Inhibition of TFG Function Causes Hereditary Axon Degeneration by Impairing Endoplasmic Reticulum Structure

Inhibition of TFG function causes hereditary axon degeneration by impairing endoplasmic reticulum structure Christian Beetza, Adam Johnsonb, Amber L. Schuhb, Seema Thakurc, Rita-Eva Vargaa, Thomas Fothergilld, Nicole Hertele, Ewa Bomba-Warczakd, Holger Thielef, Gudrun Nürnbergf, Janine Altmüllerf,g, Renu Saxenah, Edwin R. Chapmand, Erik W. Dentd, Peter Nürnbergf,g,i, and Anjon Audhyab,1 aDepartment of Clinical Chemistry and Laboratory Medicine, Jena University Hospital, 07747 Jena, Germany; bDepartment of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706; cDepartment of Genetics and Fetal Medicine, Fortis La Femme, New Delhi 110048, India; dDepartment of Neuroscience, University of Wisconsin Medical School, Madison, WI 53706; eInstitute of Anatomy I, Jena University Hospital, 07740 Jena, Germany; fCologne Centre for Genomics, University of Cologne, 50931 Cologne, Germany; gCologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, 50931 Cologne, Germany; hCentre of Medical Genetics, Sir Ganga Ram Hospital, New Delhi 110048, India; and iCenter for Molecular Medicine Cologne, University of Cologne, 50931 Cologne, Germany Edited by Charles Barlowe, Dartmouth Medical School, and accepted by the Editorial Board February 15, 2013 (received for review October 2, 2012) Hereditary spastic paraplegias are a clinically and genetically hetero- (iii) components of the early secretory pathway that control vesicle geneous group of gait disorders. Their pathological hallmark is a biogenesis and egress (10–16). Among the best-characterized HSP length-dependent distal axonopathy of nerve fibers in the cortico- genes are SPAST, ATL1,andREEP1, all of which encode proteins spinal tract. Involvement of other neurons can cause additional (spastin, atlastin-1, and REEP1, respectively) that potentially neurological symptoms, which define a diverse set of complex regulate ER organization. Mutations in SPAST were the first hereditary spastic paraplegias. We present two siblings who have to be identified as a cause for autosomal dominant, uncomplicated the unusual combination of early-onset spastic paraplegia, optic HSP (17). Multiple studies indicate that spastin functions as a atrophy, and neuropathy. Genome-wide SNP-typing, linkage anal- microtubule severing protein and can thereby regulate ER mor- GENETICS ysis, and exome sequencing revealed a homozygous c.316C>T phology (11, 18). Mutations in ATL1, which encodes a dynamin- (p.R106C) variant in the Trk-fused gene (TFG) as the only plausible related GTPase, are the second most common cause of uncom- mutation. Biochemical characterization of the mutant protein dem- plicated HSP (19). Depletion of atlastin-1 perturbs ER structure onstrated a defect in its ability to self-assemble into an oligomeric and has been shown to inhibit axon elongation in rat cortical complex, which is critical for normal TFG function. In cell lines, TFG neurons (20, 21). REEP1 mutations also cause an uncomplicated inhibition slows protein secretion from the endoplasmic reticulum form of HSP (22). Together with reticulons, which have also been (ER) and alters ER morphology, disrupting organization of periph- implicated in HSP (23), members of the REEP family are eral ER tubules and causing collapse of the ER network onto the thought to generate and/or maintain the highly curved shape of underlying microtubule cytoskeleton. The present study provides a ER membranes (7). Interestingly, spastin has been shown to unique link between altered ER architecture and neurodegeneration. interact biochemically with members of the atlastin, REEP, and reticulon families (11, 18, 24), suggesting that these proteins membrane trafficking | COPII-mediated secretion | ER exit site function together. Moreover, the consequences of mutations in the HSP-associated genes highlighted here suggest that proper ereditary spastic paraplegias (HSPs) are a diverse group of ER organization likely plays a crucial role in axonal maintenance. Hdisorders characterized by spastic weakness in the lower ex- However, it currently remains unclear which functions of the ER tremities, which results from degeneration of upper motoneuron are necessary to prevent axon degeneration as observed in HSPs. axons in the corticospinal tract (1, 2). Based on the presence or Results absence of other neurological abnormalities, HSPs are classified as complicated or pure, respectively. In addition to lower-limb We report on a family in which two of three siblings exhibit a spasticity, complicated forms of HSP may be associated with combination of several neurological symptoms that is consistent ataxia, mental retardation, dementia, extrapyramidal