Cause Of Hereditary Spastic Paraplegia Research Articles

KCNJ3 is a novel candidate gene for autosomal dominant pure hereditary spastic paraplegia identified using whole genome sequencing.

Hereditary spastic paraplegia (HSP) is a group of familial diseases characterized by progressive corticospinal tract degeneration. Clinically, patients present with lower-limb spasticity and weakness. To date, more than 80 genetic HSP types have been identified. Despite advances in molecular genetics, novel HSP gene discoveries are ongoing, with a low genetic diagnostic yield. In this study, we aimed to determine pathogenic variants in a family with HSP, which was not diagnosed through conventional genetic testing. We clinically characterized a large family and conducted whole genome sequencing (WGS) analysis of four affected and three unaffected individuals in the family to identify the genetic cause of HSP. This family had autosomal dominant pure (uncomplicated) late childhood-onset HSP. The patients' symptoms accelerated between the ages of 20 and 30. Brain magnetic resonance images typically showed white matter changes, a thin corpus callosum, and cerebellar atrophy. We identified a heterozygous missense variant, KCNJ3 c.1297T>G (p.Leu433Val), through WGS and family genetic analysis, confirmed by Sanger sequencing. We suggest that the identification of KCNJ3 c.1297T>G (p.Leu433Val) constitutes the discovery of a potential novel gene responsible for HSP in this family. This is the first study to report the possible role of a KCNJ3 variant in HSP pathogenesis. Our findings further expand the phenotypic and genotypic spectrum of HSP.

An Automated Imaging-Based Screen for Genetic Modulators of ER Organisation in Cultured Human Cells.

Hereditary spastic paraplegias (HSPs) are a heterogeneous group of mono-genetic inherited neurological disorders, whose primary manifestation is the disruption of the pyramidal system, observed as a progressive impaired gait and leg spasticity in patients. Despite the large list of genes linked to this group, which exceeds 80 loci, the number of cellular functions which the gene products engage is relatively limited, among which endoplasmic reticulum (ER) morphogenesis appears central. Mutations in genes encoding ER-shaping proteins are the most common cause of HSP, highlighting the importance of correct ER organisation for long motor neuron survival. However, a major bottleneck in the study of ER morphology is the current lack of quantitative methods, with most studies to date reporting, instead, on qualitative changes. Here, we describe and apply a quantitative image-based screen to identify genetic modifiers of ER organisation using a mammalian cell culture system. An analysis reveals significant quantitative changes in tubular ER and dense sheet ER organisation caused by the siRNA-mediated knockdown of HSP-causing genes ATL1 and RTN2. This screen constitutes the first attempt to examine ER distribution in cells in an automated and high-content manner and to detect genes which impact ER organisation.

Whole exome sequencing in Serbian patients with hereditary spastic paraplegia.

Hereditary spastic paraplegia (HSP) is a group of neurodegenerative diseases with a high genetic and clinical heterogeneity. Numerous HSP patients remain genetically undiagnosed despite screening for known genetic causes of HSP. Therefore, identification of novel variants and genes is needed. Our previous study analyzed 74 adult Serbian HSP patients from 65 families using panel of the 13 most common HSP genes in combination with a copy number variation analysis. Conclusive genetic findings were established in 23 patients from 19 families (29%). In the present study, nine patients from nine families previously negative on the HSP gene panel were selected for the whole exome sequencing (WES). Further, 44 newly diagnosed adult HSP patients from 44 families were sent to WES directly, since many studies showed WES may be used as the first step in HSP diagnosis. WES analysis of cohort 1 revealed a likely genetic cause in five (56%) of nine HSP families, including variants inthe ETHE1,ZFYVE26,RNF170,CAPN1, and WASHC5 genes. In cohort 2, possible causative variants were found in seven (16%) of 44 patients (later updated to 27% when other diagnosis were excluded), comprising six different genes:SPAST,SPG11,WASCH5,KIF1A, KIF5A,and ABCD1. These results expand the genetic spectrum of HSP patients in Serbia and the region with implications for molecular genetic diagnosis and future causative therapies. Wide HSP panel can be the first step in diagnosis, alongside with the copy number variation (CNV) analysis, while WES should be performed after.

Large-Scale Whole-Genome Analysis of HTLV-1-Associated Myelopathy Identified Hereditary Spastic Paraplegias.

