Molecular and cellular mechanisms of spastin in neural development and disease (Review).

Spastin is a microtubule (MT)­severing enzyme identified from mutations of hereditary spastic paraplegia in 1999 and extensive studies indicate its vital role in various cellular activities. In the past two decades, efforts have been made to understand the underlying molecular mechanisms of how spastin is linked to neural development and disease. Recent studies on spastin have unraveled the mechanistic processes of its MT­severing activity and revealed that spastin acts as an MT amplifier to mediate its remodeling, thus providing valuable insight into the molecular roles of spastin under physiological conditions. In addition, recent research has revealed multiple novel molecular mechanisms of spastin in cellular biological pathways, including endoplasmic reticulum shaping, calcium trafficking, fatty acid trafficking, as well as endosomal fission and trafficking. These processes are closely involved in axonal and dendritic development and maintenance. The current review presents recent biological advances regarding the molecular mechanisms of spastin at the cellular level and provides insight into how it affects neural development and disease.

A novel SPAST gene mutation identified in a Chinese family with hereditary spastic paraplegia.

BACKGROUND: Hereditary spastic paraplegia is a heterogeneous group of clinically and genetically neurodegenerative diseases characterized by progressive gait disorder. Hereditary spastic paraplegia can be inherited in various ways, and all modes of inheritance are associated with multiple genes or loci. At present, more than 76 disease-causing loci have been identified in hereditary spastic paraplegia patients. Here, we report a novel mutation in SPAST gene associated with hereditary spastic paraplegia in a Chinese family, further enriching the hereditary spastic paraplegia spectrum. METHODS: Whole genomic DNA was extracted from peripheral blood of the 15 subjects from a Chinese family using DNA Isolation Kit. The Whole Exome Sequencing of the proband was analyzed and the result was identified in the rest individuals. RaptorX prediction tool and Protein Variation Effect Analyzer were used to predict the effects of the mutation on protein tertiary structure and function. RESULTS: Spastic paraplegia has been inherited across at least four generations in this family, during which only four HSP patients were alive. The results obtained by analyzing the Whole Exome Sequencing of the proband exhibited a novel disease-associated in-frame deletion in the SPAST gene, and this mutation also existed in the rest three HSP patients in this family. This in-frame deletion consists of three nucleotides deletion (c.1710_1712delGAA) within the exon 16, resulting in lysine deficiency at the position 570 of the protein (p.K570del). This novel mutation was also predicted to result in the synthesis of misfolded SPAST protein and have the deleterious effect on the function of SPAST protein. CONCLUSION: In this case, we reported a novel mutation in the known SPAST gene that segregated with HSP disease, which can be inherited in each generation. Simultaneously, this novel discovery significantly enriches the mutation spectrum, which provides an opportunity for further investigation of genetic pathogenesis of HSP.

Structure of spastin bound to a glutamate-rich peptide implies a hand-over-hand mechanism of substrate translocation.

Many members of the AAA+ ATPase family function as hexamers that unfold their protein substrates. These AAA unfoldases include spastin, which plays a critical role in the architecture of eukaryotic cells by driving the remodeling and severing of microtubules, which are cytoskeletal polymers of tubulin subunits. Here, we demonstrate that a human spastin binds weakly to unmodified peptides from the C-terminal segment of human tubulin α1A/B. A peptide comprising alternating glutamate and tyrosine residues binds more tightly, which is consistent with the known importance of glutamylation for spastin microtubule severing activity. A cryo-EM structure of the spastin-peptide complex at 4.2 Å resolution revealed an asymmetric hexamer in which five spastin subunits adopt a helical, spiral staircase configuration that binds the peptide within the central pore, whereas the sixth subunit of the hexamer is displaced from the peptide/substrate, as if transitioning from one end of the helix to the other. This configuration differs from a recently published structure of spastin from Drosophila melanogaster, which forms a six-subunit spiral without a transitioning subunit. Our structure resembles other recently reported AAA unfoldases, including the meiotic clade relative Vps4, and supports a model in which spastin utilizes a hand-over-hand mechanism of tubulin translocation and microtubule remodeling.

Spastin tethers lipid droplets to peroxisomes and directs fatty acid trafficking through ESCRT-III.

Lipid droplets (LDs) are neutral lipid storage organelles that transfer lipids to various organelles including peroxisomes. Here, we show that the hereditary spastic paraplegia protein M1 Spastin, a membrane-bound AAA ATPase found on LDs, coordinates fatty acid (FA) trafficking from LDs to peroxisomes through two interrelated mechanisms. First, M1 Spastin forms a tethering complex with peroxisomal ABCD1 to promote LD-peroxisome contact formation. Second, M1 Spastin recruits the membrane-shaping ESCRT-III proteins IST1 and CHMP1B to LDs via its MIT domain to facilitate LD-to-peroxisome FA trafficking, possibly through IST1- and CHMP1B-dependent modifications in LD membrane morphology. Furthermore, LD-to-peroxisome FA trafficking mediated by M1 Spastin is required to relieve LDs of lipid peroxidation. M1 Spastin's dual roles in tethering LDs to peroxisomes and in recruiting ESCRT-III components to LD-peroxisome contact sites for FA trafficking may underlie the pathogenesis of diseases associated with defective FA metabolism in LDs and peroxisomes.

