Alya R. Raphael

Exome sequencing in amyotrophic lateral sclerosis identifies risk genes and pathways

New players in Lou Gehrigs disease Amyotrophic lateral sclerosis (ALS), often referred to as “Lou Gehrigs disease,” is a progressive neurodegenerative disease that affects nerve cells in the brain and the spinal cord. Cirulli et al. sequenced the expressed genes of nearly 3000 ALS patients and compared them with those of more than 6000 controls (see the Perspective by Singleton and Traynor). They identified several proteins that were linked to disease in patients. One such protein, TBK1, is implicated in innate immunity and autophagy and may represent a therapeutic target. Science, this issue p. 1436; see also p. 1422 Analysis of the expressed genes of nearly 2900 patients with amyotrophic lateral sclerosis and about 6400 controls reveals a disease predisposition–associated gene. [Also see Perspective by Singleton and Traynor] Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease with no effective treatment. We report the results of a moderate-scale sequencing study aimed at increasing the number of genes known to contribute to predisposition for ALS. We performed whole-exome sequencing of 2869 ALS patients and 6405 controls. Several known ALS genes were found to be associated, and TBK1 (the gene encoding TANK-binding kinase 1) was identified as an ALS gene. TBK1 is known to bind to and phosphorylate a number of proteins involved in innate immunity and autophagy, including optineurin (OPTN) and p62 (SQSTM1/sequestosome), both of which have also been implicated in ALS. These observations reveal a key role of the autophagic pathway in ALS and suggest specific targets for therapeutic intervention.

Therapeutic modulation of eIF2α phosphorylation rescues TDP-43 toxicity in amyotrophic lateral sclerosis disease models.

Amyotrophic lateral sclerosis (ALS) is a fatal, late-onset neurodegenerative disease primarily affecting motor neurons. A unifying feature of many proteins associated with ALS, including TDP-43 and ataxin-2, is that they localize to stress granules. Unexpectedly, we found that genes that modulate stress granules are strong modifiers of TDP-43 toxicity in Saccharomyces cerevisiae and Drosophila melanogaster. eIF2α phosphorylation is upregulated by TDP-43 toxicity in flies, and TDP-43 interacts with a central stress granule component, polyA-binding protein (PABP). In human ALS spinal cord neurons, PABP accumulates abnormally, suggesting that prolonged stress granule dysfunction may contribute to pathogenesis. We investigated the efficacy of a small molecule inhibitor of eIF2α phosphorylation in ALS models. Treatment with this inhibitor mitigated TDP-43 toxicity in flies and mammalian neurons. These findings indicate that the dysfunction induced by prolonged stress granule formation might contribute directly to ALS and that compounds that mitigate this process may represent a novel therapeutic approach.

Exome sequencing to identify de novo mutations in sporadic ALS trios

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease whose causes are still poorly understood. To identify additional genetic risk factors, we assessed the role of de novo mutations in ALS by sequencing the exomes of 47 ALS patients and both of their unaffected parents (n = 141 exomes). We found that amino acid–altering de novo mutations were enriched in genes encoding chromatin regulators, including the neuronal chromatin remodeling complex (nBAF) component SS18L1 (also known as CREST). CREST mutations inhibited activity-dependent neurite outgrowth in primary neurons, and CREST associated with the ALS protein FUS. These findings expand our understanding of the ALS genetic landscape and provide a resource for future studies into the pathogenic mechanisms contributing to sporadic ALS.

Schwann cell spectrins modulate peripheral nerve myelination

During peripheral nerve development, Schwann cells ensheathe axons and form myelin to enable rapid and efficient action potential propagation. Although myelination requires profound changes in Schwann cell shape, how neuron–glia interactions converge on the Schwann cell cytoskeleton to induce these changes is unknown. Here, we demonstrate that the submembranous cytoskeletal proteins αII and βII spectrin are polarized in Schwann cells and colocalize with signaling molecules known to modulate myelination in vitro. Silencing expression of these spectrins inhibited myelination in vitro, and remyelination in vivo. Furthermore, myelination was disrupted in motor nerves of zebrafish lacking αII spectrin. Finally, we demonstrate that loss of spectrin significantly reduces both F-actin in the Schwann cell cytoskeleton and the Nectin-like protein, Necl4, at the contact site between Schwann cells and axons. Therefore, we propose αII and βII spectrin in Schwann cells integrate the neuron–glia interactions mediated by membrane proteins into the actin-dependent cytoskeletal rearrangements necessary for myelination.