Distinguishing human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy from hereditary spastic paraplegia in patients infected with HTLV-1 is challenging due to overlapping clinical symptoms. The aim of this study was to explore the possibility that hereditary spastic paraplegia is inherently present in patients diagnosed with HTLV-1-associated myelopathy. We performed whole-genome sequencing on 315 unrelated patients registered in the HTLV-1-Associated Myelopathy patient registry "HAM-net," from 2013 to 2022 in Japan. CSF inflammatory biomarkers, including CXCL10, were measured. We identified 5 patients with pathogenic variants in the genes RTN2, SPAST, VCP, and UBAP1, which are the known causes of hereditary spastic paraplegia. These patients had no family history of hereditary spastic paraplegia. The levels of CSF inflammatory biomarkers were lower than expected in these patients, compared with disease severity. Genetic analysis is useful for the differentiation of hereditary spastic paraplegia patients from HTLV-1-associated myelopathy patients, especially for the patients with low levels of CSF inflammatory markers. Here we report the presence of hereditary spinal cord diseases in patients diagnosed with HTLV-1-associated myelopathy and provides evidence that genetic analysis would be helpful in the diagnostic workflow.

Drosophila Atlastin regulates synaptic vesicle mobilization independent of bone morphogenetic protein signaling

BackgroundThe endoplasmic reticulum (ER) contacts endosomes in all parts of a motor neuron, including the axon and presynaptic terminal, to move structural proteins, proteins that send signals, and lipids over long distances. Atlastin (Atl), a large GTPase, is required for membrane fusion and the structural dynamics of the ER tubules. Atl mutations are the second most common cause of Hereditary Spastic Paraplegia (HSP), which causes spasticity in both sexes’ lower extremities. Through an unknown mechanism, Atl mutations stimulate the BMP (bone morphogenetic protein) pathway in vertebrates and Drosophila. Synaptic defects are caused by atl mutations, which affect the abundance and distribution of synaptic vesicles (SV) in the bouton. We hypothesize that BMP signaling, does not cause Atl-dependent SV abnormalities in Drosophila.ResultsWe show that atl knockdown in motor neurons (Atl-KD) increases synaptic and satellite boutons in the same way that constitutively activating the BMP-receptor Tkv (thick veins) (Tkv-CA) increases the bouton number. The SV proteins Cysteine string protein (CSP) and glutamate vesicular transporter are reduced in Atl-KD and Tkv-CA larvae. Reducing the activity of the BMP receptor Wishful thinking (wit) can rescue both phenotypes. Unlike Tkv-CA larvae, Atl-KD larvae display altered activity-dependent distributions of CSP staining. Furthermore, Atl-KD larvae display an increased FM 1–43 unload than Control and Tkv-CA larvae. As decreasing wit function does not reduce the phenotype, our hypothesis that BMP signaling is not involved is supported. We also found that Rab11/CSP colocalization increased in Atl-KD larvae, which supports the concept that late recycling endosomes regulate SV movements.ConclusionsOur findings reveal that Atl modulates neurotransmitter release in motor neurons via SV distribution independently of BMP signaling, which could explain the observed SV accumulation and synaptic dysfunction. Our data suggest that Atl is involved in membrane traffic as well as formation and/or recycling of the late endosome.

Alu Retrotransposition Event in SPAST Gene as a Novel Cause of Hereditary Spastic Paraplegia.

To diagnose the molecular cause of hereditary spastic paraplegia (HSP) observed in a four-generation family with autosomal dominant inheritance. Multiplex ligation-dependent probe amplification (MLPA), whole-exome sequencing (WES), and RNA sequencing (RNA-seq) of peripheral blood leukocytes were performed. Reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing were used to characterize target regions of SPAST. A 121-bp AluYb9 insertion with a 30-bp poly-A tail flanked by 15-bp direct repeats on both sides was identified in the edge of intron 16 in SPAST that segregated with the disease phenotype. We identified an intronic AluYb9 insertion inducing splicing alteration in SPAST causing pure HSP phenotype that was not detected by routine WES analysis. Our findings suggest RNA-seq is a recommended implementation for undiagnosed cases by first-line diagnostic approaches. © 2023 International Parkinson and Movement Disorder Society.