Elk1 affects katanin and spastin proteins via differential transcriptional and post-transcriptional regulations.

Microtubule severing, which is highly critical for the survival of both mitotic and post-mitotic cells, has to be precisely adjusted by regulating the expression levels of severing proteins, katanin and spastin. Even though severing mechanism is relatively well-studied, there are limited studies for the transcriptional regulation of microtubule severing proteins. In this study, we identified the main regulatory region of KATNA1 gene encoding katanin-p60 as 5' UTR, which has a key role for its expression, and showed Elk1 binding to KATNA1. Furthermore, we identified that Elk1 decreased katanin-p60 and spastin protein expressions, while mRNA levels were increased upon Elk1 overexpression. In addition, SUMOylation is a known post-translational modification regulating Elk1 activity. A previous study suggested that K230, K249, K254 amino acids in the R domain are the main SUMOylation sites; however, we identified that these amino acids are neither essential nor substantial for Elk1 SUMOylation. Also, we determined that KATNA1 methylation results in the reduction of Elk1 binding whereas SPG4 methylation does not. Together, our findings emphasizing the impacts of both transcriptional and post-transcriptional regulations of katanin-p60 and spastin suggest that Elk1 has a key role for differential expression patterns of microtubule severing proteins, thereby regulating cellular functions through alterations of microtubule organization.

Missense mutation of SPAST protein (I344K) results in loss of ATPase activity and prolonged the half-life, implicated in autosomal dominant hereditary spastic paraplegia.

The spastin protein (SPAST) contains an ATPase with diverse cellular activities (AAA) domain and regulates microtubule dynamics. Missense mutations of the SPAST gene are frequently detected in patients with hereditary spastic paraplegias (HSPs) and represent the main reason of loss of SPAST function; however, the pathogenicity of mutant SPAST is heterogeneous. Here, SPAST variant with an I344K mutation (I344K-SPAST) was identified in a Korean family with autosomal dominant-type HSP. We investigated the role of the I344K-SPAST in HSP to provide a therapeutic mechanism. The I344K-SPAST mutation prolonged the half-life of the protein compared to wild-type SPAST (WT-SPAST) in cells by modulating post-translational modifications for proteasomal degradation. I344K-SPAST was localized in microtubule but defective in microtubule severing and ATPase activity compared to WT-SPAST in vitro and in cells. Mutant M87 isoform harboring the same mutation with I344K-M1 SPAST also increased protein stability and loss of MT severing activity, but the pathogenicity was not stronger than I344K-M1 SPAST in neurite outgrowth. Overexpression of I344K-SPAST resulted in microtubule accumulation following inhibited neurite growth in neuroblastoma, neural progenitor cells and mouse primary cortical neurons. Conversely, these pathogenic effects of I344K-SPAST were reduced by overexpression of WT-M1 SPAST in a dose dependent manner since WT-SPAST could interact with I344K-SPAST. Our data therefore provide proof-of-concept that gene transfer of WT-M1 SPAST may serve as a valid therapeutic option for HSPs.

The AAA protein spastin possesses two levels of basal ATPase activity.

The AAA ATPase spastin is a microtubule-severing enzyme that plays important roles in various cellular events including axon regeneration. Herein, we found that the basal ATPase activity of spastin is negatively regulated by spastin concentration. By determining a spastin crystal structure, we demonstrate the necessity of intersubunit interactions between spastin AAA domains. Neutralization of the positive charges in the microtubule-binding domain (MTBD) of spastin dramatically decreases the ATPase activity at low concentration, although the ATP-hydrolyzing potential is not affected. These results demonstrate that, in addition to the AAA domain, the MTBD region of spastin is also involved in regulating ATPase activity, making interactions between spastin protomers more complicated than expected.

[Microtubule severing proteins - structure and activity regulation]. / Bialka tnace mikrotubule - struktura i regulacja aktywnosci.

Microtubule severing proteins, katanin, spastin and fidgetin cause local destabilization of the microtubules structure. This ATP-dependent activity leads to the shortening or disassembly of the existing microtubules. The generated short microtubule fragments may serve as templates to polymerize new microtubules and in consequence, the activity of the microtubule severing proteins leads to the reorganization of the microtubular cytoskeleton. This review summarizes current knowledge concerning structural organization of the microtubule severing proteins, the molecular mechanism of their action, factors that regulate the level of the katanin and spastin within the cells and their microtubule severing activity.