RESUMEN
Cellular membranes can form two principally different involutions, which either exclude or contain cytosol. The 'classical' budding reactions, such as those occurring during endocytosis or formation of exocytic vesicles, involve proteins that assemble on the cytosol-excluding face of the bud neck. Inverse membrane involution occurs in a wide range of cellular processes, supporting cytokinesis, endosome maturation, autophagy, membrane repair and many other processes. Such inverse membrane remodelling is mediated by a heteromultimeric protein machinery known as endosomal sorting complex required for transport (ESCRT). ESCRT proteins assemble on the cytosolic (or nucleoplasmic) face of the neck of the forming involution and cooperate with the ATPase VPS4 to drive membrane scission or sealing. Here, we review similarities and differences of various ESCRT-dependent processes, with special emphasis on mechanisms of ESCRT recruitment.
Asunto(s)
Membrana Celular/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Neuronas/citología , Membrana Nuclear/metabolismo , Replicación Viral/fisiología , Animales , Citocinesis , Endosomas/metabolismo , Exosomas/metabolismo , Interacciones Huésped-Patógeno , Humanos , Neuronas/metabolismo , Fagosomas/metabolismo , Transporte de Proteínas , Espastina/metabolismoRESUMEN
Microtubule-severing enzymes are critical for the biogenesis and maintenance of complex microtubule arrays in axons, spindles, and cilia where tubulin detyrosination, acetylation, and glutamylation are abundant. These modifications exhibit stereotyped patterns suggesting spatial and temporal control of microtubule functions. Using human-engineered and differentially modified microtubules we find that glutamylation is the main regulator of the hereditary spastic paraplegia microtubule severing enzyme spastin. Glutamylation acts as a rheostat and tunes microtubule severing as a function of glutamate number added per tubulin. Unexpectedly, glutamylation is a non-linear biphasic tuner and becomes inhibitory beyond a threshold. Furthermore, the inhibitory effect of localized glutamylation propagates across neighboring microtubules, modulating severing in trans. Our work provides the first quantitative evidence for a graded response to a tubulin posttranslational modification and a biochemical link between tubulin glutamylation and complex architectures of microtubule arrays such as those in neurons where spastin deficiency causes disease.
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Adenosina Trifosfatasas/metabolismo , Microtúbulos/metabolismo , Procesamiento Proteico-Postraduccional , Paraplejía Espástica Hereditaria/metabolismo , Tubulina (Proteína)/metabolismo , Fenómenos Biomecánicos , Ácido Glutámico/metabolismo , Humanos , Neuronas/metabolismo , Ingeniería de Proteínas , Paraplejía Espástica Hereditaria/patología , EspastinaRESUMEN
Genetic variants that affect mRNA splicing are a major cause of hereditary disorders, but the spliceogenicity of variants is challenging to predict. RNA diagnostics of clinically accessible tissues enable rapid functional characterization of splice-altering variants within their natural genetic context. However, this analysis cannot be offered to all individuals as one in five human disease genes are not expressed in easily accessible cell types. To overcome this problem, we have used CRISPR activation (CRISPRa) based on a dCas9-VPR mRNA-based delivery platform to induce expression of the gene of interest in skin fibroblasts from individuals with suspected monogenic disorders. Using this ex vivo splicing assay, we characterized the splicing patterns associated with germline variants in the myelin protein zero gene (MPZ), which is exclusively expressed in Schwann cells of the peripheral nerves, and the spastin gene (SPAST), which is predominantly expressed in the central nervous system. After overnight incubation, CRISPRa strongly upregulated MPZ and SPAST transcription in skin fibroblasts, which enabled splice variant profiling using reverse transcription polymerase chain reaction, next-generation sequencing, and long-read sequencing. Our investigations show proof of principle of a promising genetic diagnostic tool that involves CRISPRa to activate gene expression in easily accessible cells to study the functional impact of genetic variants. The procedure is easy to perform in a diagnostic laboratory with equipment and reagents all readily available.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Empalme del ARN , Humanos , Empalme del ARN/genética , ARN Mensajero , Sistema Nervioso Central , EspastinaRESUMEN
The development and function of male gametes is dependent on a dynamic microtubule network, yet how this is regulated remains poorly understood. We have recently shown that microtubule severing, via the action of the meiotic AAA ATPase protein clade, plays a crucial role in this process. Here, we sought to elucidate the roles of spastin, an as-yet-unexplored member of this clade in spermatogenesis. Using a SpastKO/KO mouse model, we reveal that spastin loss resulted in a complete loss of functional germ cells. Spastin plays a crucial role in the assembly and function of the male meiotic spindle. Consistent with meiotic failure, round spermatid nuclei were enlarged, indicating aneuploidy, but were still able to enter spermiogenesis. During spermiogenesis, we observed extreme abnormalities in manchette structure, acrosome biogenesis and, commonly, a catastrophic loss of nuclear integrity. This work defines an essential role for spastin in regulating microtubule dynamics during spermatogenesis, and is of potential relevance to individuals carrying spastin variants and to the medically assisted reproductive technology industry.