Pyrroline-5-carboxylate reductase 2 (PYCR2) deficiency causes hereditary spastic paraplaegia in late childhood.

PYCR2 gene variants are extremely rare condition which is associated with hypomyelinating leukodystrophy type 10 with microcephaly (HLD10). The aim of the present study is to report the clinical findings of patients having novel PYCR2 gene variant that manifest Hereditary Spastic Paraplegia (HSP) is the only symptom without hypomyelinating leukodystrophy. This is the first study that report the PYCR2 gene variants as a cause of HSP in late childhood. We believe it can contribute to expanding the spectrum of the phenotypes associated with PYCR2. It is a retrospective study. Of the patients with similar clinical features from two related families, "patient 1" was designated as the index case, and was analyzed using Whole Exome Squence analysis (WES). The detected variation was investigated in the index case's parents, relatives, and sibling with a similar phenotype. Clinical, brain magnetic resonance (MR) images and MR spectroscopic findings of the patients were reported. A novel homozygous missense (NM_013328: c.383T>C, p.V128A) variant in the PYCR2 gene is detected in 5 patient from 2 related families. All the patients were male, their ages ranges from 6 to 26 years (15.58±8,33yrs). Developmantal milestones were normal without dysmorphic features. 4 (%80) patients exhibit mild intention tremor started at the age of approximately 6 years of age. 4 (%80) patients had gait difficulty and progressive lower limb spasticity started at the age of 8-12 years. White matter myelination was normal in all patients. Glycine peakes were detected on the MR spectroscopy in all patients. Some variants of PYCR2 gene are responsible for causing clinical features of HSP without hypomyelinating leukodystrophy in the pediatric patients.

The enhanced association between mutant CHMP2B and spastin is a novel pathological link between frontotemporal dementia and hereditary spastic paraplegias

Chromosome 3-linked frontotemporal dementia (FTD3) is caused by a gain-of-function mutation in CHMP2B, resulting in the production of a truncated toxic protein, CHMP2BIntron5. Loss-of-function mutations in spastin are the most common genetic cause of hereditary spastic paraplegias (HSP). How these proteins might interact with each other to drive pathology remains to be explored. Here we found that spastin binds with greater affinity to CHMP2BIntron5 than to CHMP2BWT and colocalizes with CHMP2BIntron5 in p62-positive aggregates. In cultured cells expressing CHMP2BIntron5, spastin level in the cytoplasmic soluble fraction is decreased while insoluble spastin level is increased. These pathological features of spastin are validated in brain neurons of a mouse model of FTD3. Moreover, genetic knockdown of spastin enhances CHMP2BIntron5 toxicity in a Drosophila model of FTD3, indicating the functional significance of their association. Thus, our study reveals that the enhanced association between mutant CHMP2B and spastin represents a novel potential pathological link between FTD3 and HSP.

Phenotypic and Genetic Heterogeneity of Adult Patients with Hereditary Spastic Paraplegia from Serbia.

Hereditary spastic paraplegia (HSP) is among the most genetically diverse of all monogenic diseases. The aim was to analyze the genetic causes of HSP among adult Serbian patients. The study comprised 74 patients from 65 families clinically diagnosed with HSP during a nine-year prospective period. A panel of thirteen genes was analyzed: L1CAM (SPG1), PLP1 (SPG2), ATL1 (SPG3A), SPAST (SPG4), CYP7B1 (SPG5A), SPG7 (SPG7), KIF5A (SPG10), SPG11 (SPG11), ZYFVE26 (SPG15), REEP1 (SPG31), ATP13A2 (SPG78), DYNC1H1, and BICD2 using a next generation sequencing-based technique. A copy number variation (CNV) test for SPAST, SPG7, and SPG11 was also performed. Twenty-three patients from 19 families (29.2%) had conclusive genetic findings, including 75.0% of families with autosomal dominant and 25.0% with autosomal recessive inheritance, and 15.7% of sporadic cases. Twelve families had mutations in the SPAST gene, usually with a pure HSP phenotype. Three sporadic patients had conclusive findings in the SPG11 gene. Two unrelated patients carried a homozygous pathogenic mutation c.233T>A (p.L78*) in SPG7 that is a founder Roma mutation. One patient had a heterozygous de novo variant in the KIF5A gene, and one had a compound heterozygous mutation in the ZYFVE26 gene. The combined genetic yield of our gene panel and CNV analysis for HSP was around 30%. Our findings broaden the knowledge on the genetic epidemiology of HSP, with implications for molecular diagnostics in this region.

