James W. Connell

Spastin Couples Microtubule Severing to Membrane Traffic in Completion of Cytokinesis and Secretion

Mutations in the gene encoding the microtubule (MT)‐severing protein spastin are the most common cause of hereditary spastic paraplegia, a genetic condition in which axons of the corticospinal tracts degenerate. We show that not only does endogenous spastin colocalize with MTs, but that it is also located on the early secretory pathway, can be recruited to endosomes and is present in the cytokinetic midbody. Spastin has two main isoforms, a 68 kD full‐length isoform and a 60 kD short form. These two isoforms preferentially localize to different membrane traffic pathways with 68 kD spastin being principally located at the early secretory pathway, where it regulates endoplasmic reticulum‐to‐Golgi traffic. Sixty kiloDalton spastin is the major form recruited to endosomes and is also present in the midbody, where its localization requires the endosomal sorting complex required for transport‐III‐interacting MIT domain. Loss of midbody MTs accompanies the abscission stage of cytokinesis. In cells lacking spastin, a MT disruption event that normally accompanies abscission does not occur and abscission fails. We suggest that this event represents spastin‐mediated MT severing. Our results support a model in which membrane traffic and MT regulation are coupled through spastin. This model is relevant in the axon, where there also is co‐ordinated MT regulation and membrane traffic.

The hereditary spastic paraplegia proteins NIPA1, spastin and spartin are inhibitors of mammalian BMP signalling

The hereditary spastic paraplegias (HSPs) are genetic conditions characterized by distal axonopathy of the longest corticospinal tract axons, and so their study provides an important opportunity to understand mechanisms involved in axonal maintenance and degeneration. A group of HSP genes encode proteins that localize to endosomes. One of these is NIPA1 (non-imprinted in Prader-Willi/Angelman syndrome 1) and we have shown recently that its Drosophila homologue spichthyin inhibits bone morphogenic protein (BMP) signalling, although the relevance of this finding to the mammalian protein was not known. We show here that mammalian NIPA1 is also an inhibitor of BMP signalling. NIPA1 physically interacts with the type II BMP receptor (BMPRII) and we demonstrate that this interaction does not require the cytoplasmic tail of BMPRII. We show that the mechanism by which NIPA1 inhibits BMP signalling involves downregulation of BMP receptors by promoting their endocytosis and lysosomal degradation. Disease-associated mutant versions of NIPA1 alter the trafficking of BMPRII and are less efficient at promoting BMPRII degradation than wild-type NIPA1. In addition, we demonstrate that two other members of the endosomal group of HSP proteins, spastin and spartin, are inhibitors of BMP signalling. Since BMP signalling is important for distal axonal function, we propose that dysregulation of BMP signalling could be a unifying pathological component in this endosomal group of HSPs, and perhaps of importance in other conditions in which distal axonal degeneration is found.

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

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.

An ESCRT–spastin interaction promotes fission of recycling tubules from the endosome

Inclusion of IST1 into the ESCRT complex allows recruitment of the microtubule-severing protein spastin to promote fission of recycling tubules from the endosome.

The AAA ATPase spastin links microtubule severing to membrane modelling.

In 1999, mutations in the gene encoding the microtubule severing AAA ATPase spastin were identified as a major cause of a genetic neurodegenerative condition termed hereditary spastic paraplegia (HSP). This finding stimulated intense study of the spastin protein and over the last decade, a combination of cell biological, in vivo, in vitro and structural studies have provided important mechanistic insights into the cellular functions of the protein, as well as elucidating cell biological pathways that might be involved in axonal maintenance and degeneration. Roles for spastin have emerged in shaping the endoplasmic reticulum and the abscission stage of cytokinesis, in which spastin appears to couple membrane modelling to microtubule regulation by severing.

Endogenous spartin (SPG20) is recruited to endosomes and lipid droplets and interacts with the ubiquitin E3 ligases AIP4 and AIP5.

The HSPs (hereditary spastic paraplegias) are genetic conditions in which there is distal degeneration of the longest axons of the corticospinal tract, resulting in spastic paralysis of the legs. The gene encoding spartin is mutated in Troyer syndrome, an HSP in which paralysis is accompanied by additional clinical features. There has been controversy over the subcellular distribution of spartin. We show here that, at steady state, endogenous spartin exists in a cytosolic pool that can be recruited to endosomes and to lipid droplets. Cytosolic endogenous spartin is mono-ubiquitinated and we demonstrate that it interacts via a PPXY motif with the ubiquitin E3 ligases AIP4 [atrophin-interacting protein 4; WWP2 (WW domain-containing E3 ubiquitin protein ligase 2] and AIP5 (WWP1). Surprisingly, the PPXY motif, AIP4 and AIP5 are not required for spartins ubiquitination, and so we propose that spartin acts as an adaptor for these proteins. Our results suggest that spartin is involved in diverse cellular functions, which may be of relevance to the complex phenotype seen in Troyer syndrome.

Defects in ER–endosome contacts impact lysosome function in hereditary spastic paraplegia

Contacts between endosomes and the endoplasmic reticulum (ER) promote endosomal tubule fission, but the mechanisms involved and consequences of tubule fission failure are incompletely understood. We found that interaction between the microtubule-severing enzyme spastin and the ESCRT protein IST1 at ER–endosome contacts drives endosomal tubule fission. Failure of fission caused defective sorting of mannose 6-phosphate receptor, with consequently disrupted lysosomal enzyme trafficking and abnormal lysosomal morphology, including in mouse primary neurons and human stem cell–derived neurons. Consistent with a role for ER-mediated endosomal tubule fission in lysosome function, similar lysosomal abnormalities were seen in cellular models lacking the WASH complex component strumpellin or the ER morphogen REEP1. Mutations in spastin, strumpellin, or REEP1 cause hereditary spastic paraplegia (HSP), a disease characterized by axonal degeneration. Our results implicate failure of the ER–endosome contact process in axonopathy and suggest that coupling of ER-mediated endosomal tubule fission to lysosome function links different classes of HSP proteins, previously considered functionally distinct, into a unifying pathway for axonal degeneration.

Quantitative Gait Analysis Using a Motorized Treadmill System Sensitively Detects Motor Abnormalities in Mice Expressing ATPase Defective Spastin

The hereditary spastic paraplegias (HSPs) are genetic conditions in which there is progressive axonal degeneration in the corticospinal tract. Autosomal dominant mutations, including nonsense, frameshift and missense changes, in the gene encoding the microtubule severing ATPase spastin are the most common cause of HSP in North America and northern Europe. In this study we report quantitative gait analysis using a motorized treadmill system, carried out on mice knocked-in for a disease-associated mutation affecting a critical residue in the Walker A motif of the spastin ATPase domain. At 4 months and at one year of age homozygous mutant mice had a number of abnormal gait parameters, including in stride length and stride duration, compared to heterozygous and wild-type littermates. Gait parameters in heterozygous animals did not differ from wild-type littermates. We conclude that quantitative gait analysis using the DigiGait system sensitively detects motor abnormalities in a hereditary spastic paraplegia model, and would be a useful method for analyzing the effects of pharmacological treatments for HSP.