Asunto(s)
Acrosoma , Microtúbulos , Animales , Ratones , Masculino , Espastina/genética , Acrosoma/metabolismo , Microtúbulos/metabolismo , Espermatogénesis/genética , Meiosis/genéticaRESUMEN
The endosomal sorting complexes required for transport (ESCRT) system is an ancient and ubiquitous membrane scission machinery that catalyzes the budding and scission of membranes. ESCRT-mediated scission events, exemplified by those involved in the budding of HIV-1, are usually directed away from the cytosol ("reverse topology"), but they can also be directed toward the cytosol ("normal topology"). The ESCRT-III subunits CHMP1B and IST1 can coat and constrict positively curved membrane tubes, suggesting that these subunits could catalyze normal topology membrane severing. CHMP1B and IST1 bind and recruit the microtubule-severing AAA+ ATPase spastin, a close relative of VPS4, suggesting that spastin could have a VPS4-like role in normal-topology membrane scission. Here, we reconstituted the process in vitro using membrane nanotubes pulled from giant unilamellar vesicles using an optical trap in order to determine whether CHMP1B and IST1 are capable of membrane severing on their own or in concert with VPS4 or spastin. CHMP1B and IST1 copolymerize on membrane nanotubes, forming stable scaffolds that constrict the tubes, but do not, on their own, lead to scission. However, CHMP1B-IST1 scaffolded tubes were severed when an additional extensional force was applied, consistent with a friction-driven scission mechanism. We found that spastin colocalized with CHMP1B-enriched sites but did not disassemble the CHMP1B-IST1 coat from the membrane. VPS4 resolubilized CHMP1B and IST1 without leading to scission. These observations show that the CHMP1B-IST1 ESCRT-III combination is capable of severing membranes by a friction-driven mechanism that is independent of VPS4 and spastin.
Asunto(s)
Membrana Celular , Complejos de Clasificación Endosomal Requeridos para el Transporte , Proteínas Oncogénicas , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Membrana Celular/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Fricción , Humanos , Proteínas Oncogénicas/metabolismo , Espastina/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
Hereditary spastic paraplegia (HSP) is a disease in which dieback degeneration of corticospinal tracts, accompanied by axonal swellings, leads to gait deficiencies. SPG4-HSP, the most common form of the disease, results from mutations of human spastin gene (SPAST), which is the gene that encodes spastin, a microtubule-severing protein. The lack of a vertebrate model that recapitulates both the etiology and symptoms of SPG4-HSP has stymied the development of effective therapies for the disease. hSPAST-C448Y mice, which express human mutant spastin at the ROSA26 locus, display corticospinal dieback and gait deficiencies but not axonal swellings. On the other hand, mouse spastin gene (Spast)-knockout (KO) mice display axonal swellings but not corticospinal dieback or gait deficiencies. One possibility is that reduced spastin function, resulting in axonal swellings, is not the cause of the disease but exacerbates the toxic effects of the mutant protein. To explore this idea, Spast-KO and hSPAST-C448Y mice were crossbred, and the offspring were compared with the parental lines via histological and behavioral analyses. The crossbred animals displayed axonal swellings as well as earlier onset, worsened gait deficiencies and corticospinal dieback compared with the hSPAST-C448Y mouse. These results, together with observations on changes in histone deacetylases 6 and tubulin modifications in the axon, indicate that each of these three transgenic mouse lines is valuable for investigating a different component of the disease pathology. Moreover, the crossbred mice are the best vertebrate model to date for testing potential therapies for SPG4-HSP.