Store-operated calcium entry is reduced in spastin-linked hereditary spastic paraplegia.

Pathogenic variants in SPAST, the gene coding for spastin, are the single most common cause of hereditary spastic paraplegia, a progressive motor neuron disease. Spastin regulates key cellular functions, including microtubule-severing and endoplasmic reticulum-morphogenesis. However, it remains unclear how alterations in these cellular functions due to SPAST pathogenic variants result in motor neuron dysfunction. Since spastin influences both microtubule network and endoplasmic reticulum structure, we hypothesized that spastin is necessary for the regulation of Ca2+ homeostasis via store-operated calcium entry.Here, we show that the lack of spastin enlarges the endoplasmic reticulum and reduces store-operated calcium entry. In addition, elevated levels of different spastin variants induced clustering of STIM1 within the endoplasmic reticulum, altered the transport of STIM1 to the plasma membrane and reduced store-operated calcium entry, which could be rescued by exogenous expression of STIM1. Importantly, store-operated calcium entry was strongly reduced in induced pluripotent stem cell-derived neurons from hereditary spastic paraplegia patients with pathogenic variants in SPAST resulting in spastin haploinsufficiency. These neurons developed axonal swellings in response to lack of spastin. We were able to rescue both store-operated calcium entry and axonal swellings in SPAST patient neurons by restoring spastin levels, using CRISPR/Cas9 to correct the pathogenic variants in SPAST.These findings demonstrate that proper amounts of spastin are a key regulatory component for store-operated calcium entry mediated Ca2+ homeostasis and suggest store-operated calcium entry as a disease relevant mechanism of spastin-linked motor neuron disease.

The emerging genetic diversity of hereditary spastic paraplegia in Korean patients

Hereditary Spastic Paraplegias (HSP) are a group of rare inherited neurological disorders characterized by progressive loss of corticospinal motor-tract function. Numerous patients with HSP remain undiagnosed despite screening for known genetic causes of HSP. Therefore, identification of novel genetic variations related to HSP is needed. In this study, we identified 88 genetic variants in 54 genes from whole-exome data of 82 clinically well-defined Korean HSP families. Fifty-six percent were known HSP genes, and 44% were composed of putative candidate HSP genes involved in the HSPome and originally reported neuron-related genes, not previously diagnosed in HSP patients. Their inheritance modes were 39, de novo; 33, autosomal dominant; and 10, autosomal recessive. Notably, ALDH18A1 showed the second highest frequency. Fourteen known HSP genes were firstly reported in Koreans, with some of their variants being predictive of HSP-causing protein malfunction. SPAST and REEP1 mutants with unknown function induced neurite abnormality. Further, 54 HSP-related genes were closely linked to the HSP progression-related network. Additionally, the genetic spectrum and variation of known HSP genes differed across ethnic groups. These results expand the genetic spectrum for HSP and may contribute to the accurate diagnosis and treatment for rare HSP.

Therapeutic Strategies for Mutant SPAST-Based Hereditary Spastic Paraplegia.

Mutations of the SPAST gene that encodes the microtubule-severing enzyme called spastin are the chief cause of Hereditary Spastic Paraplegia. Growing evidence indicates that pathogenic mutations functionally compromise the spastin protein and endow it with toxic gain-of-function properties. With each of these two factors potentially relevant to disease etiology, the present article discusses possible therapeutic strategies that may ameliorate symptoms in patients suffering from SPAST-based Hereditary Spastic Paraplegia, which is usually termed SPG4-HSP.

HACE1, GLRX5, and ELP2 gene variant cause spastic paraplegies.

