PML-II recruits ataxin-3 to PML-NBs and inhibits its deubiquitinating activity.

Promyelocytic leukemia protein (PML) nuclear bodies (NBs) are dynamic and multiprotein complexes implicated in a variety of important biochemical events. Due to alternative mRNA splicing, PML has at least six nuclear isoforms that share a common N-terminus but differ in their C-terminal regions. However, the unique role of each PML isoform is not clear. Here, we report the characterization of the deubiquitinase ataxin-3 as a specific binding partner of PML isoform II (PML-II). Ataxin-3 was identified as a potential binding protein of PML-II in a yeast-hybrid screen employing the unique C-terminal region of PML-II as bait. Ataxin-3 only binds to the C-terminal region of PML-II and not that of other PML isoforms. The interaction between ataxin-3 and PML-II was confirmed by co-immunoprecipition assays, and immunofluorescent microscopy revealed that PML-II and ataxin-3 were co-localized in PML-NBs. In addition, PML-II not only interacts with ataxin-3 with a normal range of poly-Q repeats (13Q), but also with a pathological form of ataxin-3 with extended poly-Q repeats (79Q). Importantly, the deubiquitinase activity of ataxin-3 was inhibited by PML-II. Our results suggest that PML-II may be a negative regulator of ataxin-3.

Bosutinib Attenuates Inflammation via Inhibiting Salt-Inducible Kinases in Experimental Model of Intracerebral Hemorrhage on Mice.

BACKGROUND AND PURPOSE: Intracerebral hemorrhage (ICH) is a subtype of stroke with highest mortality and morbidity. Pronounced inflammation plays a significant role in the development of the secondary brain injury after ICH. Recently, SIK-2 (salt-inducible kinase-2) was identified as an important component controlling inflammatory response. Here we sought to investigate the role of SIK-2 in post-ICH inflammation and potential protective effects of SIK-2 inhibition after ICH. METHODS: Two hundred and ninety-three male CD-1 mice were used. ICH was induced via injection of 30 µL of autologous blood. Recombinant SIK-2 was administrated 1 hour after ICH intracerebroventricularly. SIK-2 small interfering RNA was injected intracerebroventricularly 24 hours before ICH. Bosutinib, a clinically approved tyrosine kinase inhibitor with affinity to SIK-2, was given intranasally 1 hour or 6 hours after ICH. Effects of treatments were evaluated by neurological tests and brain water content calculation. Molecular pathways were investigated by Western blots and immunofluorescence studies. RESULTS: Endogenous SIK-2 was expressed in microglia and neurons. SIK-2 expression was reduced after ICH. Exogenous SIK-2 aggravated post-ICH inflammation, leading to brain edema and the neurobehavioral deficits. SIK-2 inhibition attenuated post-ICH inflammation, reducing brain edema and ameliorating neurological dysfunctions. Bosutinib inhibited SIK-2-attenuating ICH-induced brain damage. Protective effects of Bosutinib were mediated, at least partly, by CRTC3 (cyclic amp-response element binding protein-regulated transcription coactivator 3)/cyclic amp-response element binding protein/NF-κB (nuclear factor-κB) pathway. CONCLUSIONS: SIK-2 participates in inflammation induction after ICH. SIK-2 inhibition via Bosutinib or small interfering RNA decreased inflammation, attenuating brain injury. SIK-2 effects are, at least partly, mediated by CRTC3-cyclic amp-response element binding protein-NF-κB signaling pathway.

Neuroprotective Mechanisms of Melatonin in Hemorrhagic Stroke.

Hemorrhagic stroke which consists of subarachnoid hemorrhage and intracerebral hemorrhage is a dominant cause of death and disability worldwide. Although great efforts have been made, the physiological mechanisms of these diseases are not fully understood and effective pharmacological interventions are still lacking. Melatonin (N-acetyl-5-methoxytryptamine), a neurohormone produced by the pineal gland, is a broad-spectrum antioxidant and potent free radical scavenger. More importantly, there is extensive evidence demonstrating that melatonin confers neuroprotective effects in experimental models of hemorrhagic stroke. Multiple molecular mechanisms such as antioxidant, anti-apoptosis, and anti-inflammation, contribute to melatonin-mediated neuroprotection against brain injury after hemorrhagic stroke. This review article aims to summarize current knowledge regarding the beneficial effects of melatonin in experimental models of hemorrhagic stroke and explores the underlying mechanisms. We propose that melatonin is a promising neuroprotective candidate that is worthy of further evaluation for its potential therapeutic applications in hemorrhagic stroke.

Recombinant Gas6 augments Axl and facilitates immune restoration in an intracerebral hemorrhage mouse model.

Axl, a tyrosine kinase receptor, was recently identified as an essential component regulating innate immune response. Suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3 are potent Axl-inducible negative inflammatory regulators. This study investigated the role of Axl signaling pathway in immune restoration in an autologous blood-injection mouse model of intracerebral hemorrhage. Recombinant growth arrest-specific 6 (Gas6) and R428 were administrated as specific agonist and antagonist. In vivo knockdown of Axl or suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3 by siRNA was applied. After intracerebral hemorrhage, the expression of endogenous Axl, soluble Axl, and Gas6 was increased, whereas the expression of suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3 was inhibited. Recombinant growth arrest-specific 6 administration alleviated brain edema and improved neurobehavioral performances. Moreover, enhanced Axl phosphorylation with cleavage of soluble Axl (sAxl), and an upregulation of suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3 were observed. In vivo knockdown of Axl and R428 administration both abolished the effect of recombinant growth arrest-specific 6 on brain edema and also decreased the expression suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3. In vivo knockdown of suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3 aggravated cytokine releasing despite of recombinant growth arrest-specific 6. In conclusion, Axl plays essential role in immune restoration after intracerebral hemorrhage. And recombinant growth arrest-specific 6 attenuated brain injury after intracerebral hemorrhage, probably by enhancing Axl phosphorylation and production of suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3.