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Paraplejía Espástica Hereditaria , Espastina , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Mutación con Ganancia de Función , Humanos , Mutación con Pérdida de Función , Ratones , Ratones Noqueados , Ratones Transgénicos , Mutación , Espastina/genéticaRESUMEN
Hereditary spastic paraplegias (HSPs) comprise a large group of inherited neurologic disorders affecting the longest corticospinal axons (SPG1-86 plus others), with shared manifestations of lower extremity spasticity and gait impairment. Common autosomal dominant HSPs are caused by mutations in genes encoding the microtubule-severing ATPase spastin (SPAST; SPG4), the membrane-bound GTPase atlastin-1 (ATL1; SPG3A) and the reticulon-like, microtubule-binding protein REEP1 (REEP1; SPG31). These proteins bind one another and function in shaping the tubular endoplasmic reticulum (ER) network. Typically, mouse models of HSPs have mild, later onset phenotypes, possibly reflecting far shorter lengths of their corticospinal axons relative to humans. Here, we have generated a robust, double mutant mouse model of HSP in which atlastin-1 is genetically modified with a K80A knock-in (KI) missense change that abolishes its GTPase activity, whereas its binding partner Reep1 is knocked out. Atl1KI/KI/Reep1-/- mice exhibit early onset and rapidly progressive declines in several motor function tests. Also, ER in mutant corticospinal axons dramatically expands transversely and periodically in a mutation dosage-dependent manner to create a ladder-like appearance, on the basis of reconstructions of focused ion beam-scanning electron microscopy datasets using machine learning-based auto-segmentation. In lockstep with changes in ER morphology, axonal mitochondria are fragmented and proportions of hypophosphorylated neurofilament H and M subunits are dramatically increased in Atl1KI/KI/Reep1-/- spinal cord. Co-occurrence of these findings links ER morphology changes to alterations in mitochondrial morphology and cytoskeletal organization. Atl1KI/KI/Reep1-/- mice represent an early onset rodent HSP model with robust behavioral and cellular readouts for testing novel therapies.
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Modelos Animales de Enfermedad , Proteínas de la Membrana , Proteínas de Transporte de Membrana , Paraplejía Espástica Hereditaria , Animales , Axones/metabolismo , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , GTP Fosfohidrolasas/genética , Humanos , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana/genética , Ratones , Ratones Noqueados , Mutación , Paraplejía Espástica Hereditaria/genética , Espastina/genéticaRESUMEN
Spastic paraplegia type 4 (SPG4), caused by SPAST mutations, is the most predominant subtype of hereditary spastic paraplegia. Most documented SPG4 patients present as pure form, with the complex form rarely reported. We described the clinical and genetic features of 20 patients with complex phenotypes of SPG4 and further explored the genotype-phenotype correlations. We collected detailed clinical data of all SPG4 patients and assessed their phenotypes. SPAST gene mutations were identified by Multiplex ligation-dependent probe amplification in combination with whole exome sequencing. We further performed statistical analysis in genotype and phenotype among patients with various manifestations and different variants. Out of 90 SPG4 patients, 20 patients (male:female = 16:4) with additional neurologic deficits, namely complex form, were included in our study. The bimodal distribution of age of onset at 0-10 and 21-40 years old is concluded. On cranial MRI, obvious white matter lesions can be observed in five patients. We identified 9 novel and 8 reported SPAST mutations, of which 11 mutations were located in AAA (ATPase associated with various cellular activities) domain. The AAA cassette of spastin is the hottest mutated region among complex SPG4. All patients with cognitive impairment (CI) are males (n = 9/9). Additionally, 80% patients with ataxia are due to frameshift mutations (n = 4/5). Overall, our study summarized and analyzed the genetic and phenotypic characteristics of complex SPG4, making up over 1/5 of in-house SPG4 cohort, among which CI and ataxia are the most common features. Further studies are expected to explore the underlying mechanisms.
