Schwann cells ER-associated degradation contributes to myelin maintenance in adult nerves and limits demyelination in CMT1B mice.

In the peripheral nervous system (PNS) myelinating Schwann cells synthesize large amounts of myelin protein zero (P0) glycoprotein, an abundant component of peripheral nerve myelin. In humans, mutations in P0 cause the demyelinating Charcot-Marie-Tooth 1B (CMT1B) neuropathy, one of the most diffused genetic disorders of the PNS. We previously showed that several mutations, such as the deletion of serine 63 (P0-S63del), result in misfolding and accumulation of P0 in the endoplasmic reticulum (ER), with activation of the unfolded protein response (UPR). In addition, we observed that S63del mouse nerves display the upregulation of many ER-associated degradation (ERAD) genes, suggesting a possible involvement of this pathway in the clearance of the mutant P0. In ERAD in fact, misfolded proteins are dislocated from the ER and targeted for proteasomal degradation. Taking advantage of inducible cells that express the ER retained P0, here we show that the P0-S63del glycoprotein is degraded via ERAD. Moreover, we provide strong evidence that the Schwann cell-specific ablation of the ERAD factor Derlin-2 in S63del nerves exacerbates both the myelin defects and the UPR in vivo, unveiling a protective role for ERAD in CMT1B neuropathy. We also found that lack of Derlin-2 affects adult myelin maintenance in normal nerves, without compromising their development, pinpointing ERAD as a previously unrecognized player in preserving Schwann cells homeostasis in adulthood. Finally, we provide evidence that treatment of S63del peripheral nerve cultures with N-Acetyl-D-Glucosamine (GlcNAc), known to enhance protein quality control pathways in C.elegans, ameliorates S63del nerve myelination ex vivo. Overall, our study suggests that potentiating adaptive ER quality control pathways might represent an appealing strategy to treat both conformational and age-related PNS disorders.

Polyglutamine Atrophin provokes neurodegeneration in Drosophila by repressing fat.

Large alterations in transcription accompany neurodegeneration in polyglutamine (polyQ) diseases. These pathologies manifest both general polyQ toxicity and mutant protein-specific effects. In this study, we report that the fat tumour suppressor gene mediates neurodegeneration induced by the polyQ protein Atrophin. We have monitored early transcriptional alterations in a Drosophila model of Dentatorubral-pallidoluysian Atrophy and found that polyQ Atrophins downregulate fat. Fat protects from neurodegeneration and Atrophin toxicity through the Hippo kinase cascade. Fat/Hippo signalling does not provoke neurodegeneration by stimulating overgrowth; rather, it alters the autophagic flux in photoreceptor neurons, thereby affecting cell homeostasis. Our data thus provide a crucial insight into the specific mechanism of a polyQ disease and reveal an unexpected neuroprotective role of the Fat/Hippo pathway.

Treatment with IFB-088 Improves Neuropathy in CMT1A and CMT1B Mice.

Charcot-Marie-Tooth disease type 1A (CMT1A), caused by duplication of the peripheral myelin protein 22 (PMP22) gene, and CMT1B, caused by mutations in myelin protein zero (MPZ) gene, are the two most common forms of demyelinating CMT (CMT1), and no treatments are available for either. Prior studies of the MpzSer63del mouse model of CMT1B have demonstrated that protein misfolding, endoplasmic reticulum (ER) retention and activation of the unfolded protein response (UPR) contributed to the neuropathy. Heterozygous patients with an arginine to cysteine mutation in MPZ (MPZR98C) develop a severe infantile form of CMT1B which is modelled by MpzR98C/ + mice that also show ER stress and an activated UPR. C3-PMP22 mice are considered to effectively model CMT1A. Altered proteostasis, ER stress and activation of the UPR have been demonstrated in mice carrying Pmp22 mutations. To determine whether enabling the ER stress/UPR and readjusting protein homeostasis would effectively treat these models of CMT1B and CMT1A, we administered Sephin1/IFB-088/icerguestat, a UPR modulator which showed efficacy in the MpzS63del model of CMT1B, to heterozygous MpzR98C and C3-PMP22 mice. Mice were analysed by behavioural, neurophysiological, morphological and biochemical measures. Both MpzR98C/ + and C3-PMP22 mice improved in motor function and neurophysiology. Myelination, as demonstrated by g-ratios and myelin thickness, improved in CMT1B and CMT1A mice and markers of UPR activation returned towards wild-type values. Taken together, our results demonstrate the capability of IFB-088 to treat a second mouse model of CMT1B and a mouse model of CMT1A, the most common form of CMT. Given the recent benefits of IFB-088 treatment in amyotrophic lateral sclerosis and multiple sclerosis animal models, these data demonstrate its potential in managing UPR and ER stress for multiple mutations in CMT1 as well as in other neurodegenerative diseases. (Left panel) the accumulation of overexpressed PMP22 or misfolded mutant P0 in the Schwann cell endoplasmic reticulum (ER) leads to overwhelming of the degradative capacity, activation of ER-stress mechanisms, and myelination impairment. (Right panel) by prolonging eIF2α phosphorylation, IFB-088 reduces the amount of newly synthesized proteins entering the ER, allowing the protein quality control systems to better cope with the unfolded/misfolded protein and allowing myelination to progress.

