Efficient optical clearance is fundamental for whole brain imaging. In particular, clearance of the brain without membrane damage is required for the imaging of lipophilic tracer-labeled neural tracts. Relying on an ascending gradient of fructose solutions, SeeDB can achieve sufficient transparency of the mouse brain while ensuring that the plasma membrane remains intact. However, it is challenging to extend this method to larger mammalian brains due to the extremely high viscosity of the saturated fructose solution. Here we report a SeeDB-derived optical clearing method, termed FRUIT, which utilizes a cocktail of fructose and urea. As demonstrated in the adult mouse brain, combination of these two highly water-soluble clearing agents exerts a synergistic effect on clearance. More importantly, the final FRUIT solution has low viscosity so as to produce transparency of the whole adult rabbit brain via arterial perfusion, which is impossible to achieve with a saturated fructose solution. In addition to good compatibility with enhanced yellow fluorescent protein, the cocktail also preserves the fluorescence of the lipophilic tracer DiI. This work provides a volume-independent optical clearing method which retains the advantages of SeeDB, particularly compatibility with lipophilic tracers.

Neddylation, the covalent attachment of ubiquitin-like protein Nedd8, of the Cullin-RING E3 ligase family regulates their ubiquitylation activity. However, regulation of HECT ligases by neddylation has not been reported to date. Here we show that the C2-WW-HECT ligase Smurf1 is activated by neddylation. Smurf1 physically interacts with Nedd8 and Ubc12, forms a Nedd8-thioester intermediate, and then catalyses its own neddylation on multiple lysine residues. Intriguingly, this autoneddylation needs an active site at C426 in the HECT N-lobe. Neddylation of Smurf1 potently enhances ubiquitin E2 recruitment and augments the ubiquitin ligase activity of Smurf1. The regulatory role of neddylation is conserved in human Smurf1 and yeast Rsp5. Furthermore, in human colorectal cancers, the elevated expression of Smurf1, Nedd8, NAE1 and Ubc12 correlates with cancer progression and poor prognosis. These findings provide evidence that neddylation is important in HECT ubiquitin ligase activation and shed new light on the tumour-promoting role of Smurf1.

BACKGROUND AND PURPOSE WNK kinases, including WNK3, and the associated downstream SPAK and OSR1 kinases, comprise an important signaling cascade that regulates the cation-chloride cotransporters. Ischemia-induced stimulation of the bumetanide-sensitive Na+-K+-Cl- cotransporter (NKCC1) plays an important role in the pathophysiology of experimental stroke, but the mechanism of its regulation in this context is unknown. Here, we investigated the WNK3-SPAK/OSR1 pathway as a regulator of NKCC1 stimulation and their collective role in ischemic brain damage. METHOD Wild-type WNK3 (WT) and WNK3 knockout (KO) mice were subjected to ischemic stroke via transient middle cerebral artery (MCA) occlusion. Infarct volume, brain edema, blood brain barrier (BBB) damage, white matter demyelination, and neurological deficits were assessed. Total and phosphorylated forms of WNK3 and SPAK/OSR1 were assayed by immunobloting and immunostaining. In vitro ischemia studies in cultured neurons and immature oligodendrocytes were conducted using the oxygen-glucose deprivation/reoxygenation method. RESULTS WNK3 KO mice exhibited significantly decreased infarct volume and axonal demyelination, less cerebral edema, and accelerated neurobehavioral recovery compared to WNK3 WT mice subjected to MCA occlusion. The neuroprotective phenotypes conferred by WNK3 KO were associated with a decrease in stimulatory hyper-phosphorylations of the SPAK/OSR1 catalytic T-loop and of NKCC1 stimulatory sites Thr203/Thr207/Thr212, as well as with decreased cell surface expression of NKCC1. Genetic inhibition of WNK3 or siRNA knockdown of SPAK/OSR1 increased the tolerance of cultured primary neurons and oligodendrocytes to in vitro ischemia. CONCLUSION These data identify a novel role for the WNK3-SPAK/OSR1-NKCC1 signaling pathway in ischemic neuroglial injury, and suggest the WNK3-SPAK/OSR1 kinase pathway as a therapeutic target for neuroprotection following ischemic stroke.