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Hypoxia-inducible factor prolyl 4-hydroxylases (HIF-P4Hs) regulate the hypoxic induction of300 genes required for survival and adaptation under oxygen deprivation. Inhibition of HIF-P4H-2 has been shown to be protective in focal cerebral ischemia rodent models, while that of HIF-P4H-1 has no effects and inactivation of HIF-P4H-3 has adverse effects. A transmembrane prolyl 4-hydroxylase (P4H-TM) is highly expressed in the brain and contributes to the regulation of HIF, but the outcome of its inhibition on stroke is yet unknown. To study this, we subjected WT and P4htm

Summary The circadian clock and the hypoxia-signaling pathway are regulated by an integrated interplay of positive and negative feedback limbs that incorporate energy homeostasis and carcinogenesis. We show that the negative circadian regulator CRY1 is also a negative regulator of hypoxia-inducible factor (HIF). Mechanistically, CRY1 interacts with the basic-helix-loop-helix domain of HIF-1α via its tail region. Subsequently, CRY1 reduces HIF-1α half-life and binding of HIFs to target gene promoters. This appeared to be CRY1 specific because genetic disruption of CRY1, but not CRY2, affected the hypoxia response. Furthermore, CRY1 deficiency could induce cellular HIF levels, proliferation, and migration, which could be reversed by CRISPR/Cas9- or short hairpin RNA-mediated HIF knockout. Altogether, our study provides a mechanistic explanation for genetic association studies linking a disruption of the circadian clock with hypoxia-associated processes such as carcinogenesis., Graphical Abstract, Highlights • Hypoxia and HIFs affect the circadian rhythm • CRY1 directly interacts with both HIF-1α and HIF-2α • CRY1 inhibits binding of HIFs to its target gene promoters • The CRY1-HIFα interaction has opposite roles on cellular growth and migration, Biological Sciences; Biochemistry; Molecular Biology; Cell Biology

The transcription factor Hypoxia-inducible factor 1 (HIF-1) plays a pivotal role in tissue regeneration. HIF-1 is negatively controlled by O2-dependent prolyl hydroxylases with a predominant role of prolyl hydroxylase 2 isoform (Phd2). Transgenic mice, hypomorphic for this isoform, accumulate more HIF-1 under normoxic conditions. Using these mice, we investigated the influence of Phd2 and HIF-1 on the regenerative capability of skeletal muscle tissue after myotrauma. Phd2-hypomorphic and wild type mice (on C57Bl/6 background) were grouped with regeneration times from 6 to 168 hours after closed mechanic muscle trauma to the hind limb. Tissue samples were analysed by immuno-staining and real-time PCR. Bone marrow derived macrophages of wild type and Phd2-hypomorphic mice were isolated and analysed via flow cytometry and quantitative real-time PCR. Phd2 reduction led to a higher regenerative capability due to enhanced activation of myogenic factors accompanied by induction of genes responsible for glucose and lactate metabolism in Phd2-hypomorphic mice. Macrophage infiltration into the trauma areas in hypomorphic mice started earlier and was more pronounced compared to wild type mice. Phd2-hypomorphic mice also showed higher numbers of macrophages in areas with sustained trauma 72 hours after myotrauma application. In conclusion, we postulate that the HIF-1 pathway is activated secondary to a Phd2 reduction which may lead to i) higher activation of myogenic factors, ii) increased number of positive stem cell proliferation markers, and iii) accelerated macrophage recruitment to areas of trauma, resulting in faster muscle tissue regeneration after myotrauma. With the current development of prolyl hydroxylase domain inhibitors, our findings point towards a potential clinical benefit after myotrauma. CA - Fandrey

