In the Drosophila larva, blood cells or hemocytes are formed in the lymph gland. The major blood cell type, called plasmatocyte, is small, non-adhesive and phagocytic. Plasmatocytes differentiate into adhesive lamellocytes to form multilayered capsules around foreign substances or, in mutant melanotic tumor strains, around self tissue. Mutations in cactus or Toll, or constitutive expression of dorsal can induce lamellocyte differentiation and cause the formation of melanotic capsules. As maternally encoded proteins, Toll, Cactus and Dorsal, along with Tube and Pelle, participate in a common signal transduction pathway to specify the embryonic dorsal-ventral axis. Using the maternal pathway as a paradigm, we investigated if these proteins have additional roles in larval hemocyte formation and differentiation. Analysis of cactus mutants that lack Cactus protein revealed that almost all of these animals have an overabundance of hemocytes, carry melanotic capsules and die before reaching pupal stages. In addition, the lymph glands of cactus larvae are considerably enlarged. The number of mitotic cells in the cactus and TollD hemolymph is higher than that in the wild-type hemolymph. The hemocyte density of mutant Toll, tube or pelle hemolymph is significantly lower than that of the wild type. Lethality of mutant cactus animals could be rescued either by the selective expression of wild-type Cactus protein in the larval lymph gland or by the introduction of mutations in Toll, tube or pelle. Cactus, Toll, Tube and Pelle proteins are expressed in the nascent hemocytes of the larval lymph gland. Our results suggest that the Toll/Cactus signal transduction pathway plays a significant role in regulating hemocyte proliferation and hemocyte density in the Drosophila larva. These findings are discussed in light of similar hematopoietic functions of Rel/I(kappa)B-family proteins in mice.
Background/Aims: Metformin, the common medication for type II diabetes, has protective effects on cerebral ischemia. However, the molecular mechanisms are far from clear. Mitotic arrest deficient 2-like protein 2 (MAD2B), an inhibitor of the anaphase-promoting complex (APC), is widely expressed in hippocampal and cortical neurons and plays an important role in mediating high glucose-induced neurotoxicity. The present study investigated whether metformin modifies the expression of MAD2B and to exert its neuroprotective effects in primary cultured cortical neurons during oxygen-glucose deprivation/reoxygenation (OGD/R), a widely used in vitro model of ischemia/reperfusion. Methods: Primary cortical neurons were cultured, deprived of oxygen-glucose for 1 h, and then recovered with oxygen-glucose for 12 h and 24 h. Cell viability was measured by detecting the levels of lactate dehydrogenase (LDH) in culture medium. The levels of MAD2B, cyclin B and p-histone 3 were measured by Western blot. Results: Cell viability of neurons was reduced under oxygen-glucose deprivation/reoxygenation (OGD/R). The expression of MAD2B was increased under OGD/R. The levels of cyclin B1, which is a substrate of APC, were also increased. Moreover, OGD/R up-regulated the phosphorylation levels of histone 3, which is the induction of aberrant re-entry of post-mitotic neurons. However, pretreatment of neurons with metformin alleviated OGD/R-induced injury. Metformin further decreased the expression of MAD2B, cyclin B1 and phosphorylation levels of histone 3. Conclusion: Metformin exerts its neuroprotective effect through regulating the expression of MAD2B in neurons under OGD/R.
Although our recent study has demonstrated that mitotic spindle assembly checkpoint protein (MAD2B) mediates high glucose‐induced neuronal apoptosis, the mechanisms for MAD2B degradation under hyperglycaemia have not yet been elucidated. In this study, we first found that the activation of adenosine 5′‐monophosphate (AMP)‐activated protein kinase (AMPK) was decreased in neurons, accompanied with the increased expression of MAD2B. Mechanistically, we demonstrated that activation of AMPK with its activators such as AICAR and metformin decreased the expression of MAD2B, indicating a role of AMPK in regulating the expression of MAD2B. Moreover, activation of AMPK prevented neuronal cells from high glucose‐induced injury as demonstrated by the reduced expression of cyclin B1 and percentage of apoptosis as detected by TUNEL. We further found that when total protein synthesis was suppressed by chlorhexidine, the degradation of MAD2B was slower in high glucose‐treated neurons and was mainly dependent on the ubiquitin–proteasome system. Finally, it was indicated that high glucose inhibited the ubiquitination of MAD2B, which could be reversed by activation of AMPK. Collectively, this study demonstrates that AMPK acts as a key regulator of MAD2B expression, suggesting that activation of AMPK signalling might be crucial for the treatment of high glucose‐induced neuronal injury.
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