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Progressive degeneration of dopaminergic (DA) neurons in the substantia nigra is a hallmark of Parkinson’s disease (PD). Dysregulation of developmental transcription factors is implicated in dopaminergic neurodegeneration, but the underlying molecular mechanisms remain largely unknown. Drosophila Fer2 is a prime example of a developmental transcription factor required for the birth and maintenance of midbrain DA neurons. Using an approach combining ChIP-seq, RNA-seq, and genetic epistasis experiments with PD-linked genes, here we demonstrate that Fer2 controls a transcriptional network to maintain mitochondrial structure and function, and thus confers dopaminergic neuroprotection against genetic and oxidative insults. We further show that conditional ablation of Nato3 , a mouse homolog of Fer2 , in differentiated DA neurons causes mitochondrial abnormalities and locomotor impairments in aged mice. Our results reveal the essential and conserved role of Fer 2 homologs in the mitochondrial maintenance of midbrain DA neurons, opening new perspectives for modeling and treating PD. Mitochondrial dysfunction in dopaminergic neurons is a pathological hallmark of Parkinson’s disease. Here, the authors find a conserved mechanism by which a single transcription factor controls mitochondrial health in dopaminergic neurons during the aging process.

Neural circuits are refined by both functional and structural changes. Structural remodeling by large-scale pruning occurs where relatively long neuronal branches are cut away from their parent neuron and removed by local degeneration. Until now, the molecular mechanisms executing such branch severing events have remained poorly understood. Here, we reveal a role for the Endosomal Sorting Complex Required for Transport (ESCRT) machinery during neuronal remodeling. Our data show that a specific ESCRT pruning module, including members of the ESCRT-I and ESCRT-III complexes, but not ESCRT-0 or ESCRT-II, are required for the neurite scission event during pruning. Furthermore we show that this ESCRT module requires a direct, in vivo , interaction between Shrub/CHMP4B and the accessory protein Myopic/HD-PTP.

Forkhead box (FOXO) proteins are evolutionarily conserved, stress-responsive transcription factors (TFs) that can promote or counteract cell death. Mutations in FOXO genes are implicated in numerous pathologies, including age-dependent neurodegenerative disorders, such as Parkinson's disease (PD). However, the complex regulation and downstream mechanisms of FOXOs present a challenge in understanding their roles in the pathogenesis of PD. Here, we investigate the involvement of FOXO in the death of dopaminergic (DA) neurons, the key pathological feature of PD, in Drosophila. We show that dFOXO null mutants exhibit a selective loss of DA neurons in the subgroup crucial for locomotion, the protocerebral anterior medial (PAM) cluster, during development as well as in adulthood. PAM neuron-targeted adult-restricted knockdown demonstrates that dFOXO in adult PAM neurons tissue-autonomously promotes neuronal survival during aging. We further show that dFOXO and the bHLH-TF 48-related-2 (FER2) act in parallel to protect PAM neurons from different forms of cellular stress. Remarkably, however, dFOXO and FER2 share common downstream processes leading to the regulation of autophagy and mitochondrial morphology. Thus, overexpression of one can rescue the loss of function of the other. These results indicate a role of dFOXO in neuroprotection and highlight the notion that multiple genetic and environmental factors interact to increase the risk of DA neuron degeneration and the development of PD.

Dysregulation of CDK8 (Cyclin-Dependent Kinase 8) and its regulatory partner CycC (Cyclin C), two subunits of the conserved Mediator (MED) complex, have been linked to diverse human diseases such as cancer. Thus, it is essential to understand the regulatory network modulating the CDK8-CycC complex in both normal development and tumorigenesis. To identify upstream regulators or downstream effectors of CDK8, we performed a dominant modifier genetic screen in Drosophila based on the defects in vein patterning caused by specific depletion or overexpression of CDK8 or CycC in developing wing imaginal discs. We identified 26 genomic loci whose haploinsufficiency can modify these CDK8- or CycC-specific phenotypes. Further analysis of two overlapping deficiency lines and mutant alleles led us to identify genetic interactions between the CDK8-CycC pair and the components of the Decapentaplegic (Dpp, the Drosophila homolog of TGFβ, or Transforming Growth Factor-β) signaling pathway. We observed that CDK8-CycC positively regulates transcription activated by Mad (Mothers against dpp), the primary transcription factor downstream of the Dpp/TGFβ signaling pathway. CDK8 can directly interact with Mad in vitro through the linker region between the DNA-binding MH1 (Mad homology 1) domain and the carboxy terminal MH2 (Mad homology 2) transactivation domain. Besides CDK8 and CycC, further analyses of other subunits of the MED complex have revealed six additional subunits that are required for Mad-dependent transcription in the wing discs: Med12, Med13, Med15, Med23, Med24, and Med31. Furthermore, our analyses confirmed the positive roles of CDK9 and Yorkie in regulating Mad-dependent gene expression in vivo. These results suggest that CDK8 and CycC, together with a few other subunits of the MED complex, may coordinate with other transcription cofactors in regulating Mad-dependent transcription during wing development in Drosophila.