Hereditary spastic paraplegias (HSPs) are a clinically and genetically heterogeneous group of conditions that are characterized by lower limb spasticity and weakness. Considering the clinical overlap between metabolic causes, genetic diseases, and autosomal recessive HSP, differentiation between these types can be difficult based solely on their clinical characteristics. This study aimed to investigate the genetic etiology of patients with clinically suspected HSP. The study group was composed of seven Turkish families who each had two affected children and three families who each had a single affected child (17 total patients). The 17 probands (14 males, 3 females) underwent whole exome sequencing. Five typical HSP genes (FA2H, AP4M1, AP4E1, CYP7B1, and MAG) and three genes not previously related to HSP (HACE1, GLRX5, ad ELP2) were identified in 14 probands. Eight novel variants were identified in seven families: c.653T > C (p.Leu218Pro) in the FA2H gene, c.347G > A (p.Gly116Asp) in the GLRX5 gene, c.2581G > C (p.Ala861Pro) in the HACE1 gene, c.1580G > A (p.Arg527Gln) and c.1189-1G > A in the ELP2 gene, c.10C > T (p.Gln4*) and c.1025 + 1G > A in the AP4M1 gene, c.1291delG (p.Gly431Alafs*3) and c.3250delA (p.Ile1084*) in the AP4E1 gene, and c.475T > G (p.Cys159Gly) in the MAG gene. The growing use of next-generation sequencing improved diagnosis but also led to the continual identification of new causal genes for neurogenetic diseases associated with lower limb spasticity. The increasing number of HSP genes identified thus far highlights the extreme genetic heterogeneity of these disorders and their clinical and functional overlap with other neurological conditions. Our findings suggest that the HACE1, GLRX5, and ELP2 genes are genetic causes of HSP.

Description of clinical features and genetic analysis of one ultra-rare (SPG64) and two common forms (SPG5A and SPG15) of hereditary spastic paraplegia families

Hereditary spastic paraplegia (HSP) is a clinically and genetically heterogeneous neurodegenerative disorder, characterized by lower-limb spasticity and weakness. To date, more than 82 loci/genes (SPG1-SPG82) have been identified that contribute to the cause of HSP. Despite the use of next-generation sequencing-based methods, genetic-analysis has failed in the finding of causative genes in more than 50% of HSP patients, indicating a more significant heterogeneity and absence of a given phenotype-genotype correlation. Here, we performed whole-exome sequencing (WES) to identify HSP-causing genes in three unrelated-Iranian probands. Candidate variants were detected and confirmed in the probands and co-segregated in the family members. The phenotypic data gathered and compared with earlier cases with the same sub-types of disease. Three novel homozygous variants, c.978delT; p.Q327Kfs*39, c.A1208G; p.D403G and c.3811delT; p.S1271Lfs*44, in known HSP-causing genes including ENTPD1, CYP7B1, and ZFYVE26 were identified, respectively. Intra and interfamilial clinical variability were observed among affected individuals. Mutations in CYP7B1 and ZFYVE26 are relatively common causes of HSP and associated with SPG5A and SPG15, respectively. However, mutations in ENTPD1 are related to SPG64 which is an ultra-rare form of HSP. The research affirmed more complexities of phenotypic manifestations and allelic heterogeneity in HSP. Due to these complexities, it is not feasible to show a clear phenotype-genotype correlation in HSP cases. Identification of more families with mutations in HSP-causing genes may help the establishment of this correlation, further understanding of the molecular basis of the disease, and would provide an opportunity for genetic-counseling in these families.

Clinico‐genetic study of two Japanese pedigrees with hereditary spastic paraparesis and Alzheimer's disease

