Exposure of the inner mitochondrial membrane triggers apoptotic mitophagy.

During apoptosis mediated by the intrinsic pathway, BAX/BAK triggers mitochondrial permeabilization and the release of cytochrome-c, followed by a dramatic remodelling of the mitochondrial network that results in mitochondrial herniation and the subsequent release of pro-inflammatory mitochondrial components. Here, we show that mitochondrial herniation and subsequent exposure of the inner mitochondrial membrane (IMM) to the cytoplasm, initiates a unique form of mitophagy to deliver these damaged organelles to lysosomes. IMM-induced mitophagy occurs independently of canonical PINK1/Parkin signalling and is driven by ubiquitination of the IMM. Our data suggest IMM-induced mitophagy is an additional safety mechanism that cells can deploy to contain damaged mitochondria. It may have particular relevance in situations where caspase activation is incomplete or inhibited, and in contexts where PINK1/Parkin-mitophagy is impaired or overwhelmed.

Loss of NEDD4 causes complete XY gonadal sex reversal in mice.

Gonadogenesis is the process wherein two morphologically distinct organs, the testis and the ovary, arise from a common precursor. In mammals, maleness is driven by the expression of Sry. SRY subsequently upregulates the related family member Sox9 which is responsible for initiating testis differentiation while repressing factors critical to ovarian development such as FOXL2 and ß-catenin. Here, we report a hitherto uncharacterised role for the ubiquitin-protein ligase NEDD4 in this process. XY Nedd4-deficient mice exhibit complete male-to-female gonadal sex reversal shown by the ectopic upregulation of Foxl2 expression at the time of gonadal sex determination as well as insufficient upregulation of Sox9. This sex reversal extends to germ cells with ectopic expression of SYCP3 in XY Nedd4-/- germ cells and significantly higher Sycp3 transcripts in XY and XX Nedd4-deficient mice when compared to both XY and XX controls. Further, Nedd4-/- mice exhibit reduced gonadal precursor cell formation and gonadal size as a result of reduced proliferation within the developing gonad as well as reduced Nr5a1 expression. Together, these results establish an essential role for NEDD4 in XY gonadal sex determination and development and suggest a potential role for NEDD4 in orchestrating these cell fate decisions through the suppression of the female pathway to ensure proper testis differentiation.

Identification of novel interacting partners of the NEDD4 ubiquitin ligase in mouse testis.

Posttranslational modification by ubiquitination targets proteins for degradation, recycling, stabilization or altered trafficking, and as such can alter cellular signaling pathways. The substrate specificity of this multistep process is controlled by ubiquitin ligases, including those of the HECT domain-containing NEDD4 family. In the testis, ubiquitination of many proteins contributes to organ development and maturation of spermatozoa and NEDD4 is known to be important in the control of spermatogonial stem cell homeostasis. However, a comprehensive understanding of NEDD4 substrates in testis development is lacking. Here we demonstrate high expression of Nedd4 in somatic cells of the mouse testis and in the murine Leydig cell-like cell line TM3. Immunoprecipitation of NEDD4 tagged with GFP at either the amino or carboxyl terminus was subjected to proteomic analysis for interacting proteins. We identified a substantial list of potential interaction partners, including known NEDD4 substrates, proteins involved in ubiquitination and proteins important for testis development and spermatogenesis. We confirmed the interaction of NEDD4 with a subset of these putative interacting proteins, validating the integrity of the dataset. These potential interactors may be further explored to reveal important roles of NEDD4-mediated ubiquitination in the testis. SIGNIFICANCE: Ubiquitination is important for testis development and function, and NEDD4 is known to ubiquitinate various proteins to affect cellular signaling and development, including those implicated in spermatogenesis. However, substrates of NEDD4 that are important during testis development remain to be identified. Here we report NEDD4 expression in the developing testis and TM3 testicular cell line. This study identifies a substantial list of NEDD4 interacting proteins in the TM3 testicular cell line, with validation of some of these interactions. Hence, this provides novel NEDD4 targets that may contribute to testis development and function that may be further explored.

Signaling Pathways Involved in Mammalian Sex Determination and Gonad Development.

The development of any organ system requires a complex interplay of cellular signals to initiate the differentiation and development of the heterogeneous cell and tissue types required to carry out the organs' functions. In this way, an extracellular stimulus is transmitted to an intracellular target through an array of interacting protein intermediaries, ultimately enabling the target cell to elicit a response. Surprisingly, only a small number of signaling pathways are implicated throughout embryogenesis and are used over and over again. Gonadogenesis is a unique process in that 2 morphologically distinct organs, the testes and ovaries, arise from a common precursor, the bipotential genital ridge. Accordingly, most of the signaling pathways observed throughout embryogenesis also have been shown to be important for mammalian sex determination and gonad development. Here, we review the mechanisms of signal transduction within these pathways and the importance of these pathways throughout mammalian gonad development, mainly concentrating on data obtained in mouse but including other species where appropriate.