Asunto(s)
Estudios de Asociación Genética , Mutación , Fenotipo , Paraplejía Espástica Hereditaria , Espastina , Adolescente , Adulto , Niño , Femenino , Humanos , Masculino , Persona de Mediana Edad , Edad de Inicio , China/epidemiología , Estudios de Cohortes , Pueblos del Este de Asia/genética , Secuenciación del Exoma , Predisposición Genética a la Enfermedad , Genotipo , Paraplejía , Paraplejía Espástica Hereditaria/genética , Paraplejía Espástica Hereditaria/patología , Espastina/genética , Recién NacidoRESUMEN
BACKGROUND: The aim of the study was to characterize molecular diagnoses in patients with childhood-onset progressive neurological disorders of suspected genetic etiology. METHODS: We studied 48 probands (age range from newborn to 17 years old) with progressive neurological disorders of unknown etiology from the largest pediatric neurology clinic in Finland. Phenotypes included encephalopathy (54%), neuromuscular disorders (33%), movement disorders (11%), and one patient (2%) with hemiplegic migraine. All patients underwent whole-exome sequencing and disease-causing genes were analyzed. RESULTS: We found 20 (42%) of the patients to have variants in genes previously associated with disease. Of these, 12 were previously reported disease-causing variants, whereas eight patients had a novel variant on a disease-causing gene: ATP7A, CHD2, PURA, PYCR2, SLC1A4, SPAST, TRIT1, and UPF3B. Genetics also enabled us to define atypical clinical presentations of Rett syndrome (MECP2) and Menkes disease (ATP7A). Except for one deletion, all findings were single-nucleotide variants (missense 72%, truncating 22%, splice-site 6%). Nearly half of the variants were de novo. CONCLUSIONS: The most common cause of childhood encephalopathies are de novo variants. Whole-exome sequencing, even singleton, proved to be an efficient tool to gain specific diagnoses and in finding de novo variants in a clinically heterogeneous group of childhood encephalopathies. IMPACT: Whole-exome sequencing is useful in heterogeneous pediatric neurology cohorts. Our article provides further evidence for and novel variants in several genes. De novo variants are an important cause of childhood encephalopathies.
Asunto(s)
Encefalopatías , Enfermedades del Sistema Nervioso , Neurología , Síndrome de Rett , Recién Nacido , Humanos , Niño , Adolescente , Enfermedades del Sistema Nervioso/genética , Fenotipo , Espastina/genética , Proteínas de Unión al ARN/genéticaRESUMEN
This cohort study aimed to characterize the prodromal phase of hereditary spastic paraplegia type 4 (SPG4) using biomarkers and clinical signs and symptoms that develop before manifest gait abnormalities. Fifty-six first-degree relatives at risk of developing SPG4 underwent blinded genotyping and standardized phenotyping, including the Spastic Paraplegia Rating Scale (SPRS), complicating symptoms, non-motor affection, Three-Minute Walk, and neurophysiological assessment. Automated MR image analysis was used to compare volumetric properties. CSF of 33 probands was analysed for neurofilament light chain (NfL), tau, and amyloid-ß (Aß). Thirty participants turned out to be SPAST mutation carriers, whereas 26 did not inherit a SPAST mutation. Increased reflexes, ankle clonus, and hip abduction weakness were more frequent in prodromal mutation carriers but were also observed in non-mutation carriers. Only Babinski's sign differentiated reliably between the two groups. Timed walk and non-motor symptoms did not differ between groups. Whereas most mutation carriers had total SPRS scores of 2 points or more, only two non-mutation carriers reached more than 1 point. Motor evoked potentials revealed no differences between mutation and non-mutation carriers. We found NfL but not tau or Aß to rise in CSF of mutation carriers when approaching the time point of predicted disease manifestation. Serum NfL did not differ between groups. Volumetric MRI analyses did not reveal group differences apart from a smaller cingulate gyrus in mutation carriers. This study depicts subtle clinical signs which develop before gait abnormalities in SPG4. Long-term follow-up is needed to study the evolution of SPG4 in the prodromal stage and conversion into manifest disease. NfL in CSF is a promising fluid biomarker that may indicate disease activity in prodromal SPG4 but needs further evaluation in longitudinal studies.
