers of eggs laid were recorded every 3 hours, and the day following exposure the numbers of hatched larvae were counted. Re sults: While fecundity and fertility were typically impaired by teratogens, unexpectedly, many teratogens initially increased egg-laying at the earliest interval compared to control but not at later intervals. However, egg quality, as assessed by embryo viability, remained the same because many of the eggs (<50%) did not hatch. Conclusions: Chronic, low dose exposures to teratogens during early larval development have subtle, long-term effects on egg laying and egg quality.

Adenylyl cyclase 6 is required for maintaining acid-base homeostasis

Adenylyl cyclase (AC) isoform 6 (AC6) is highly expressed throughout the renal tubule and collecting duct (CD), catalyzes the synthesis of cAMP and contributes to various aspects of renal transport. Several proteins involved in acid-base homeostasis are regulated by cAMP. In the present study, we assess the relative contribution of AC6 to overall acid-base regulation using mice with global deletion of AC6 (AC6-/-) or newly generated mice lacking AC6 in the renal tubule and CD (AC6loxloxPax8Cre). Higher energy expenditure in AC6-/- relative to wild-type (WT) mice, was associated with lower urinary pH, mild alkalosis in conjunction with elevated blood HCO3- concentrations, and significantly higher renal abundance of the H+-ATPase B1 subunit. In contrast with WT mice, AC6-/- mice have a less pronounced increase in urinary pH after 8 days of HCO3- challenge, which is associated with increased blood pH and HCO3- concentrations. Immunohistochemistry demonstrated that AC6 was expressed in intercalated cells (IC), but subcellular distribution of the H+-ATPase B1 subunit, pendrin, and the anion exchangers 1 and 2 in AC6-/- mice was normal. In the AC6-/- mice, H+-ATPase B1 subunit levels after HCO3- challenge were greater, which correlated with a higher number of type A IC. In contrast with the AC6-/- mice, AC6loxloxPax8Cre mice had normal urinary pH under baseline conditions but higher blood HCO3- than controls after HCO3- challenge. In conclusion, AC6 is required for maintaining normal acid-base homeostasis and energy expenditure. Under baseline conditions, renal AC6 is redundant for acid-base balance but becomes important under alkaline conditions.

Insights into the Phenotypic and Behavioral Effects of Teratogenic Drugs in Caenorhabditis elegans

Environmental toxicants, chemical substances produced or introduced into the environment directly by humans or their activities, can act as teratogens during development that negatively impact health. Long-term ramifications of environmental exposures to sublethal doses of teratogens are often unrecognized and unknown. The round worm, Caenorhabditis elegans, is an emerging model organism to investigate the long-term impacts of environmental teratogens upon health. This chapter describes a toxicant exposure paradigm integrated with phenotyping assays to screen adult worms, and their progeny, for effects on reproduction, growth and development, behavior, and energy balance.Environmental toxicants, chemical substances produced or introduced into the environment directly by humans or their activities, can act as teratogens during development that negatively impact health. Long-term ramifications of environmental exposures to sublethal doses of teratogens are often unrecognized and unknown. The round worm, Caenorhabditis elegans, is an emerging model organism to investigate the long-term impacts of environmental teratogens upon health. This chapter describes a toxicant exposure paradigm integrated with phenotyping assays to screen adult worms, and their progeny, for effects on reproduction, growth and development, behavior, and energy balance.

Abstract 2248: Dynamic changes in transposon expression and epigenetic regulators detected during early breast cancer transition to malignancy

Introduction: Breast cancer is the second leading cause of cancer-related deaths in women. In 2017, about 315,000 women were diagnosed with breast cancer, with approximately 63,000 new cases of ductal carcinoma in situ (DCIS), the earliest form of breast cancer. Little is known as to why some patients diagnosed with atypia and DCIS remain cancer-free while others progresses to invasive ductal carcinoma (IDC). Hypothesis: While mutagenic role of transposonable elements (TEs) in cancer is known, we test the novel role of long terminal repeat (LTR) TEs as potential drivers promoting cell de-differentiation during early stages of breast cancer. Objective: To identify molecular signatures driving cell fate decisions at atypia and DCIS to transformation, we investigated expression of TEs in atypia, DCIS and IDC. Methods: We created a TE Enrichment Set Analysis (TESA) to identify TEs in RNA sequencing datasets across four stages of breast cancer, normal atypia, DCIS, IDC (n= 8-23 samples per stage). Results: After quality control steps to remove outliers, TEs, compared to genes, exhibit substantially less variation in their expression because the first principal component accounted for over 80% expression variation compared to 20% in transcript expression variation (p 0.10). Six general patterns of TE expression were identified using k-means clustering algorithm. In general, the majority of TEs were upregulated during atypia or DCIS stages or both stages. To identify potential molecular mechanisms of TE de-repression, which induce LTR expression during atypia and DCIS, we measured the correlation between specific LTRs, including HERV-H, HERV-K, LTR7Y, and expression of epigenetic regulators, TETs, DNMTs, and TRIM28/KAP1 across all four stages. As expected, normal tissue has positive and negative correlations between LTRs and epigenetic regulators, however, at atypia there was a loss of some negative correlations and at DCIS all negative correlations between LTRs and DNMTS were lost (p Citation Format: Isaac D. Raplee, Alexei Evsikov, Caralina Marin de Evsikova. Dynamic changes in transposon expression and epigenetic regulators detected during early breast cancer transition to malignancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2248.