signs, visual with complicated HSP (Fig. 1A). Deliveries were uneventful and dysfunction, and/or epilepsy. HSPs are typically progressive, but early developmental milestones were achieved on time. Pro- age of onset is highly variable. The heterogeneity in clinical pre- nounced leg spasticity, however, precluded the ability to walk sentation is accompanied by genetic heterogeneity. To date, more independently. Vision problems were noted at 2.5 y of age and than 40 different genetic loci, which include nearly all modes of result from optic atrophy. Wasting of hand and leg muscles indi- inheritance, have been linked to HSPs (1–5). However, in nearly cated additional neuropathy. In particular, clinical electrophysi- half of these cases, the identities of the causative genes remain ology confirmed an axonal demyelinating motor neuropathy and unknown. The identification of additional HSP genes and, more revealed evidence for mild sensory involvement (Table S1). MRI importantly, the functional characterization of their encoded performed on the older patient revealed no significant abnor- products, will both contribute to our understanding of the path- malities (Table S2). At current ages of 16 and 12 y, both patients omechanisms underlying HSPs and reveal the general require- ments for lifelong axonal maintenance. More than half the HSP cases in North America and Northern Author contributions: C.B. and A.A. designed research; C.B., A.J., A.L.S., S.T., R.-E.V., T.F., Europe can be attributed to defects in organelle dynamics (6). In N.H., E.B.-W., J.A., and A.A. performed research; C.B., R.S., E.R.C., E.W.D., P.N., and A.A. particular, mutations that impact the architecture of the endo- contributed new reagents/analytic tools; C.B., A.J., A.L.S., H.T., G.N., and A.A. analyzed plasmic reticulum (ER) are common in patients with HSP. The data; and C.B. and A.A. wrote the paper. ER is comprised of a network of membrane tubules and sheet- The authors declare no conflict of interest. like cisternae that extend throughout the cytoplasm and encase This article is a PNAS Direct Submission. C.B. is a guest editor invited by the Editorial the nucleus (7–9). Several factors contribute to this architecture, Board. including (i) membrane-bending proteins of the REEP and 1To whom correspondence should be addressed. E-mail: [email protected]. reticulon families; (ii) regulators of the microtubule cytoskele- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ton, which governs the spatial patterning of the ER network; and 1073/pnas.1217197110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1217197110 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 Further analysis of the genotyping data indicated that all three siblings harbor numerous long runs of homozygosity (Table S7). Although the parents did not report consanguinity, run of homozygosity size and number strongly indicates recent parental relatedness (26). Consistent with this possibility, ancestors of both parents originate from a small geographic region in Northern India and belong to the same caste. We therefore introduced a consanguineous loop into the family, an approach successfully used in previous positional cloning projects in which consanguinity was only inferred (27), and performed genome-wide linkage analysis assuming recessive inheritance of a homozygous mutation. This revealed four regions with the maximum possible logarithm of odds score of 2.53 (Fig. 1B). Based on frequent association of mitochondrial dysfunction with optic atrophy (28, 29), we picked TOMM70A (chromosome 3 region; encoding a mitochondrial import trans- locator) (30) and C6ORF66 (chromosome 6 region; previously implicated in mitochondrial complex I deficiency) (31) as candidates, but sequencing failed to identify potentially pathogenic variants. To define the causative mutation, we performed whole-exome sequencing. A single homozygous variant was found in the regions of linkage as defined earlier. It affects the Trk-fused gene (TFG; c.316C>T) and was shown by Sanger sequencing to be homozygous in both patients, but heterozygous in the parents and the unaffected sibling (Fig. 1C). TFG was recently identified as a conserved regulator of protein secretion that assembles into an oligomeric matrix at the interface between the ER and ER– Golgi intermediate compartments in metazoans cells. It facili- tates assembly of the COPII coat complex, required for membrane deformation and vesicle biogenesis from the ER, at least in part via its ability to form octamers in vivo (32). Interestingly, the variant identified alters a highly conserved residue within the TFG coiled-coil domain (p.R106C; Fig. 1 D and E), which plays an important role in TFG oligomerization (33). Analysis of mRNA

Load more