Asunto(s)
Paraplejía Espástica Hereditaria , Humanos , Paraplejía Espástica Hereditaria/genética , Estudios de Cohortes , Paraplejía/genética , Mutación/genética , Péptidos beta-Amiloides/genética , Espastina/genéticaRESUMEN
PURPOSE: To provide an overview of outcome and complications of selective dorsal rhizotomy (SDR) and intrathecal baclofen pump implantation (ITB) for spasticity treatment in children with hereditary spastic paraplegia (HSP). METHODS: Retrospective study including children with HSP and SDR or ITB. Gross motor function measure (GMFM-66) scores and level of spasticity were assessed. RESULTS: Ten patients were included (most had mutations in ATL1 (n = 4) or SPAST (n = 3) genes). Four walked without and two with walking aids, four were non-walking children. Six patients underwent SDR, three patients ITB, and one both. Mean age at surgery was 8.9 ± 4.5 years with a mean follow-up of 3.4 ± 2.2 years. Five of the SDR patients were walking. Postoperatively spasticity in the legs was reduced in all patients. The change in GMFM-66 score was + 8.0 (0-19.7 min-max). The three ITB patients treated (SPAST (n = 2) and PNPLA6 (n = 1) gene mutation) were children with a progressive disease course. No complications of surgery occurred. CONCLUSIONS: SDR is a feasible treatment option in carefully selected children with HSP, especially in walking patients. The majority of patients benefit with respect to gross motor function, complication risk is low. ITB was used in children with severe and progressive disease.
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Parálisis Cerebral , Paraplejía Espástica Hereditaria , Niño , Humanos , Adolescente , Preescolar , Estudios Retrospectivos , Paraplejía Espástica Hereditaria/genética , Paraplejía Espástica Hereditaria/cirugía , Paraplejía Espástica Hereditaria/complicaciones , Parálisis Cerebral/complicaciones , Espasticidad Muscular/genética , Espasticidad Muscular/cirugía , Baclofeno/uso terapéutico , Rizotomía/métodos , Resultado del Tratamiento , EspastinaRESUMEN
Our previous studies have suggested that spastin, which aggregates on spindle microtubules in oocytes, may promote the assembly of mouse oocyte spindles by cutting microtubules. This action may be related to CRMP5, as knocking down CRMP5 results in reduced spindle microtubule density and maturation defects in oocytes. In this study, we found that, after knocking down CRMP5 in oocytes, spastin distribution shifted from the spindle to the spindle poles and errors in microtubule-kinetochore attachment appeared in oocyte spindles. However, CRMP5 did not interact with the other two microtubule-severing proteins, katanin-like-1 (KATNAL1) and fidgetin-like-1 (FIGNL1), which aggregate at the spindle poles. We speculate that, in oocytes, due to the reduction of spastin distribution on chromosomes after knocking down CRMP5, microtubule-kinetochore errors cannot be corrected through severing, resulting in meiotic division abnormalities and maturation defects in oocytes. This finding provides new insights into the regulatory mechanisms of spastin in oocytes and important opportunities for the study of meiotic division mechanisms.