Zika Virus Replication in Dorsal Root Ganglia Explants from Interferon Receptor1 Knockout Mice Causes Myelin Degeneration.

Zika virus (ZIKV) is a neurotropic agent that targets the developing fetal brain in women infected during pregnancy. In addition to the developing central nervous system, ZIKV has been recently shown to infect cells of the peripheral nervous system (PNS), highlighting its potential to cause acute peripheral neuropathies in adults, such as Guillain-Barré Syndrome (GBS). Here we show that myelinating dorsal root ganglia (DRG) explants obtained from interferon-alpha/beta receptor knock-out mice are productively infected by ZIKV. Virus replication is cytopathic in both peripheral neurons and myelinating Schwann cells leading to myelin disruption. These results confirm and extend previous observations suggesting that the PNS is indeed a potential site of ZIKV infection, replication and cytopathicity.

Endoplasmic Reticulum Protein Quality Control Failure in Myelin Disorders.

Reaching the correct three-dimensional structure is crucial for the proper function of a protein. The endoplasmic reticulum (ER) is the organelle where secreted and transmembrane proteins are synthesized and folded. To guarantee high fidelity of protein synthesis and maturation in the ER, cells have evolved ER-protein quality control (ERQC) systems, which assist protein folding and promptly degrade aberrant gene products. Only correctly folded proteins that pass ERQC checkpoints are allowed to exit the ER and reach their final destination. Misfolded glycoproteins are detected and targeted for degradation by the proteasome in a process known as endoplasmic reticulum-associated degradation (ERAD). The excess of unstructured proteins in the ER triggers an adaptive signal transduction pathway, called unfolded protein response (UPR), which in turn potentiates ERQC activities in order to reduce the levels of aberrant molecules. When the situation cannot be restored, the UPR drives cells to apoptosis. Myelin-forming cells of the central and peripheral nervous system (oligodendrocytes and Schwann cells) synthesize a large amount of myelin proteins and lipids and therefore are particularly susceptible to ERQC failure. Indeed, deficits in ERQC and activation of ER stress/UPR have been implicated in several myelin disorders, such as Pelizaeus-Merzbacher and Krabbe leucodystrophies, vanishing white matter disease and Charcot-Marie-Tooth neuropathies. Here we discuss recent evidence underlying the importance of proper ERQC functions in genetic disorders of myelinating glia.

Single Drosophila ommatidium dissection and imaging.

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative diseases because of its powerful genetics(1). The fly eye in particular has been the organ of choice for neurodegeneration research, being the most accessible and life-dispensable part of the Drosophila nervous system. However the major caveat of intact eyes is the difficulty, because of the intense autofluorescence of the pigment, in imaging intracellular events, such as autophagy dynamics(2), which are paramount to understanding of neurodegeneration. We have recently used the dissection and culture of single ommatidia(3) that has been essential for our understanding of autophagic dysfunctions in a fly model of Dentatorubro-Pallidoluysian Atrophy (DRPLA)(3, 4). We now report a comprehensive description of this technique (Fig. 1), adapted from electrophysiological studies(5), which is likely to expand dramatically the possibility of fly models for neurodegeneration. This method can be adapted to image live subcellular events and to monitor effective drug administration onto photoreceptor cells (Fig. 2). If used in combination with mosaic techniques(6-8), the responses of genetically different cells can be assayed in parallel (Fig. 2).