Mediator (MED) is a conserved multisubunit complex bridging transcriptional activators and repressors to the general RNA polymerase II initiation machinery. In yeast, MED is organized in three core modules and a separable ‘Cdk8 module’ consisting of the cyclin‐dependent kinase Cdk8, its partner CycC, Med12 and Med13. This regulatory module, specifically required for cellular adaptation to environmental cues, is thought to act through the Cdk8 kinase activity. Here we have investigated the functions of the four Cdk8 module subunits in the metazoan model Drosophila . Physical interactions detected among the four fly subunits provide support for a structurally conserved Cdk8 module. We analyzed the in vivo functions of this module using null mutants for Cdk8 , CycC, Med12 and Med13 . Each gene is required for the viability of the organism but not of the cell. Cdk8–CycC and Med12–Med13 act as pairs, which share some functions but also have distinct roles in developmental gene regulation. These data reveal functional attributes of the Cdk8 module, apart from its regulated kinase activity, that may contribute to the diversification of genetic programs.

Dysregulation of CDK8 (Cyclin-Dependent Kinase 8) and its regulatory partner CycC (Cyclin C), two subunits of the conserved Mediator (MED) complex, have been linked to diverse human diseases such as cancer. Thus, it is essential to understand the regulatory network modulating the CDK8-CycC complex in both normal development and tumorigenesis. To identify upstream regulators or downstream effectors of CDK8, we performed a dominant modifier genetic screen in Drosophila based on the defects in vein patterning caused by specific depletion or overexpression of CDK8 or CycC in developing wing imaginal discs. We identified 26 genomic loci whose haploinsufficiency can modify these CDK8- or CycC-specific phenotypes. Further analysis of two overlapping deficiency lines and mutant alleles led us to identify genetic interactions between the CDK8-CycC pair and the components of the Decapentaplegic (Dpp, the Drosophila homolog of TGFβ, or Transforming Growth Factor-β) signaling pathway. We observed that CDK8-CycC positively regulates transcription activated by Mad (Mothers against dpp), the primary transcription factor downstream of the Dpp/TGFβ signaling pathway. CDK8 can directly interact with Mad in vitro through the linker region between the DNA-binding MH1 (Mad homology 1) domain and the carboxy terminal MH2 (Mad homology 2) transactivation domain. Besides CDK8 and CycC, further analyses of other subunits of the MED complex have revealed six additional subunits that are required for Mad-dependent transcription in the wing discs: Med12, Med13, Med15, Med23, Med24, and Med31. Furthermore, our analyses confirmed the positive roles of CDK9 and Yorkie in regulating Mad-dependent gene expression in vivo. These results suggest that CDK8 and CycC, together with a few other subunits of the MED complex, may coordinate with other transcription cofactors in regulating Mad-dependent transcription during wing development in Drosophila.

Mediator (MED) is a conserved multisubunit complex bridging transcriptional activators and repressors to the general RNA polymerase II initiation machinery. In yeast, MED is organized in three core modules and a separable ‘Cdk8 module' consisting of the cyclin-dependent kinase Cdk8, its partner CycC, Med12 and Med13. This regulatory module, specifically required for cellular adaptation to environmental cues, is thought to act through the Cdk8 kinase activity. Here we have investigated the functions of the four Cdk8 module subunits in the metazoan model Drosophila. Physical interactions detected among the four fly subunits provide support for a structurally conserved Cdk8 module. We analyzed the in vivo functions of this module using null mutants for Cdk8, CycC, Med12 and Med13. Each gene is required for the viability of the organism but not of the cell. Cdk8–CycC and Med12–Med13 act as pairs, which share some functions but also have distinct roles in developmental gene regulation. These data reveal functional attributes of the Cdk8 module, apart from its regulated kinase activity, that may contribute to the diversification of genetic programs.