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Cinetocoros , Huso Acromático , Ratones , Animales , Cinetocoros/metabolismo , Espastina/genética , Espastina/metabolismo , Huso Acromático/fisiología , Microtúbulos/metabolismo , Meiosis , Oocitos/fisiologíaRESUMEN
The most common form of hereditary spastic paraplegia (HSP), SPG4 is caused by single nucleotide variants and microrearrangements in the SPAST gene. The high percentage of multi-exonic deletions or duplications observed in SPG4 patients is predisposed by the presence of a high frequency of Alu sequences in the gene sequence. In the present study, we analyzed DNA and RNA samples collected from patients with different microrearrangements in SPAST to map gene breakpoints and evaluate the mutation mechanism. The study group consisted of 69 individuals, including 50 SPG4 patients and 19 healthy relatives from 18 families. Affected family members from 17 families carried varying ranges of microrearrangements in the SPAST gene, while one individual had a single nucleotide variant in the 5'UTR of SPAST. To detect the breakpoints of the SPAST gene, long-range PCR followed by sequencing was performed. The breakpoint sequence was detected for five different intragenic SPAST deletions and one duplication, revealing Alu-mediated microhomology at breakpoint junctions resulting from non-allelic homologous recombination in these patients. Furthermore, SPAST gene expression analysis was performed using patient RNA samples extracted from whole blood. Quantitative real-time PCR tests performed in 14 patients suggest no expression of transcripts with microrearrangements in 5 of them. The obtained data indicate that nonsense-mediated decay degradation is not the only mechanism of hereditary spastic paraplegia in patients with SPAST microrearrangements.
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Haploinsuficiencia , Paraplejía Espástica Hereditaria , Espastina , Humanos , Espastina/genética , Paraplejía Espástica Hereditaria/genética , Masculino , Femenino , Haploinsuficiencia/genética , Linaje , Variaciones en el Número de Copia de ADN , Adulto , Elementos Alu/genética , Persona de Mediana Edad , Adolescente , Adulto Joven , Degradación de ARNm Mediada por Codón sin SentidoRESUMEN
Spastic paraplegia type 4 (SPG4) is the most common type of autosomally inherited spastic paraplegia. Its main clinical features include typical simple hereditary spastic paraplegia, with neurological impairments limited to lower limb spasticity, hypertonic bladder dysfunction, and mild weakening of lower limb vibration sensation, without accompanying features such as nerve atrophy, ataxia, cognitive impairment, seizures, and muscle tone disorders. SPAST is the main pathogenic gene underlying SPG4, and various pathogenic SPAST variants have been discovered. This disease has featured a high degree of clinical heterogeneity, and the same pathogenic variant can have different age of onset and severity among different patients and even within the same family. There is a lack of systematic research on the correlation between the genotype and phenotype of SPG4, and the pathogenic mechanism has remained controversial. This article has provided a review for the clinical characteristics, pathogenic gene characteristics, correlation between the genotype and phenotype, and pathogenic mechanism of this disease, with an aim to provide reference for its clinical diagnosis and treatment.
Asunto(s)
Paraplejía Espástica Hereditaria , Humanos , Paraplejía Espástica Hereditaria/genética , Mutación , Espastina/genética , Paraplejía/genética , FenotipoRESUMEN
Investigating novel mechanisms of neurite outgrowth via cytoskeleton is critical for developing therapeutic strategies against neural disorders. Rab3A is a vesicle-related protein distributed throughout the nervous system, but the detailed mechanism related to cytoskeleton remains largely unknown. Our previous reports show that spastin serves microtubule to regulate neurite outgrowth. Here, we asked whether Rab3A could function via modulating spastin during neuronal development. The results revealed that Rab3A colocalized with spastin in cultured hippocampal neurons. Immunoprecipitation assays showed that Rab3A physically interacted with spastin in rat brain lysates. Rab3A overexpression significantly induced spastin degradation; this effect was reversed by leupeptin- or MG-132- administration, suggesting the lysosomal and ubiquitin-mediated degradation system. Immunofluorescence staining further confirmed that Rab3A and spastin immune-colocalized with the lysosome marker lysotracker. In COS7 cells, Rab3A overexpression significantly downregulated spastin expression and abolished the spastin-mediated microtubule severing. Furthermore, overexpression inhibited while genetic knockdown of Rab3A promoted neurite outgrowth. However, this inhibitory effect on neurite outgrowth in hippocampal neurons could be reversed via co-transfection of spastin, indicating that Rab3A functions via its interaction protein spastin. In general, our data identify an interaction between Rab3A and spastin, and this interaction affects the protein stability of spastin and eliminates its microtubule severing function, thereby modulating neurite outgrowth.