ErbB signaling has a role in radial sorting independent of Schwann cell number.

In the peripheral nervous system, Schwann cells make myelin, a specialized sheath that is essential for rapid axonal conduction of action potentials. Immature Schwann cells initially interact with many axons, but, through a process termed radial sorting, eventually interact with one segment of a single axon as promyelinating Schwann cells. Previous studies have identified genes that are required for Schwann cell process extension and proliferation during radial sorting. Previous analyses also show that ErbB signaling is required for Schwann cell proliferation, myelination, radial sorting, and the proper formation of unmyelinated Remak bundles. Because ErbB signaling and Schwann cell proliferation are both required during radial sorting, we sought to determine if the primary function of ErbB signaling in this process is to regulate Schwann cell proliferation or if ErbB signaling also controls other aspects of radial sorting. To address this question, we applied small molecule inhibitors in vivo in zebrafish to independently block ErbB signaling and proliferation. Ultrastructural analysis of treated animals revealed that both ErbB signaling and Schwann cell proliferation are required for radial sorting in vivo. ErbB signaling, however, is required for Schwann cell process extension, while Schwann cell proliferation is not. These results provide in vivo evidence that ErbB signaling plays a direct role in process extension during radial sorting, in addition to its role in regulating Schwann cell proliferation.

New insights into signaling during myelination in zebrafish.

Myelin is a vertebrate adaptation that allows for the rapid propagation of action potentials along axons. Specialized glial cells-oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS)-form myelin by repeatedly wrapping axon segments. Debilitating diseases result from the disruption of myelin, including multiple sclerosis and Charcot-Marie-Tooth peripheral neuropathies. The process of myelination involves extensive communication between glial cells and the associated neurons. The past few years have seen important progress in understanding the molecular basis of the signals that coordinate the development of these fascinating cells. This review highlights recent advances in myelination deriving from studies in the zebrafish model system, with a primary focus on the PNS. While Neuregulin1-ErbB signaling has long been known to play important roles in peripheral myelin development, work in zebrafish has elucidated its roles in Schwann cell migration and radial sorting of axons in vivo. Forward genetic screens in zebrafish have also uncovered new genes required for development of myelinated axons, including gpr126, which encodes a G-protein coupled receptor required for Schwann cells to progress beyond the promyelinating stage. In addition, work in zebrafish uncovered new roles for Schwann cells themselves, including in regulating the boundary between the PNS and CNS and positioning a nerve after its initial outgrowth.

Targeted exon capture and sequencing in sporadic amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that results in progressive degeneration of motor neurons, ultimately leading to paralysis and death. Approximately 10% of ALS cases are familial, with the remaining 90% of cases being sporadic. Genetic studies in familial cases of ALS have been extremely informative in determining the causative mutations behind ALS, especially as the same mutations identified in familial ALS can also cause sporadic disease. However, the cause of ALS in approximately 30% of familial cases and in the majority of sporadic cases remains unknown. Sporadic ALS cases represent an underutilized resource for genetic information about ALS; therefore, we undertook a targeted sequencing approach of 169 known and candidate ALS disease genes in 242 sporadic ALS cases and 129 matched controls to try to identify novel variants linked to ALS. We found a significant enrichment in novel and rare variants in cases versus controls, indicating that we are likely identifying disease associated mutations. This study highlights the utility of next generation sequencing techniques combined with functional studies and rare variant analysis tools to provide insight into the genetic etiology of a heterogeneous sporadic disease.