The degeneration of dopaminergic (DA) neurons in the substantia nigra is a hallmark of Parkinson's disease (PD). Dysregulation of developmental transcription factors is implicated in dopaminergic neurodegeneration, but the underlying molecular mechanisms remain largely unknown. Drosophila Fer2 is a prime example of a developmental transcription factor required for the birth and maintenance of midbrain DA neurons. Using an approach combining ChIP-seq, RNA-seq, and genetic epistasis experiments with PD-linked genes, here we demonstrate that Fer2 controls a transcriptional network to maintain mitochondrial structure and function, and thus confers dopaminergic neuroprotection against genetic and oxidative insults. We further show that conditional ablation of Nato3, a mouse homolog of Fer2, in differentiated DA neurons results in locomotor impairments and mitochondrial abnormality in aged mice. Our results reveal the essential and conserved role of Fer2 homologs in the mitochondrial maintenance of midbrain DA neurons, opening new perspectives for modelling and treating PD. Competing Interest Statement The authors have declared no competing interest.

Dysregulations of CDK8 and its regulatory partner CycC, two subunits of the conserved Mediator complex, have been linked to diverse human diseases such as cancer, thus it is essential to understand the regulatory network mobilizing the CDK8-CycC complex in both normal development and tumorigenesis. To identify upstream regulators or downstream effectors of CDK8, we performed a dominant modifier genetic screen in Drosophila based on the defects in vein patterning caused by specific depletion or overexpression of CDK8 or CycC in wing imaginal discs. We identified 26 genomic loci whose haploinsufficiency can modify these CDK8-specific phenotypes. Further analysis of two deficiency lines and mutant alleles led us to identify interactions between CDK8-CycC and the components of the Decapentaplegic (Dpp, the Drosophila homolog of TGF ) signaling pathway. We observed that CDK8-CycC positively regulates transcription activated by Mad (Mothers against dpp), the primary transcription factor downstream of the Dpp/TGF signaling pathway. CDK8 can directly interact with Mad in vitro through the linker region between the DNA-binding MH1 (Mad homology 1) domain and the carboxy terminal MH2 transactivation domain. Besides CDK8 and CycC, further analyses of other subunits of the Mediator complex have revealed six additional Mediator subunits that are required for Mad-dependent transcription in the wing discs, including Med12, Med13, Med15, Med23, Med24, and Med31. Furthermore, CDK9 and Yorkie also positively regulate Mad-dependent gene expression in vivo. These results suggest that the Mediator complex may coordinate with other transcription cofactors in regulating Mad-dependent transcription during the wing vein patterning in Drosophila.

Pancreatic ductal adenocarcinoma is one of the most intractable and fatal cancer. The decreased blood vessel density displayed by this tumor not only favors its resistance to chemotherapy but also participates in its aggressiveness due to the consequent high degree of hypoxia. It is indeed clear that hypoxia promotes selective pressure on malignant cells that must develop adaptive metabolic responses to reach their energetic and biosynthetic demands. Here, using a well-defined mouse model of pancreatic cancer, we report that hypoxic areas from pancreatic ductal adenocarcinoma are mainly composed of epithelial cells harboring epithelial-mesenchymal transition features and expressing glycolytic markers, two characteristics associated with tumor aggressiveness. We also show that hypoxia increases the “glycolytic” switch of pancreatic cancer cells from oxydative phosphorylation to lactate production and we demonstrate that increased lactate efflux from hypoxic cancer cells favors the growth of normoxic cancer cells. In addition, we show that glutamine metabolization by hypoxic pancreatic tumor cells is necessary for their survival. Metabolized glucose and glutamine converge toward a common pathway, termed hexosamine biosynthetic pathway, which allows O-linked N-acetylglucosamine modifications of proteins. Here, we report that hypoxia increases transcription of hexosamine biosynthetic pathway genes as well as levels of O-glycosylated proteins and that O-linked N-acetylglucosaminylation of proteins is a process required for hypoxic pancreatic cancer cell survival. Our results demonstrate that hypoxia-driven metabolic adaptive processes, such as high glycolytic rate and hexosamine biosynthetic pathway activation, favor hypoxic and normoxic cancer cell survival and correlate with pancreatic ductal adenocarcinoma aggressiveness.