AbstractBackgroundMutations in the SPG4/SPAST gene are the major cause of hereditary spastic paraplegias (HSPs), a group of genetic disorders leading to progressive spasticity and weakness of the lower limbs. Alzheimer's disease (AD) is a degenerative disorder of the central nervous system. Early‐onset familial AD (EOFAD) accounts for 3‐5% of all AD cases and denotes families in which onset is reliably before age 60 to 65 years and often before age 55 years. The present study reports a second Japanese pedigree in which a SPG4/SPAST mutation co‐segregates with HSP and EOFAD.MethodWhole exome sequencing (WES), Sanger sequencing, Multiplex Ligation dependent Probe Amplification (MLPA) analysis, bioinformatics, neurological evaluation, diagnostic imaging, and pathological assessment. An SPG4/SPAST Japanese family with similar genotype‐phenotype findings was already described by our research team. Haplotype analysis was conducted on the two pedigrees.ResultOne Japanese man was diagnosed as certainly affected by slowly progressive autosomal dominant HSP, according to the Harding criteria. The age at onset of the first motor symptoms was 31 years. At 52 years, phenotype was complicated by cognitive impairment; a diagnosis of probable EOFAD following the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s Disease and Related Disorders Association (NINCDS‐ADRDA) criteria was formulated. The case was definite Alzheimer’s disease (autopsy proven): the brain pathology revealed plaques with a congophilic core and neuritic pathology. Both the brother and the son of the proband revealed the same initial symptoms of dementia and HSP was diagnosed, respectively, at age 55 and 34 years. WES revealed in all the patients a heterozygous change in SPAG4/SPAST. MLPA analysis excluded the presence of deletions/duplications in SPG4/SPAST. Haplotype analysis disclosed a founder effect for the mutation in the two families sharing the same clinico‐genetic characteristics. Bioinformatic analyses and population study confirm a pathogenetic role of such mutation. Screening of PS1, PS2, and bAPP genes did not reveal any coding mutation. No affected subject carried out APOE genotype ε4/ε3 or ε4/ε4.ConclusionThis work describes the second Japanese family with a SPAG4/SPAST mutation and association of HSP and EOFAD. A possible founder effect was confirmed by haplotype analysis.

Mitochondrial Function in Hereditary Spastic Paraplegia: Deficits in SPG7 but Not SPAST Patient-Derived Stem Cells.

Mutations in SPG7 and SPAST are common causes of hereditary spastic paraplegia (HSP). While some SPG7 mutations cause paraplegin deficiency, other SPG7 mutations cause increased paraplegin expression. Mitochondrial function has been studied in models that are paraplegin-deficient (human, mouse, and Drosophila models with large exonic deletions, null mutations, or knockout models) but not in models of mutations that express paraplegin. Here, we evaluated mitochondrial function in olfactory neurosphere-derived cells, derived from patients with a variety of SPG7 mutations that express paraplegin and compared them to cells derived from healthy controls and HSP patients with SPAST mutations, as a disease control. We quantified paraplegin expression and an extensive range of mitochondrial morphology measures (fragmentation, interconnectivity, and mass), mitochondrial function measures (membrane potential, oxidative phosphorylation, and oxidative stress), and cell proliferation. Compared to control cells, SPG7 patient cells had increased paraplegin expression, fragmented mitochondria with low interconnectivity, reduced mitochondrial mass, decreased mitochondrial membrane potential, reduced oxidative phosphorylation, reduced ATP content, increased mitochondrial oxidative stress, and reduced cellular proliferation. Mitochondrial dysfunction was specific to SPG7 patient cells and not present in SPAST patient cells, which displayed mitochondrial functions similar to control cells. The mitochondrial dysfunction observed here in SPG7 patient cells that express paraplegin was similar to the dysfunction reported in cell models without paraplegin expression. The p.A510V mutation was common to all patients and was the likely species associated with increased expression, albeit seemingly non-functional. The lack of a mitochondrial phenotype in SPAST patient cells indicates genotype-specific mechanisms of disease in these HSP patients.

Mutation of Spastin Affects Microtubule Dynamics Through Differential Distribution

SPG4 gene encodes Spastin, and its mutations are the main cause of hereditary spastic paraplegia (HSP). There are more than 50 gene mutations in HSP, and mutations of Spastin account for more than 40%, so SPG4 is the key gene that causes the disease. In order to clarify the effect on microtubule stability after inactivation of AAA functional domain caused by mutations of Spastin, we carried out PCR amplification and C413Y site-directed mutation of the target gene according to the sequence of SPG4 in PUBMED gene bank and constructed GFP-Spastin and GFP-Spastin C413Y recombinant plasmids. The recombinant plasmids were introduced into COS7 cells, and the expression of recombinant plasmids in COS7 cells and changes of microtubule stability were observed. The results of colony PCR and gene identification showed that recombinant plasmids were successfully constructed. Western blotting showed that GFP-Spastin and GFP-Spastin C413Y could be expressed normally in COS7 cells. Immunofluorescence assay showed that the distribution of GFP-Spastin was punctate in the cells and GFP-Spastin had strong microtubule cleavage ability, while GFP-Spastin C413Y was almost distributing in the nucleus, and the ability of microtubule cleavage was weakened and the microtubule was in a stable state. Therefore, we concluded that Spastin C413Y changed the distribution of Spastin in cells and weakened the ability of microtubule cleavage. Spastin C413Y accumulated in the nucleus which could not cut microtubules effectively.

