Mutations in the ER-shaping protein reticulon 2 cause the axon-degenerative disorder hereditary spastic paraplegia type 12

Abstract

Hereditary spastic paraplegias (HSPs) are a group of genetically heterogeneous neurodegenerative conditions. They are characterized by progressive spastic paralysis of the legs as a result of selective, length-dependent degeneration of the axons of the corticospinal tract. Mutations in 3 genes encoding proteins that work together to shape the ER into sheets and tubules - receptor accessory protein 1 (REEP1), atlastin-1 (ATL1), and spastin (SPAST) - have been found to underlie many cases of HSP in Northern Europe and North America. Applying Sanger and exome sequencing, we have now identified 3 mutations in reticulon 2 (RTN2), which encodes a member of the reticulon family of prototypic ER-shaping proteins, in families with spastic paraplegia 12 (SPG12). These autosomal dominant mutations included a complete deletion of RTN2 and a frameshift mutation predicted to produce a highly truncated protein. Wild-type reticulon 2, but not the truncated protein potentially encoded by the frameshift allele, localized to the ER. RTN2 interacted with spastin, and this interaction required a hydrophobic region in spastin that is involved in ER localization and that is predicted to form a curvature-inducing/sensing hairpin loop domain. Our results directly implicate a reticulon protein in axonopathy, show that this protein participates in a network of interactions among HSP proteins involved in ER shaping, and further support the hypothesis that abnormal ER morphogenesis is a pathogenic mechanism in HSP.

Figure 1. Identification of RTN2 as the SPG12 gene.

(A) By candidate gene screening of the linked chromosomal locus, we identified a mutation in RTN2. A schematic of the RTN2 gene with the corresponding conserved protein domains is drawn to scale. Subsequently, we identified another missense change and a gene deletion. Exons are represented by rectangles. The letters “A,” “B,” “C,” and “D” indicate the location of the copy number variation assays (see D below). TM, transmembrane domain; tel, telomere; cen, centromere. (B) Sanger sequence traces of the identified mutations. The variant nucleotide is highlighted by an “N” in a black rectangle. Fam., family. (C) The missense mutation (S367F) is highly conserved and immediately flanks the transmembrane domain. Letters in lighter font represent not fully conserved amino acids. (D) Four copy number variant assays across the gene (referred to as “A,” “B,” “C,” and “D” on the bars of the graph) identified a patient with a heterozygous loss of RTN2 (green bars). Gray bars indicate samples that had 2 copies of RTN2. The orange bar (X) indicates a control locus on a different chromosome tested in the patient with the RTN2 deletion.

Figure 2. Wild-type, but not truncated RTN2B, localizes to the ER.

HEK293T cells were cotransfected with GFP-tagged RTN2B constructs and pmKate2-ER, which expresses a fluorescent protein fused to an ER-targeting signal and the ER retention signal KDEL. Images show cells transfected with (A) RTN2B-WT, (B) RTN2B-R60f, or (C) RTN2B-S294F. Scale bars: 10 μm.

Figure 3. RTN2B interacts with spastin.

(A) HeLa cells were transfected with the constructs indicated and immunoprecipitated with spastin86-340 antibody or preimmune serum (PI), and then the immunoprecipitates were immunoblotted with the antibodies shown. Syntaxin-17 is an ER membrane protein used to demonstrate specificity of the immunoprecipitation between spastin and RTN2. The M87A mutation of spastin prevents transcription of M87 spastin from the M1 transcript but does not alter function of the M1 form. (B) Quantitation of immunoblot band strength in 3 experiments in which HeLa cells were cotransfected with Myc-M1 spastin M87A and RTN2B-V5 and then immunoprecipitated with spastin86-340 antibody or preimmune serum. Immunoprecipitates were immunoblotted with antibodies against the V5-tag or syntaxin-17, and the relevant band density was quantified. P values were calculated using 2-tailed paired t tests. Values plotted are mean ± SEM. (C and D) Confocal micrographs of representative HeLa cells cotransfected with RTN2B-V5 and either Myc-M1 spastin or ATPase-defective Myc-M1 spastin K388R. Some smaller colocalized structures are indicated with arrowheads in the higher-magnification boxes. The color of the words in each black and white image correlates with the color of that image in the corresponding color panel. Scale bars: 10 μm.