Aprataxin Research Articles

Functional role for senataxin, defective in ataxia oculomotor apraxia type 2, in transcriptional regulation

Ataxia oculomotor apraxia type 2 (AOA2) is an autosomal recessive neurodegenerative disorder characterized by cerebellar ataxia and oculomotor apraxia. The gene mutated in AOA2, SETX, encodes senataxin, a putative DNA/RNA helicase which shares high homology to the yeast Sen1p protein and has been shown to play a role in the response to oxidative stress. To investigate further the function of senataxin, we identified novel senataxin-interacting proteins, the majority of which are involved in transcription and RNA processing, including RNA polymerase II. Binding of RNA polymerase II to candidate genes was significantly reduced in senataxin deficient cells and this was accompanied by decreased transcription of these genes, suggesting a role for senataxin in the regulation/modulation of transcription. RNA polymerase II-dependent transcription termination was defective in cells depleted of senataxin in keeping with the observed interaction of senataxin with poly(A) binding proteins 1 and 2. Splicing efficiency of specific mRNAs and alternate splice-site selection of both endogenous genes and artificial minigenes were altered in senataxin depleted cells. These data suggest that senataxin, similar to its yeast homolog Sen1p, plays a role in coordinating transcriptional events, in addition to its role in DNA repair.

Ataxia oculomotor apraxia type 2: course over 27 years and a novel stop mutation in the senataxin gene

Sirs,Ataxia with oculomotor apraxia type 2 (AOA2) is anautosomal recessive cerebellar ataxia associated withmutations in the senataxin (SETX) gene coding for theortholog of a yeast DNA/RNA helicase [7]. The disorder ischaracterized by adolescent age at onset, spinocerebellargait ataxia, cerebellar atrophy, peripheral sensorimotorneuropathy, areflexia, elevated a-fetoprotein, saccadicocular pursuit, and occasionally oculomotor apraxia. Mildcognitive impairment, involuntary movements, c-globulin,and creatine phosphokinase elevation may also occur [1–7]. Here we report the documented course over 27 years ofa case of AOA2 and a novel homozygous stop mutationp.R1778X in the SETX gene.This 47-year-old woman was born to consanguineousparents (first cousins). After normal delivery and childhoodshe first noticed gait problems at the age of 15 years.Unsteadiness gradually worsened and she has essentiallybeen wheelchair-bound from the age of 30. Mild mental andpsychomotor decline became evident after graduating fromjunior high school at age 16. Severe myopia evolved fromthe age of 8. As reported in other AOA2 cases [2, 5, 6],this patient developed secondary amenorrhoea in earlyadulthood,indicating animportant roleofsenataxin notonlyin neuron survival but also in germ line cell survival [2].The patient was examined in our institute at age 20(1981) and at age 47 (2008). On both occasions, mildcognitive impairment was obvious. Dysarthria was ratedmild at age 20, and moderate at age 47. Eye movementswere full with marked saccadic pursuit at both dates, andthere was no oculomotor apraxia. There were no muscleweakness or foot deformities. Deep tendon reflexes werenormal at age 20 but absent at age 47. There was noextensor plantar response. Vibration and position senseswere not documented at age 20 but impaired in her upperand absent in her lower extremities at age 47. Touch sen-sation was normal. Finger-to-nose and heel-to-shin testingwas mildly dysmetric at age 20 and 47 without majorprogression. Choreic movements were apparent in 1977and a severe kinetic tremor described in 1981 decreasedover the course of the disease. At age 20, her gait waswide-based and Romberg’s sign was positive. At age 47,our patient presented with severe gait ataxia and standinginstability, and was confined to a wheelchair.At age 20, electromyography (EMG) and muscle biopsyfrom tibialis anterior muscle showed severe neurogenicchanges while nerve conduction velocity (NCV) wasspared (peroneal nerve: 42 m/s). At age 47, neurophysio-logical testing revealed a sensorimotor neuropathy. EMGshowed chronic neurogenic remodelling with single motorunit discharge patterns, pathologic spontaneous activity,and pseudomyotonic discharges. Median nerve showedprolonged distal latency (6.0 ms), and reduced NCV(33.4 m/s) and amplitude (1.9 mV). Sensory NCV was notreproducible in the radial, median, and ulnar nerves. BrainCT at age 47 revealed severe cerebellar atrophy affectingpredominantly the vermis (Fig. 1). Her general physicaland gynaecological examination was unremarkable without

Synergistic decrease of DNA single-strand break repair rates in mouse neural cells lacking both Tdp1 and aprataxin