Expression of recombinant mouse spastin in E. coli

Mutations of the gene SPAST that encodes a microtubule severing enzyme, spastin, are the most frequent cause of Hereditary spastic paraplegia (HSP) disease. HSP is heterogeneous group of inherited neurodegenerative disorders characterized predominantly by progressive lower limb spasticity and weakness. Spastin belongs ATPase associated with various cellular activities (AAA) protein family and catalyzes microtubule severing. Spastin in mouse and human are highly identical in protein sequence and several spastin mutation models in mice have been generated in order to evaluate the significance of spastin loss-of-function in mammals. Expression and purification of spastin and the mutant variants determined in patients will facilitate the structure-function relationship study of spastin. Here I systemically optimized the expression condition of a truncated version of mouse spastin in E. coli. The recombinant protein and a mutant were further purified for ATPase activity assay.

Spastin MIT Domain Disease-Associated Mutations Disrupt Lysosomal Function.

The hereditary spastic paraplegias (HSPs) are genetic motor neuron diseases characterized by progressive degeneration of corticospinal tract axons. Mutations in SPAST, encoding the microtubule-severing ATPase spastin, are the most common causes of HSP. The broad SPAST mutational spectrum indicates a haploinsufficiency pathogenic mechanism in most cases. Most missense mutations cluster in the ATPase domain, where they disrupt the protein's ability to sever microtubules. However, several putative missense mutations in the protein's microtubule interacting and trafficking (MIT) domain have also been described, but the pathogenicity of these mutations has not been verified with functional studies. Spastin promotes endosomal tubule fission, and defects in this lead to lysosomal enzyme mistrafficking and downstream lysosomal abnormalities. We investigated the function of three disease-associated spastin MIT mutants and found that none was able to promote normal endosomal tubule fission, lysosomal enzyme receptor trafficking, or lysosomal morphology. One of the mutations affected recruitment of spastin to endosomes, a property that requires the canonical function of the MIT domain in binding endosomal sorting complex required for transport (ESCRT)-III proteins. However, the other mutants did not affect spastin's endosomal recruitment, raising the possibility of pathologically important non-canonical roles for the MIT domain. In conclusion, we demonstrate that spastin MIT mutants cause functional abnormalities related to the pathogenesis of HSP. These mutations do not directly affect spastin's microtubule-severing capacity, and so we identify a new molecular pathological mechanism by which spastin mutations may cause disease.

Novel mutations in the SPAST gene cause hereditary spastic paraplegia

BackgroundMutations in the SPAST gene are the most frequent cause of hereditary spastic paraplegia (HSP). We aim to extend the mutation spectrum of spastic paraplegia 4 (SPG4) and carried out experiment in vitro to explore the influence of the SPAST gene mutation on the function of corresponding protein. MethodsWhole-exome sequencing (WES) combined with multiplex ligation-dependent probe amplification (MLPA) were performed in a cohort of 150 patients clinically diagnosed with HSP. We focus on screening for mutations in SPAST gene and carrying out functional experiments to assess the effects of the novel variants. ResultsA total of 34 different mutations in the SPAST gene were identified, of which 10 were novel, including 1 missense (c.1479T > A), 1 nonsense (c.766G > T), 3 splicing (c.1413 + 1_1413+4delGTAA, c.1729-1G > A and c.1536+2T > G) and 5 frameshift mutations (c.1094delC, c.885dupA, c.517_518delAG, c.280delG and c.908dupC). For 7 novel non-splicing mutations, functional study showed that accumulated M1 spastin colcocalized with microtubules which was different from a uniformly diffused M87 spastin. While an impairment in severing activity was observed in both mutant M1 and mutant M87, except for c.280delG. All 3 novel splicing variants w ere predicted to affect splicing by using bioinformatic programs. However, only c.1536+2T > G had no influence on splice site in vitro, which conflicts with the in-silico analysis. ConclusionWe genetically diagnosed 40 SPG4 patients. All the novel non-splicing mutations except for c.280delG were certified to exert an effect on the microtubule-severing and all the novel splicing mutations other than c.1536+2T > G would cause abnormal splicing of the spastin.