Congenital Muscular Dystrophy and Generalized Epilepsy Caused by GMPPB Mutations

The alpha-dystroglycanopathies are genetically heterogeneous muscular dystrophies that result from hypoglycosylation of alpha-dystroglycan (α-DG). Alpha-dystroglycan is an essential link between the extracellular matrix and the muscle fiber sarcolemma, and proper glycosylation is critical for its ability to bind to ligands in the extracellular matrix. We sought to identify the genetic basis of alpha-dystroglycanopathy in a family wherein the affected individuals presented with congenital muscular dystrophy, brain abnormalities and generalized epilepsy. We performed whole exome sequencing and identified compound heterozygous GMPPB mutations in the affected children. GMPPB is an enzyme in the glycosylation pathway, and GMPPB mutations were recently linked to eight cases of alpha-dystroglycanopathy with a range of symptoms. We identified a novel mutation in GMPPB (p.I219T) as well as a previously published mutation (p.R287Q). Thus, our work further confirms a role for GMPPB defects in alpha-dystroglycanopathy, and suggests that glycosylation may play a role in the neuronal membrane channels or networks involved in the physiology of generalized epilepsy syndromes. This article is part of a Special Issue entitled RNA Metabolism 2013.

Schwann Cells Inhibit Ectopic Clustering of Axonal Sodium Channels

The clustering of voltage-gated sodium channels at the axon initial segment (AIS) and nodes of Ranvier is essential for the initiation and propagation of action potentials in myelinated axons. Sodium channels localize to the AIS through an axon-intrinsic mechanism driven by ankyrin G, while clustering at the nodes requires cues from myelinating glia that interact with axonal neurofascin186 (Sherman et al., 2005; Dzhashiashvili et al., 2007; Yang et al., 2007). Here, we report that in zebrafish mutants lacking Schwann cells in peripheral nerves (erbb2, erbb3, and sox10/colorless), axons form numerous aberrant sodium channel clusters throughout their length. Morpholino knockdown of ankyrin G, but not neurofascin, reduces the number of sodium channel clusters in Schwann cell-deficient mutants, suggesting that these aberrant clusters form by an axon-intrinsic mechanism. We also find that gpr126 mutants, in which Schwann cells are arrested at the promyelinating stage (Monk et al., 2009), are deficient in the clustering of neurofascin at the nodes of Ranvier. When Schwann cell migration in gpr126 mutants is blocked, there is an increase in the number of neurofascin clusters in peripheral axons. Our results suggest that Schwann cells inhibit the ability of ankyrin G to cluster sodium channels at ectopic locations, restricting its activity to the AIS and nodes of Ranvier.

Schwann cells reposition a peripheral nerve to isolate it from postembryonic remodeling of its targets

Although much is known about the initial construction of the peripheral nervous system (PNS), less well understood are the processes that maintain the position and connections of nerves during postembryonic growth. Here, we show that the posterior lateral line nerve in zebrafish initially grows in the epidermis and then rapidly transitions across the epidermal basement membrane into the subepidermal space. Our experiments indicate that Schwann cells, which myelinate axons in the PNS, are required to reposition the nerve. In mutants lacking Schwann cells, the nerve is mislocalized and the axons remain in the epidermis. Transplanting wild-type Schwann cells into these mutants rescues the position of the nerve. Analysis of chimeric embryos suggests that the process of nerve relocalization involves two discrete steps – the degradation and recreation of the epidermal basement membrane. Although the outgrowth of axons is normal in mutants lacking Schwann cells, the nerve becomes severely disorganized at later stages. In wild-type embryos, exclusion of the nerve from the epidermis isolates axons from migration of their targets (sensory neuromasts) within the epidermis. Without Schwann cells, axons remain within the epidermis and are dragged along with the migrating neuromasts. Our analysis of the posterior lateral line system defines a new process in which Schwann cells relocate a nerve beneath the epidermal basement membrane to insulate axons from the postembryonic remodeling of their targets.