Ataxia oculomotor apraxia-1 (AOA1) is an autosomal recessive neurodegenerative disease that results from mutations of aprataxin (APTX). APTX associates with the DNA single- and double-strand break repair machinery and is able to remove AMP from 5′-termini at DNA strand breaks in vitro. However, attempts to establish a DNA strand break repair defect in APTX-defective cells have proved conflicting and unclear. We reasoned that this may reflect that DNA strand breaks with 5′-AMP represent only a minor subset of breaks induced in cells, and/or the availability of alternative mechanisms for removing AMP from 5′-termini. Here, we have attempted to increase the dependency of chromosomal single- and double-strand break repair on aprataxin activity by slowing the rate of repair of 3′-termini in aprataxin-defective neural cells, thereby increasing the likelihood that the 5′-termini at such breaks become adenylated and/or block alternative repair mechanisms. To do this, we generated a mouse model in which APTX is deleted together with tyrosyl DNA phosphodiesterase (TDP1), an enzyme that repairs 3′-termini at a subset of single-strand breaks (SSBs), including those with 3′-topoisomerase-1 (Top1) peptide. Notably, the global rate of repair of oxidative and alkylation-induced SSBs was significantly slower in Tdp1−/−/Aptx−/− double knockout quiescent mouse astrocytes compared with Tdp1−/− or Aptx−/− single knockouts. In contrast, camptothecin-induced Top1-SSBs accumulated to similar levels in Tdp1−/− and Tdp1−/−/Aptx−/− double knockout astrocytes. Finally, we failed to identify a measurable defect in double-strand break repair in Tdp1−/−, Aptx−/− or Tdp1−/−/Aptx−/− astrocytes. These data provide direct evidence for a requirement for aprataxin during chromosomal single-strand break repair in primary neural cells lacking Tdp1.

Molecular Mechanism of DNA Deadenylation by the Neurological Disease Protein Aprataxin

The human neurological disease known as ataxia with oculomotor apraxia 1 is caused by mutations in the APTX gene that encodes Aprataxin (APTX) protein. APTX is a member of the histidine triad superfamily of nucleotide hydrolases and transferases but is distinct from other family members in that it acts upon DNA. The target of APTX is 5'-adenylates at DNA nicks or breaks that result from abortive DNA ligation reactions. In this work, we show that APTX acts as a nick sensor, which provides a mechanism to assess the adenylation status of unsealed nicks. When an adenylated nick is encountered by APTX, base pairing at the 5' terminus of the nick is disrupted as the adenylate is accepted into the active site of the enzyme. Adenylate removal occurs by a two-step process that proceeds through a transient AMP-APTX covalent intermediate. These results pinpoint APTX as the first protein to adopt canonical histidine triad-type reaction chemistry for the repair of DNA.

Ataxia With Oculomotor Apraxia Type 1 (AOA1): Clinical and Neuropsychological Features in 2 New Patients and Differential Diagnosis

Ataxia with oculomotor apraxia type 1 (AOA1) is an autosomal recessive disease characterized by early-onset and slowly progressive cerebellar ataxia, areflexia, and peripheral neuropathy. Ocular apraxia is most prominent in the early stage of the disease, by contrast, hypoalbuminemia, hypercholesterolemia, and cognitive impairment are present in the adult stage. AOA1 is caused by a mutation in the APTX gene (9p13.3) encoding a nuclear protein named aprataxin, which is involved in the mechanism of DNA repair. We report here the clinical features of 2 patients with mutations in the APTX gene, and we discuss the differential diagnosis with other forms of hereditary ataxia.

Complete deletion of the aprataxin gene: ataxia with oculomotor apraxia type 1 with severe phenotype and cognitive deficit

Ataxia with oculomotor apraxia type 1 (AOA1) is a recently described autosomal-recessive neurodegenerative condition of childhood onset. It is caused by mutations in the APTX gene, which encodes the protein...

Neurodegeneration in xeroderma pigmentosum

The inability to repair damage to DNA clearly can have effects on post-mitotic neurons and cause severe CNS symptoms. Ataxia telangiectasia (A-T), ataxia oculomotor apraxia type 1 (AOA1) and spinocerebellar ataxia with axonal neuropathy (SCAN1) all show progressive neurodegeneration. While it is known that these disorders show defective DNA double or single strand break repair, the precise mechanism of the neurodegeneration is still a mystery (reviewed in McKinnon and Caldecott, 2007). For these and other disorders like Parkinson's disease, oxidative stress may play an important role in damaging the neuronal DNA. In the case of xeroderma pigmentosum (XP), a very rare recessively inherited skin disorder showing predisposition to skin cancer as a result of a severe sensitivity to UV light, neurodegeneration is perhaps an unexpected feature. Nevertheless, this has been known for many years (Andrews et al. , 1978; Kraemer et al. , 1987). Unravelling the function of the genes causing XP was important in understanding the process of nucleotide excision repair (NER), one of several processes involved in repairing damaged DNA (reviewed in Lehmann, 2003; Cleaver, 2005). NER is particularly relevant to the repair of thymine dimers caused by UV light. The cellular response to the thymine dimer involves its removal and replacement with normal …

