Thomas R. Gawriluk

Autophagy: Regulation and role in development

Autophagy is an evolutionarily conserved cellular process through which long-lived proteins and damaged organelles are recycled to maintain energy homeostasis. These proteins and organelles are sequestered into a double-membrane structure, or autophagosome, which subsequently fuses with a lysosome in order to degrade the cargo. Although originally classified as a type of programmed cell death, autophagy is more widely viewed as a basic cell survival mechanism to combat environmental stressors. Autophagy genes were initially identified in yeast and were found to be necessary to circumvent nutrient stress and starvation. Subsequent elucidation of mammalian gene counterparts has highlighted the importance of this process to normal development. This review provides an overview of autophagy, the types of autophagy, its regulation and its known impact on development gleaned primarily from murine models.Autophagy is an evolutionarily conserved cellular process through which long-lived proteins and damaged organelles are recycled to maintain energy homeostasis. These proteins and organelles are sequestered into a double-membrane structure, or autophagosome, which subsequently fuses with a lysosome in order to degrade the cargo. Although originally classified as a type of programmed cell death, autophagy is more widely viewed as a basic cell survival mechanism to combat environmental stressors. Autophagy genes were initially identified in yeast and were found to be necessary to circumvent nutrient stress and starvation. Subsequent elucidation of mammalian gene counterparts has highlighted the importance of this process to normal development. This review provides an overview of autophagy, the types of autophagy, its regulation and its known impact on development gleaned primarily from murine models.

Autophagy is a cell survival program for female germ cells in the murine ovary

It is estimated that infertility affects 15-20% of couples and can arise from female or male reproductive defects. Mouse models have ascribed roles to over 100 genes in the maintenance of female fertility. Although previous models have determined roles for apoptosis in male and female fertility, we find that compromised autophagy within the perinatal ovary, through the loss of Becn1 or Atg7, results in the premature loss of female germ cells. Becn1(+/-) ovaries have a 56% reduction of germ cells compared with control ovaries at post-natal day 1, whereas Atg7(-/-) ovaries lack discernable germ cells at this stage. Thus autophagy appears to be a cell survival mechanism to maintain the endowment of female germ cells prior to establishing primordial follicle pools in the ovary.

Beclin-1 Deficiency in the Murine Ovary Results in the Reduction of Progesterone Production to Promote Preterm Labor

Significance The success of mammalian reproduction is contingent upon the production of hormones within the female to not only promote germ cell development, but to establish and maintain pregnancy. We demonstrate that abrogating autophagy, a cellular process to maintain energy stores, can lead to reproductive defects that prevent a successful pregnancy in mice. Females that lack the crucial autophagy gene Beclin1 (Becn1) in the progesterone-producing cells of the ovary demonstrate reduced circulating progesterone and a preterm birth phenotype concurrent with the loss of litters, which is rescued by the administration of exogenous progesterone. Because progesterone is a necessary hormone for mammalian pregnancy, these data suggest that autophagy may play a role in steroidogenesis and, thus, in successful human reproduction. Autophagy is an important cellular process that serves as a companion pathway to the ubiquitin-proteasome system to degrade long-lived proteins and organelles to maintain cell homeostasis. Although initially characterized in yeast, autophagy is being realized as an important regulator of development and disease in mammals. Beclin1 (Becn1) is a putative tumor suppressor gene that has been shown to undergo a loss of heterozygosity in 40–75% of human breast, ovarian, and prostate cancers. Because Becn1 is a key regulator of autophagy, we sought to investigate its role in female reproduction by using a conditional knockout approach in mice. We find that pregnant females lacking Becn1 in the ovarian granulosa cell population have a defect in progesterone production and a subsequent preterm labor phenotype. Luteal cells in this model exhibit defective autophagy and a failure to accumulate lipid droplets needed for steroidogenesis. Collectively, we show that Becn1 provides essential functions in the ovary that are essential for mammalian reproduction.

Macrophages are necessary for epimorphic regeneration in African spiny mice

How the immune system affects tissue regeneration is not well understood. In this study, we used an emerging mammalian model of epimorphic regeneration, the African spiny mouse, to examine cell-based inflammation and tested the hypothesis that macrophages are necessary for regeneration. By directly comparing inflammatory cell activation in a 4 mm ear injury during regeneration (Acomys cahirinus) and scarring (Mus musculus), we found that both species exhibited an acute inflammatory response, with scarring characterized by stronger myeloperoxidase activity. In contrast, ROS production was stronger and more persistent during regeneration. By depleting macrophages during injury, we demonstrate a functional requirement for these cells to stimulate regeneration. Importantly, the spatial distribution of activated macrophage subtypes was unique during regeneration with pro-inflammatory macrophages failing to infiltrate the regeneration blastema. Together, our results demonstrate an essential role for inflammatory cells to regulate a regenerative response. DOI: http://dx.doi.org/10.7554/eLife.24623.001

BECN1, corpus luteum function, and preterm labor.

Progesterone is a steroid hormone that is necessary to maintain pregnancy in mammals. We recently found that mice with a conditional deletion of Becn1/Beclin 1 specifically in the progesterone-synthesizing cells of the corpus luteum, had reduced progesterone synthesis and these mice failed to maintain pregnancy.1 Furthermore, we identified that lipid storage and feedback through PRLR (prolactin receptor) and LHCGR (luteinizing hormone/choriogonadotropin receptor) were negatively affected by Becn1 deletion. BECN1 is necessary for the interaction of the 2 catalytic subunits of the class III phosphatidylinositol 3-kinase complex, PIK3C3, and PIK3R4, which are responsible for the generation of phosphatidylinositol 3-phosphate that is required for nucleation of the phagophore. Work from Sun et al. and Itakura et al. demonstrated that this BECN1 complex is also necessary for the fusion of autophagosomes and endosomes with lysosomes. Therefore, we suspected that ablating Becn1 in luteal cells would inhibit macroautophagy, hereafter referred to as autophagy. In support, we provide evidence that autophagic flux is reduced in our model. Thus, this study provides evidence that Becn1 is necessary for steroid production in murine luteal cells.

Altering Autophagy: Mouse Models of Human Disease

Since the advent of knockout technologies using mouse embryonic stem cells in the late 1980s, there has been an explosion of murine models to profile human diseases. The understanding of the genetic contribution to these diseases has been further enhanced with the incorporation of tissue-specific gene deletion strategies through the use of the Cre-lox and FLP-FRT sitespecific recombination systems. Autophagy, a crucial regulator of cell energy homeostasis, is also a companion process to the ubiquitin-proteasome system to assist in the turnover of proteins. Two distinct types of mouse models have been engineered to characterize autophagy. The first type is based on the reporter model system to both detect and quantitate the in vivo levels of autophagy in all tissues and organs. The second type is based on genomic modification to perform global or tissue-specific gene deletions for generation of pathological disease conditions. A wide array of human diseases and conditions have been shown to be intimately linked to alterations in autophagy and include: 1) cancer, 2) heart disease, 3) neurodegenerative diseases (e.g. Alzheimer’s and Parkinson’s disease), 4) aging, 5) lysosomal storage disorders, 6) infectious disease and immunity (e.g. Crohn’s disease), 7) muscle atrophy, 8) stroke, 9) type 2 diabetes, and 10) reproductive infertility. This article will address the role of autophagy in human disease progression by reviewing the strengths and weaknesses of current murine models, as well as discussing their utility as therapeutic models for disease prevention and amelioration.