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Adenosina Trifosfatasas , Paraplejía Espástica Hereditaria , Animales , Ratas , Adenosina Trifosfatasas/metabolismo , Neuritas/metabolismo , Proyección Neuronal , Neuronas/metabolismo , Proteína de Unión al GTP rab3A , Paraplejía Espástica Hereditaria/genética , Paraplejía Espástica Hereditaria/metabolismo , Espastina/metabolismo , Espastina/farmacologíaRESUMEN
Hereditary spastic paraplegias (HSPs) are a heterogeneous group of neurodegenerative disorders characterized by progressive spasticity and weakness in the lower extremities. To date, a total of 88 types of SPG are known. To diagnose HSP, multiple technologies, including microarray, direct sequencing, multiplex ligation-dependent probe amplification, and short-read next-generation sequencing, are often chosen based on the frequency of HSP subtypes. Exome sequencing (ES) is commonly used. We used ES to analyze ten cases of HSP from eight families. We identified pathogenic variants in three cases (from three different families); however, we were unable to determine the cause of the other seven cases using ES. We therefore applied long-read sequencing to the seven undetermined HSP cases (from five families). We detected intragenic deletions within the SPAST gene in four families, and a deletion within PSEN1 in the remaining family. The size of the deletion ranged from 4.7 to 12.5 kb and involved 1-7 exons. All deletions were entirely included in one long read. We retrospectively performed an ES-based copy number variation analysis focusing on pathogenic deletions, but were not able to accurately detect these deletions. This study demonstrated the efficiency of long-read sequencing in detecting intragenic pathogenic deletions in ES-negative HSP patients.
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Adenosina Trifosfatasas , Paraplejía Espástica Hereditaria , Humanos , Adenosina Trifosfatasas/genética , Exoma/genética , Mutación , Variaciones en el Número de Copia de ADN , Estudios Retrospectivos , Espastina/genética , Paraplejía Espástica Hereditaria/diagnóstico , Paraplejía Espástica Hereditaria/genética , Paraplejía/genéticaRESUMEN
Copy number variations (CNV) may represent a significant proportion of SPG4 and SPG3A diagnosis, the most frequent autosomal dominant subtypes of hereditary spastic paraplegias (HSP). We aimed to assess the frequency of CNVs in SPAST and ATL1 and to update the molecular epidemiology of HSP families in southern Brazil. A cohort study that included 95 Brazilian index cases with clinical suspicion of HSP was conducted between April 2011 and September 2022. Multiplex Ligation Dependent Probe Amplification (MLPA) was performed in 41 cases without defined diagnosis by different massive parallel sequencing techniques (MPS). Diagnosis was obtained in 57/95 (60%) index cases, 15/57 (26.3%) being SPG4. Most frequent autosomal recessive HSP subtypes were SPG7 followed by SPG11, SPG76 and cerebrotendinous xanthomatosis. No CNVs in SPAST and ATL1 were found. Copy number variations are rare among SPG4 and SPG3A families in Brazil. Considering the possibility of CNVs detection by specific algorithms with MPS data, we consider that this is likely the most cost-effective approach to investigate CNVs in these genes in low-risk populations, with MLPA being reserved as an orthogonal confirmatory test.
