WldS Reduces Paraquat-Induced Cytotoxicity via SIRT1 in Non-Neuronal Cells by Attenuating the Depletion of NAD

WldS is a fusion protein with NAD synthesis activity, and has been reported to protect axonal and synaptic compartments of neurons from various mechanical, genetic and chemical insults. However, whether WldS can protect non-neuronal cells against toxic chemicals is largely unknown. Here we found that WldS significantly reduced the cytotoxicity of bipyridylium herbicides paraquat and diquat in mouse embryonic fibroblasts, but had no effect on the cytotoxicity induced by chromium (VI), hydrogen peroxide, etoposide, tunicamycin or brefeldin A. WldS also slowed down the death of mice induced by intraperitoneal injection of paraquat. Further studies demonstrated that WldS markedly attenuated mitochondrial injury including disruption of mitochondrial membrane potential, structural damage and decline of ATP induced by paraquat. Disruption of the NAD synthesis activity of WldS by an H112A or F116S point mutation resulted in loss of its protective function against paraquat-induced cell death. Furthermore, WldS delayed the decrease of intracellular NAD levels induced by paraquat. Similarly, treatment with NAD or its precursor nicotinamide mononucleotide attenuated paraquat-induced cytotoxicity and decline of ATP and NAD levels. In addition, we showed that SIRT1 was required for both exogenous NAD and WldS-mediated cellular protection against paraquat. These findings suggest that NAD and SIRT1 mediate the protective function of WldS against the cytotoxicity induced by paraquat, which provides new clues for the mechanisms underlying the protective function of WldS in both neuronal and non-neuronal cells, and implies that attenuation of NAD depletion may be effective to alleviate paraquat poisoning

PLAA Mutations Cause a Lethal Infantile Epileptic Encephalopathy by Disrupting Ubiquitin-Mediated Endolysosomal Degradation of Synaptic Proteins.

During neurotransmission, synaptic vesicles undergo multiple rounds of exo-endocytosis, involving recycling and/or degradation of synaptic proteins. While ubiquitin signaling at synapses is essential for neural function, it has been assumed that synaptic proteostasis requires the ubiquitin-proteasome system (UPS). We demonstrate here that turnover of synaptic membrane proteins via the endolysosomal pathway is essential for synaptic function. In both human and mouse, hypomorphic mutations in the ubiquitin adaptor protein PLAA cause an infantile-lethal neurodysfunction syndrome with seizures. Resulting from perturbed endolysosomal degradation, Plaa mutant neurons accumulate K63-polyubiquitylated proteins and synaptic membrane proteins, disrupting synaptic vesicle recycling and neurotransmission. Through characterization of this neurological intracellular trafficking disorder, we establish the importance of ubiquitin-mediated endolysosomal trafficking at the synapse.This work was supported by core funding from the MRC (MC_UU_12018/26 to E.A.H., G.R.M., and P.M.; MC_PC_U127527200 to P.B., M.A.K., L.M., R.L.M., and I.J.J.; and MC_UU_12016/6 to Y. Kulathu and Y. Kristaryanto), MRCMICA grant (to M.S.N.), Muscular Dystrophy Association (MDA294433) (to L.M.M.), Science Foundation Ireland (13/SIRG/2174) (to A.G.-M. and A.v.K.), NIHR Cambridge BRC 2012 (to C.G.W.), MRC, Wellcome, and WellChild (to E.R.M.), and King Salman Center for Disability Research (to F.S.A.)

A rise in NAD precursor nicotinamide mononucleotide (NMN) after injury promotes axon degeneration.

NAD metabolism regulates diverse biological processes, including ageing, circadian rhythm and axon survival. Axons depend on the activity of the central enzyme in NAD biosynthesis, nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2), for their maintenance and degenerate rapidly when this activity is lost. However, whether axon survival is regulated by the supply of NAD or by another action of this enzyme remains unclear. Here we show that the nucleotide precursor of NAD, nicotinamide mononucleotide (NMN), accumulates after nerve injury and promotes axon degeneration. Inhibitors of NMN-synthesising enzyme NAMPT confer robust morphological and functional protection of injured axons and synapses despite lowering NAD. Exogenous NMN abolishes this protection, suggesting that NMN accumulation within axons after NMNAT2 degradation could promote degeneration. Ectopic expression of NMN deamidase, a bacterial NMN-scavenging enzyme, prolongs survival of injured axons, providing genetic evidence to support such a mechanism. NMN rises prior to degeneration and both the NAMPT inhibitor FK866 and the axon protective protein Wld(S) prevent this rise. These data indicate that the mechanism by which NMNAT and the related Wld(S) protein promote axon survival is by limiting NMN accumulation. They indicate a novel physiological function for NMN in mammals and reveal an unexpected link between new strategies for cancer chemotherapy and the treatment of axonopathies

Restricted growth of Schwann cells lacking Cajal bands slows conduction in myelinated nerves

Nerve impulses are propagated at nodes of Ranvier in the myelinated nerves of vertebrates. Internodal distances have been proposed to affect the velocity of nerve impulse conduction; however, direct evidence is lacking, and the cellular mechanisms that might regulate the length of the myelinated segments are unknown. Ramon y Cajal described longitudinal and transverse bands of cytoplasm or trabeculae in internodal Schwann cells and suggested that they had a nutritive function. Here we show that internodal growth in wild-type nerves is precisely matched to nerve extension, but disruption of the cytoplasmic bands in Periaxin-null mice impairs Schwann cell elongation during nerve growth. By contrast, myelination proceeds normally. The capacity of wild-type and mutant Schwann cells to elongate is cell-autonomous, indicating that passive stretching can account for the lengthening of the internode during limb growth. As predicted on theoretical grounds, decreased internodal distances strikingly decrease conduction velocities and so affect motor function.We propose that microtubule-based transport in the longitudinal bands of Cajal permits internodal Schwann cells to lengthen in response to axonal growth, thus ensuring rapid nerve impulse transmission