The Finnish-variant late infantile neuronal ceroid lipofuscinosis, also known as CLN5 disease, is caused by mutations in the CLN5 gene. Cln5 is strongly expressed in the developing brain and expression continues into adulthood. CLN5, a protein of unknown function, is implicated in neurodevelopment but detailed investigation is lacking. Using Cln5−/− embryos of various ages and cells harvested from Cln5−/− brains we investigated the hitherto unknown role of Cln5 in the developing brain. Loss of Cln5 results in neuronal differentiation deficits and delays in interneuron development during in utero period. Specifically, the radial thickness of dorsal telencephalon was significantly decreased in Cln5−/− mouse embryos at embryonic day 14.5 (E14.5), and expression of Tuj1, an important neuronal marker during development, was down-regulated. An interneuron marker calbindin and a mitosis marker p-H3 showed down-regulation in ganglionic eminences. Neurite outgrowth was compromised in primary cortical neuronal cultures derived from E16 Cln5−/− embryos compared with WT embryos. We show that the developmental deficits of interneurons may be linked to increased levels of the repressor element 1-silencing transcription factor, which we report to bind to glutamate decarboxylase (Gad1), which encodes GAD67, a rate-limiting enzyme in the production of gamma-aminobutyric acid (GABA). Indeed, adult Cln5−/− mice presented deficits in hippocampal parvalbumin-positive interneurons. Furthermore, adult Cln5−/− mice presented deficits in hippocampal parvalbumin-positive interneurons and showed age-independent cortical hyper excitability as measured by electroencephalogram and auditory-evoked potentials. This study highlights the importance of Cln5 in neurodevelopment and suggests that in contrast to earlier reports, CLN5 disease is likely to develop during embryonic stages.

The circadian clock and the hypoxia signaling pathway are regulated by an integrated interplay of positive and negative feedback limbs that incorporate energy homeostasis and carcinogenesis. We show that the negative circadian regulator CRY1 is also a negative regulator of hypoxiainducible factor (HIF). Mechanistically, CRY1 interacts with the basic helixloop-helix domain of HIF-1α via its tail region. Subsequently, CRY1 reduces HIF-1α half-life and binding of HIFs to target gene promoters. This appeared to be CRY1 specific since genetic disruption of CRY1 but not CRY2 affected the hypoxia response. Further, CRY1-deficiency could induce cellular HIF levels, proliferation and migration, which could be reversed by CRISPR/Cas9 or shRNA mediated HIF knock-out. Altogether, our study provides a mechanistic explanation for genetic association studies linking a disruption of the circadian clock with hypoxia-associated processes such as carcinogenesis.

Thrombopoietin (TPO), a hematopoietic growth factor regulating platelet production, and its receptor (TPOR) were recently shown to be expressed in the brain where they exert proapoptotic activity. Here we used PC12 cells, an established model of neuronal differentiation, to investigate the effects of TPO on neuronal survival and differentiation. These cells expressed TPOR mRNA. TPO increased cell death in neuronally differentiated PC12 cells but had no effect in undifferentiated cells. Surprisingly, TPO inhibited nerve growth factor (NGF)-induced differentiation of PC12 cells in a dose- and time-dependent manner. This inhibition was dependent on the activity of Janus kinase-2 (JAK2). Using phospho-kinase arrays and Western blot we found downregulation of the NGF-stimulated phosphorylation of the extracellular signal-regulated kinase p42ERK by TPO with no effect on phosphorylation of Akt or stress kinases. NGF-induced phosphorylation of ERK-activating kinases, MEK1/2 and C-RAF was also reduced by TPO while NGF-induced RAS activation was not attenuated by TPO treatment. In contrast to its inhibitory effects on NGF signalling, TPO had no effect on epidermal growth factor (EGF)-stimulated ERK phosphorylation or proliferation of PC12 cells. Our data indicate that TPO via activation of its receptor-bound JAK2 delays the NGF-dependent acquisition of neuronal phenotype and decreases neuronal survival by suppressing NGF-induced ERK activity.

