scholarly journals Truncating mutations of SPAST associated with hereditary spastic paraplegia indicate greater accumulation and toxicity of the M1 isoform of spastin

2017 ◽  
Vol 28 (13) ◽  
pp. 1728-1737 ◽  
Author(s):  
Joanna M. Solowska ◽  
Anand N. Rao ◽  
Peter W. Baas
Keyword(s):  
Neurite Outgrowth ◽  
Hereditary Spastic Paraplegia ◽  
Pathogenic Mutation ◽  
Full Length ◽  
Start Codon ◽  
Spastic Paraplegia ◽  
Wild Type ◽  
Microtubule Severing ◽  
Corticospinal Tracts ◽  
Over Time

The SPAST gene, which produces two isoforms (M1 and M87) of the microtubule-severing protein spastin, is the chief gene mutated in hereditary spastic paraplegia. Haploinsufficiency is a popular explanation for the disease, in part because most of the >200 pathogenic mutations of the gene are truncating and expected to produce only vanishingly small amounts of shortened proteins. Here we studied two such mutations, N184X and S245X, and our results suggest another possibility. We found that the truncated M1 proteins can accumulate to notably higher levels than their truncated M87 or wild-type counterparts. Reminiscent of our earlier studies on a pathogenic mutation that generates full-length M1 and M87 proteins, truncated M1 was notably more detrimental to neurite outgrowth than truncated M87, and this was true for both N184X and S245X. The greater toxicity and tendency to accumulate suggest that, over time, truncated M1 could damage the corticospinal tracts of human patients. Curiously, the N184X mutation triggers the reinitiation of translation at a third start codon in SPAST, resulting in synthesis of a novel M187 spastin isoform that is able to sever microtubules. Thus microtubule severing may not be as reduced as previously assumed in the case of that mutation.

scholarly journals Different fusion tags affect the activity of ubiquitin overexpression on spastin protein stability

2021 ◽  
Vol 65 (4) ◽  
Author(s):  
Jianyu Zou ◽  
Zhenbin Cai ◽  
Zhi Liang ◽  
Yaozhong Liang ◽  
Guowei Zhang ◽  
...  
Keyword(s):  
Protein Stability ◽  
Neurite Outgrowth ◽  
Hereditary Spastic Paraplegia ◽  
Hippocampal Neurons ◽  
Fluorescent Protein ◽  
The Novel ◽  
Spastic Paraplegia ◽  
Promoting Effect ◽  
Microtubule Severing ◽  
Green Fluorescent

Spastin is one of the proteins which lead to hereditary spastic paraplegia (HSP), whose dysfunction towards microtubule severing and membrane transporting is critically important. The present study is to elucidate the mechanisms of the protein stability regulation of spastin. The ubiquitin encoding plasmids are transfected into COS-7 cells with different fusion tags including Green Fluorescent Protein (GFP), mCherry and Flag. The expression level of spastin was detected, microtubule severing activity and neurite outgrowth were quantified. The data showed that ubiquitin overexpression significantly induced the decreased expression of spastin, suppressed the activity of microtubule severing in COS-7 cells and inhibited the promoting effect on neurite outgrowth in cultured hippocampal neurons. Furthermore, when modulating the overexpression experiments of ubiquitin, it was found that relatively small tag like Flag, but not large tags such as GFP or mCherry fused with ubiquitin, retained the activity on spastin stability. The present study investigated the effects of small/large tags addition to ubiquitin and the novel mechanisms of post-transcriptional modifications of spastin on regulating neurite outgrowth, in the attempt to experimentally elucidate the mechanisms that control the level or stability of spastin in hereditary spastic paraplegia.

Aminoglycosides are efficient reagents to induce readthrough of premature termination codon in mutant B4GALNT1 genes found in families of hereditary spastic paraplegia

2020 ◽  
Vol 168 (2) ◽  
pp. 103-112 ◽  
Author(s):  
Farhana Yesmin ◽  
Robiul H Bhuiyan ◽  
Yuhsuke Ohmi ◽  
Yuki Ohkawa ◽  
Orie Tajima ◽  
...  
Keyword(s):  
Hereditary Spastic Paraplegia ◽  
Premature Termination ◽  
Premature Termination Codon ◽  
Full Length ◽  
Termination Codon ◽  
Dependent Manner ◽  
Spastic Paraplegia ◽  
Before And After ◽  
Faint Band ◽  
First Time