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Variaciones en el Número de Copia de ADN , Paraplejía Espástica Hereditaria , Espastina , Humanos , Brasil/epidemiología , Estudios de Cohortes , Variaciones en el Número de Copia de ADN/genética , Mutación , Proteínas/genética , Paraplejía Espástica Hereditaria/epidemiología , Paraplejía Espástica Hereditaria/genética , Paraplejía Espástica Hereditaria/diagnóstico , Espastina/genéticaRESUMEN
OBJECTIVES: To diagnose the molecular cause of hereditary spastic paraplegia (HSP) observed in a four-generation family with autosomal dominant inheritance. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
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Paraplejía Espástica Hereditaria , Humanos , Paraplejía Espástica Hereditaria/genética , Paraplejía Espástica Hereditaria/diagnóstico , Espastina/genética , Adenosina Trifosfatasas/genética , Fenotipo , Intrones/genética , MutaciónRESUMEN
Mutations in the gene encoding the microtubule-severing protein spastin (spastic paraplegia 4 [SPG4]) cause hereditary spastic paraplegia (HSP), associated with neurodegeneration, spasticity, and motor impairment. Complicated forms (complicated HSP [cHSP]) further include cognitive deficits and dementia; however, the etiology and dysfunctional mechanisms of cHSP have remained unknown. Here, we report specific working and associative memory deficits upon spastin depletion in mice. Loss of spastin-mediated severing leads to reduced synapse numbers, accompanied by lower miniature excitatory postsynaptic current (mEPSC) frequencies. At the subcellular level, mutant neurons are characterized by longer microtubules with increased tubulin polyglutamylation levels. Notably, these conditions reduce kinesin-microtubule binding, impair the processivity of kinesin family protein (KIF) 5, and reduce the delivery of presynaptic vesicles and postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Rescue experiments confirm the specificity of these results by showing that wild-type spastin, but not the severing-deficient and disease-associated K388R mutant, normalizes the effects at the synaptic, microtubule, and transport levels. In addition, short hairpin RNA (shRNA)-mediated reduction of tubulin polyglutamylation on spastin knockout background normalizes KIF5 transport deficits and attenuates the loss of excitatory synapses. Our data provide a mechanism that connects spastin dysfunction with the regulation of kinesin-mediated cargo transport, synapse integrity, and cognition.
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Ácido Glutámico/metabolismo , Cinesinas/metabolismo , Trastornos de la Memoria/metabolismo , Trastornos de la Memoria/fisiopatología , Memoria a Corto Plazo , Neuronas/metabolismo , Espastina/deficiencia , Tubulina (Proteína)/metabolismo , Potenciales de Acción , Animales , Membrana Celular/metabolismo , Espinas Dendríticas/metabolismo , Espinas Dendríticas/ultraestructura , Potenciales Postsinápticos Excitadores , Hipocampo/patología , Hipocampo/fisiopatología , Ratones Noqueados , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Actividad Motora , Neuronas/patología , Neuronas/ultraestructura , Transporte de Proteínas , Espastina/metabolismo , Sinapsis/metabolismo , Sinapsis/ultraestructura , Vesículas Sinápticas/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Microtubule defects are a common feature in several neurodegenerative disorders, including hereditary spastic paraplegia. The most frequent form of hereditary spastic paraplegia is caused by mutations in the SPG4/SPAST gene, encoding the microtubule severing enzyme spastin. To date, there is no effective therapy available but spastin-enhancing therapeutic approaches are emerging; thus prognostic and predictive biomarkers are urgently required. METHODS: An automated, simple, fast and non-invasive cell imaging-based method was developed to quantify microtubule cytoskeleton organization changes in lymphoblastoid cells and peripheral blood mononuclear cells. RESULTS: It was observed that lymphoblastoid cells and peripheral blood mononuclear cells from individuals affected by SPG4-hereditary spastic paraplegia show a polarized microtubule cytoskeleton organization. In a pilot study on freshly isolated peripheral blood mononuclear cells, our method discriminates SPG4-hereditary spastic paraplegia from healthy donors and other hereditary spastic paraplegia subtypes. In addition, it is shown that our method can detect the effects of spastin protein level changes. CONCLUSIONS: These findings open the possibility of a rapid, non-invasive, inexpensive test useful to recognize SPG4-hereditary spastic paraplegia subtype and evaluate the effects of spastin-enhancing drug in non-neuronal cells.