CRL4 antagonizes SCFFbxo7-mediated turnover of cereblon and BK channel to regulate learning and memory

Intellectual disability (ID), one of the most common human developmental disorders, can be caused by genetic mutations in Cullin 4B (Cul4B) and cereblon (CRBN). CRBN is a substrate receptor for the Cul4A/B-DDB1 ubiquitin ligase (CRL4) and can target voltage- and calcium-activated BK channel for ER retention. Here we report that ID-associated CRL4CRBNmutations abolish the interaction of the BK channel with CRL4, and redirect the BK channel to the SCFFbxo7ubiquitin ligase for proteasomal degradation. Glioma cell lines harbouring CRBN mutations record density-dependent decrease of BK currents, which can be restored by blocking Cullin ubiquitin ligase activity. Importantly, mice with neuron-specific deletion of DDB1 or CRBN express reduced BK protein levels in the brain, and exhibit similar impairment in learning and memory, a deficit that can be partially rescued by activating the BK channel. Our results reveal a competitive targeting of the BK channel by two ubiquitin ligases to achieve exquisite control of its stability, and support changes in neuronal excitability as a common pathogenic mechanism underlying CRL4CRBN–associated ID

Kidney development: roles of Sprouty, Wnt2b and type XVIII collagen in the ureteric bud morphogenesis

Abstract The mammalian metanephric kidney develops through ureteric bud branching morphogenesis and tubule formation and involves secreted inductive signals and possibly their antagonists to regulate the process. Sprouty (spry) genes encode antagonists of FGFs and the EGF signalling pathways. To get an insight to potential developmental roles of the spry genes, the expression of spry1, 2 and 4 was analyzed in developing kidney. Spry1 is expressed in the ureteric bud, and spry2 and 4 in the ureteric bud, the kidney mesenchyme and the nephrons deriving from it suggesting developmental roles for the sprys in kidney development. Spry function was addressed in vivo in the kidney by targeting hspry2 expression to the ureteric bud with a Pax2 promoter. Hspry2 expression led to development of small, ectopic and cystic kidneys. Ureter branching was reduced and there was less glomeruli in a smaller kidney compared to the wild type controls. Spry2 may antagonize signalling of FGF2 and lead to changes in FGFR1 and FGFR3 expression. In organ culture ectopic FGFs restored ureteric branching of the hSpry2 transgenic kidneys suggesting that hSpry2 may antagonize FGF signalling in embryonic kidney. In addition to changes in FGFs, hspry2 expression also lead to downregulation of GDNF and BMP4. We conclude that the Sprouty-FGFs-FGFR signaling is important for kidney development. Wnt2b is a recently identified member of the Wnt family of secreted growth factors, but its function in organogenesis is unknown. In the kidney Wnt2b is localized to the perinephric mesenchymal cells at the initiation of organogenesis. Wnt2b signalling supported ureteric bud growth and branching in vitro. Ureteric bud that was co-cultured with Wnt2b expressive cells or incubated with a known Wnt pathway regulator lithium, and then recombined with isolated kidney mesenchyme led to recovery of the expression of some ureteric epithelial marker genes and reconstitution of early kidney development. Hence, Wnt2b signalling is critical for induction of ureteric branching in vitro. Type XVIII collagen is a matrix molecule and may be involved in Wnt signalling. Roles of type XVIII collagen in kidney and lung organogenesis was analysed. Type XVIII collagen expression correlated with the differences in epithelial branching in both of these organs and its expression in the epithelial tissue was mutually exclusive. In recombinants of ureteric bud and lung mesenchyme, type XVIII collagen expression pattern shifted from kidney to lung type and was accompanied by a shift in epithelial Sonic Hedgehog (Shh) expression and by ectopic lung Surfactant Protein C in the ureteric bud. Blocking of type XVIII collagen function prevented ureteric development with lung mesenchyme and associated with reduction in the expression of Wnt2. Taken together, the findings suggest critical roles for Sprouty2, Wnt2b and type XVIII collagen in controlling pattern formation and the mode of ureteric bud branching in the embryonic kidney

KSHV-induced notch components render endothelial and mural cell characteristics and cell survival

Kaposi sarcoma–associated herpesvirus (KSHV) infection is essential to the development of Kaposi sarcoma (KS). Notch signaling is also known to play a pivotal role in KS cell survival and lytic phase entrance of KSHV. In the current study, we sought to determine whether KSHV regulates Notch components. KSHV-infected lymphatic endothelial cells showed induction of receptors Notch3 and Notch4, Notch ligands Dll4 and Jagged1, and activated Notch receptors in contrast to uninfected lymphatic endothelial cells. In addition, KSHV induced the expression of endothelial precursor cell marker (CD133) and mural cell markers (calponin, desmin, and smooth muscle alpha actin), suggesting dedifferentiation and trans-differentiation. Overexpression of latency proteins (LANA, vFLIP) and lytic phase proteins (RTA, vGPCR, viral interleukin-6) further supported the direct regulatory capacity of KSHV viral proteins to induce Notch receptors (Notch2, Notch3), ligands (Dll1, Dll4, Jagged1), downstream targets (Hey, Hes), and endothelial precursor CD133. Targeting Notch pathway with γ-secretase inhibitor and a decoy protein in the form of soluble Dll4 inhibited growth of KSHV-transformed endothelial cell line. Soluble Dll4 was also highly active in vivo against KS tumor xenograft. It inhibited tumor cell growth, induced tumor cell death, and reduced vessel perfusion. Soluble Dll4 is thus a candidate for clinical investigation