Abstract The readthrough of premature termination codon (PTC) by ribosome sometimes produces full-length proteins. We previously reported a readthrough of PTC of glycosyltransferase gene B4GALNT1 with hereditary spastic paraplegia (HSP). Here we featured the readthrough of B4GALNT1 of two mutants, M4 and M2 with PTC by immunoblotting and flow cytometry after transfection of B4GALNT1 cDNAs into cells. Immunoblotting showed a faint band of full-length mutant protein of M4 but not M2 at a similar position with that of wild-type B4GALNT1. AGC sequences at immediately before and after the PTC in M4 were critical for the readthrough. Treatment of cells transfected with mutant M4 cDNA with aminoglycosides resulted in increased readthrough of PTC. Furthermore, treatment of transfectants of mutant M2 cDNA with G418 also resulted in the induction of readthrough of PTC. Both M4 and M2 cDNA transfectants showed increased/induced bands in immunoblotting and GM2 expression in a dose-dependent manner of aminoglycosides. Results of mass spectrometry supported this effect. Here, we showed for the first time the induction and/or enhancement of the readthrough of PTCs of B4GALNT1 by aminoglycoside treatment, suggesting that aminoglycosides are efficient for patients with HSP caused by PTC of B4GALNT1, in which gradual neurological disorders emerged with aging.

scholarly journals Tau interferes with axonal neurite stabilization and cytoskeletal composition independently of its ability to associate with microtubules

Biology Open ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. bio052530
Author(s):  
Edward F. Boumil ◽  
Rishel B. Vohnoutka ◽  
Sangmook Lee ◽  
Thomas B. Shea
Keyword(s):  
Neurite Outgrowth ◽  
Axonal Transport ◽  
Full Length ◽  
Axonal Outgrowth ◽  
Wild Type

ABSTRACTTau impacts overall axonal transport particularly when overexpressed by interfering with translocation of kinesin along microtubules (MTs) and/or as a cargo of kinesin by outcompeting other kinesin cargo. To discern between which of these mechanisms was more robust during axonal outgrowth, we overexpressed phosphomimetic (E18; which is incapable of MT binding), phospho-null (A18) or wild-type (WT) full-length human tau conjugated to EGFP, the latter two of which bind MTs. Expression of WT and A18 displayed increased acetylated MTs and resistance to colchicine, while expression of E18 did not, indicating that E18 did not contribute to MT stabilization. Expression of all tau constructs reduced overall levels of neurofilaments (NFs) within axonal neurites, and distribution of NFs along neurite lengths. Since NFs are another prominent cargo of kinesin during axonal neurite outgrowth, this finding is consistent with WT, A18 and E18 inhibiting NF transport to the same extent by competing as cargo of kinesin. These findings indicate that tau can impair axonal transport independently of association with MTs in growing axonal neurites.

scholarly journals Microtubule-dependent and independent roles of spastin in lipid droplet dispersion and biogenesis

2020 ◽  
Vol 3 (6) ◽  
pp. e202000715
Author(s):  
Nimesha Tadepalle ◽  
Lennart Robers ◽  
Matteo Veronese ◽  
Peter Zentis ◽  
Felix Babatz ◽  
...  
Keyword(s):  
Hereditary Spastic Paraplegia ◽  
Mammalian Cells ◽  
Lipid Droplets ◽  
Neutral Lipids ◽  
Plastic Behavior ◽  
Glucose Starvation ◽  
Spastic Paraplegia ◽  
Microtubule Severing ◽  
Tag Accumulation

Lipid droplets (LDs) are metabolic organelles that store neutral lipids and dynamically respond to changes in energy availability by accumulating or mobilizing triacylglycerols (TAGs). How the plastic behavior of LDs is regulated is poorly understood. Hereditary spastic paraplegia is a central motor axonopathy predominantly caused by mutations in SPAST, encoding the microtubule-severing protein spastin. The spastin-M1 isoform localizes to nascent LDs in mammalian cells; however, the mechanistic significance of this targeting is not fully explained. Here, we show that tightly controlled levels of spastin-M1 are required to inhibit LD biogenesis and TAG accumulation. Spastin-M1 maintains the morphogenesis of the ER when TAG synthesis is prevented, independent from microtubule binding. Moreover, spastin plays a microtubule-dependent role in mediating the dispersion of LDs from the ER upon glucose starvation. Our results reveal a dual role of spastin to shape ER tubules and to regulate LD movement along microtubules, opening new perspectives for the pathogenesis of hereditary spastic paraplegia.

scholarly journals Coexistence of Hereditary Spastic Paraplegia Type 4 and Narcolepsy: A Case Report

2021 ◽  
pp. 84-91
Author(s):  
Takahiro Nagai ◽  
Yoko Sunami ◽  
Risa Kato ◽  
Megumi Sugai ◽  
Makoto Takahara ◽  
...  
Keyword(s):  
Cognitive Decline ◽  
Hereditary Spastic Paraplegia ◽  
Clinical Symptoms ◽  
Splice Site Mutation ◽  
Motor Symptom ◽  
Spastic Paraplegia ◽  
Site Mutation ◽  
Neuronal Morphogenesis ◽  
Microtubule Severing

Spastic paraplegia type 4 (SPG4) is the most common type of hereditary spastic paraplegia (HSP) caused by the mutations in the <i>SPAST</i> gene, which encodes a microtubule-severing protein named spastin. Spastin regulates the number and mobility of microtubules and is essential for axonal outgrowth and neuronal morphogenesis. Herein, we report a patient with SPG4 harboring a novel donor splice site mutation in the <i>SPAST</i> gene (c.1616+1dupG). Although SPG4 usually manifests itself as a pure form of HSP, this patient exhibited a slow progressive cognitive decline and also developed narcolepsy type 2 (narcolepsy without cataplexy) prior to the onset of SPG4. Recently, cognitive decline has attracted attention as a main non-motor symptom of SPG4. However, this is the first reported case of a patient developing both SPG4 and narcolepsy, although it remains unclear whether the manifestation of the two diseases is a coincidence or an association. In this report, we describe the clinical symptoms and genetic background of the patient.