The adaptor protein regulator for ubiquitous kinase/c-Cbl-interacting protein of 85kDa (Ruk/CIN85) was found to modulate HER1/EGFR signaling and processes like cell adhesion and apoptosis. Although these features imply a role in carcinogenesis, it is so far unknown how and by which molecular mechanisms Ruk/CIN85 could affect a certain tumor phenotype. By analyzing samples from breast cancer patients, we found high levels of Ruk(l)/CIN85 especially in lymph node metastases from patients with invasive breast adenocarcinomas, suggesting that Ruk(l)/CIN85 contributes to malignancy. Expression of Ruk(l)/CIN85 in weakly invasive breast adenocarcinoma cells deficient of Ruk(l)/CIN85 indeed converted them into more malignant cells. In particular, Ruk(l)/CIN85 reduced the growth rate, decreased cell adhesion, enhanced anchorage-independent growth, increased motility in both transwell migration and wound healing assays as well as affected the response to epidermal growth factor. Thereby, Ruk(l)/CIN85 led to a more rapid and prolonged epidermal growth factor-dependent activation of Src, Akt and ERK1/2 and treatment with the Src inhibitor PP2 and the PI3K inhibitor LY294002 abolished the Ruk(l)/CIN85-dependent changes in cell motility. Together, this study indicates that high levels of Ruk(l)/CIN85 contribute to the conversion of breast adenocarcinoma cells into a more malignant phenotype via modulation of the Src/Akt pathway.

The tissue protective functions of the hematopoietic growth factor erythropoietin (EPO) are independent of its action on erythropoiesis. EPO and its receptors (EPOR) are expressed in multiple brain cells during brain development and upregulated in the adult brain after injury. Peripherally administered EPO crosses the blood-brain barrier and activates in the brain anti-apoptotic, anti-oxidant and anti-inflammatory signaling in neurons, glial and cerebrovascular endothelial cells and stimulates angiogenesis and neurogenesis. These mechanisms underlie its potent tissue protective effects in experimental models of stroke, cerebral hemorrhage, traumatic brain injury, neuroinflammatory and neurodegenerative disease. The preclinical data in support of the use of EPO in brain disease have already been translated to first clinical pilot studies with encouraging results with the use of EPO as a neuroprotective agent.

The transcription factors signal transducer and activator of transcription 5a and 5b (Stat5) are activated by the neuroprotective and neurotrophic cytokines, erythropoietin (EPO) and growth hormone (GH). Here, we show a dissociation of the intracellular pathway mediating the protective effect of EPO against glutamate toxicity from that needed for its neurotrophic activity using hippocampal neuronal cultures from Stat5a/b-knockout (Stat5(-/-)) mouse fetuses. Both pretreatment and post-treatment with EPO counteracted glutamate-induced cell death in Stat5(+/+) and Stat5(-/-) neurons. Acute pharmacological inhibition of Janus kinase 2 (JAK2)/Stat signalling had no effect on EPO neuroprotection, whereas inhibition of phosphatidylinositol-3' kinase (PI3K)/Akt pathway abolished the protective effect of EPO in both Stat5(+/+) and Stat5(-/-) neurons. GH effectively protected Stat5(+/+) cells against glutamate toxicity but had no effect in Stat5(-/-) neurons or in Stat5(+/+) neurons treated with JAK2/Stat or PI3K inhibitor. EPO and GH stimulated neurite outgrowth and branching of Stat5(+/+) neurons by activating PI3K/Akt signalling but had no trophic effect in Stat5(-/-) cells. We conclude that in hippocampal neurons, Stat5 is not required for neuroprotection by EPO but is together with Akt essential for its neurotrophic activity. Both Stat5 and Akt are needed for neuroprotective and neurotrophic signalling of GH in neurons.

Correct timing and spatial location of growth factor expression is critical for undisturbed brain development and functioning. In terminally differentiated cells distinct biological responses to growth factors may depend on cell type specific activation of signalling cascades. We show that the hematopoietic growth factors thrombopoietin (TPO) and granulocyte colony-stimulating factor (GCSF) exert cell type specific effects on survival, proliferation and the degree of phosphorylation of Akt1, ERK1/2 and STAT3 in rat hippocampal neurons and cortical astrocytes. In neurons, TPO induced cell death and selectively activated ERK1/2. GCSF protected neurons from TPO- and hypoxia-induced cell death via selective activation of Akt1. In astrocytes, neither TPO nor GCSF had any effect on cell viability but inhibited proliferation. This effect was accompanied by activation of ERK1/2 and inhibition of STAT3 activity. A balance between growth factors, their receptors and signalling proteins may play an important role in regulation of neural cell survival.