Complicated hereditary spastic paraplegia with thin corpus callosum: Variation of phenotypic expression over time

2004 ◽  
Vol 251 (10) ◽  
pp. 1285-1287 ◽  
Author(s):  
Anne-D�rte Sperfeld ◽  
Jan Kassubek ◽  
Andrew H. Crosby ◽  
Beate Winner ◽  
Albert C. Ludolph ◽  
...  
Keyword(s):  
Corpus Callosum ◽  
Hereditary Spastic Paraplegia ◽  
Phenotypic Expression ◽  
Spastic Paraplegia ◽  
Thin Corpus Callosum ◽  
Over Time

A Novel Factor XII Mutation, FXII Tokyo (R84P), Causing Factor XII Deficiency In a Patient with Hereditary Spastic Paraplegia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1404-1404
Author(s):  
Eri Matsuki ◽  
Yoshitaka Miyakawa ◽  
Shinichiro Okamoto
Keyword(s):  
Hereditary Spastic Paraplegia ◽  
Intracellular Transport ◽  
Protein Transport ◽  
Conflicts Of Interest ◽  
Genetic Diagnosis ◽  
Autosomal Recessive Disorder ◽  
Factor Xii ◽  
Spastic Paraplegia ◽  
Wild Type ◽  
Her Family

Abstract Abstract 1404 Hereditary factor XII (FXII) deficiency is a clinically asymptomatic, autosomal recessive disorder. We have experienced a rare case of FXII deficiency in a patient with hereditary spastic paraplegia (HSP). This is the first report in the literature of the coexistence of these two disorders together with the analysis of their genetic cause. The patient, who had no major bleeding episodes, presented with a prolonged APTT of 104.9 seconds. Further evaluation revealed decreased FXII activity of less than 10% and FXII antigen level of 17% with negative results on mixing study. Based upon these findings, she was diagnosed as having hereditary FXII deficiency. Her family history was positive for HSP on her mother's side, the genetic diagnosis of which had been confirmed in her younger daughter. Pedigree analysis showed that her two daughter's coagulation profiles were all within normal limit. Sequencing of the 14 exons and intron/exon boundaries of the FXII gene revealed a novel missense mutation at exon 4 that substitutes arginine 84 to proline (R84P), which we named FXII Tokyo. The mutation status was also confirmed by restriction fragment length polymorphism using restriction enzyme BtgZI. To elucidate the molecular mechanism of FXII deficiency, wild type and R84P mutant FXII cDNA were transiently expressed in CHO cells. We performed Western blot analysis, and found that secretion but not synthesis of R84P mutant protein was markedly reduced compared to wild type. Altogether, these results indicated that R84P mutation might impair the intracellular transport or secretion of FXII protein of the cells and could be a useful tool for the analysis of structure-function relationship and intracellular protein transport of FXII protein in future. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Recognition of C-terminal amino acids in tubulin by pore loops in Spastin is important for microtubule severing

2007 ◽  
Vol 176 (7) ◽  
pp. 995-1005 ◽  
Author(s):  
Susan Roehl White ◽  
Katia J. Evans ◽  
Jeffrey Lary ◽  
James L. Cole ◽  
Brett Lauring
Keyword(s):  
Amino Acids ◽  
Conformational Changes ◽  
Hereditary Spastic Paraplegia ◽  
Spastic Paraplegia ◽  
Central Cavity ◽  
Aaa Atpase ◽  
Microtubule Severing ◽  
Microtubule Binding Domain ◽  
Terminal Amino ◽  
Form Ring

Spastin, an AAA ATPase mutated in the neurodegenerative disease hereditary spastic paraplegia, severs microtubules. Many other AAA proteins form ring-shaped hexamers and contain pore loops, which project into the ring's central cavity and act as ratchets that pull on target proteins, leading, in some cases, to conformational changes. We show that Spastin assembles into a hexamer and that loops within the central pore recognize C-terminal amino acids of tubulin. Key pore loop amino acids are required for severing, including one altered by a disease-associated mutation. We also show that Spastin contains a second microtubule binding domain that makes a distinct interaction with microtubules and is required for severing. Given that Spastin engages the MT in two places and that both interactions are required for severing, we propose that severing occurs by forces exerted on the C-terminal tail of tubulin, which results in a conformational change in tubulin, which releases it from the polymer.

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