A 2-gene classifier for predicting response to the farnesyltransferase inhibitor tipifarnib in acute myeloid leukemia

At present, there is no method available to predict response to farnesyltransferase inhibitors (FTIs). We analyzed gene expression profiles from the bone marrow of patients from a phase 2 study of the FTI tipifarnib in older adults with previously untreated acute myeloid leukemia (AML). The RASGRP1/APTX gene expression ratio was found to predict response to tipifarnib with the greatest accuracy using a "leave one out" cross validation (LOOCV; 96%). RASGRP1 is a guanine nucleotide exchange factor that activates RAS, while APTX (aprataxin) is involved in DNA excision repair. The utility of this classifier for predicting response to tipifarnib was validated in an independent set of 58 samples from relapsed or refractory AML, with a negative predictive value (NPV) and positive predictive value (PPV) of 92% and 28%, respectively (odds ratio of 4.4). The classifier also predicted for improved overall survival (154 vs 56 days; P < .001), which was independent of other covariates, including a previously described prognostic gene expression classifier. Therefore, these data indicate that a 2-gene expression assay may have utility in categorizing a population of patients with AML who are more likely to respond to tipifarnib.

A Two-Gene Classifier for Predicting Response to the Farnesyltransferase Inhibitor Tipifarnib in Acute Myeloid Leukemia.

Currently there is no method available to predict response to farnesyltransferase inhibitors (FTI). We analyzed gene expression profiles from the bone marrow of patients from a phase 2 study of the FTI tipifarnib (R115777, ZARNESTRA), in older adults with previously untreated acute myeloid leukemia (AML). The RASGRP1:APTX gene expression ratio was found to predict response to tipifarnib with the greatest accuracy. This two-gene ratio was validated by quantitative PCR in the newly diagnosed AML cohort. We further demonstrated that this classifier could predict response to tipifarnib in an independent set of 54 samples from relapsed or refractory AML, with a negative predictive value and positive predictive value of 92% and 28%, respectively (odds ratio of 4.4). The classifier also predicted for improved overall survival (154 vs 56 days, p = 0.0001), which was shown to be independent of other prognostic factors including a previously described gene expression classifier predictive of prognosis. Therefore, these data indicate that a two-gene expression assay may have utility in categorizing a population of AML patients who are more likely to respond to tipifarnib.

Investigation of Recessive Ataxia Loci in Patients with Young Age of Onset

Autosomal recessive cerebellar ataxias are a phenotypically and genetically heterogeneous group of diseases. Major forms can be distinguished on the basis of clinical signs, age of onset, biochemical parameters or genotypes. To develop rational diagnostic strategies, phenotypic information, e.g., age of onset combined with population-specific disease frequencies could be highly favourable. We tested this hypothesis for single candidate loci and mutations in North European ataxia patients with juvenile and early adult onset. While we could prove that Friedreich ataxia (FRDA) is frequent in Germany, only few patients with ataxia-oculomotor apraxia type 1 (AOA1) and type 2 (AOA2) were diagnosed. The frequency of the mitochondrial recessive ataxia syndrome (MIRAS) and the infantile onset spinocerebellar ataxia (IOSCA) in this population remains unknown since no case with the common mutation of the corresponding gene was detected.

A novel mutation of aprataxin associated with ataxia ocular apraxia type 1: Phenotypical and genotypical characterization

Ataxia oculomotor apraxia type 1 (AOA1) is the most common form of autosomal recessive ataxia in Japan, and the second in Portugal after Friedreich ataxia. AOA1 is typically characterized by early-onset cerebellar ataxia, oculomotor apraxia, hypoalbuminemia, hypercholesterolemia and late axonal sensori-motor neuropathy. AOA1 is associated with the aprataxin gene ( APTX) encoding a protein involved in DNA repair. We characterized a novel homozygous missense mutation of APTX in a 34 year-old female patient born from consanguineous parents. The mutation, a Val230Gly caused by a c.689 T>G substitution, involved the histidine-triad (HIT) domain of the protein, affected a phylogenetically conserved amino acid and was absent in the control population. We described the clinical and neurophysiological features, the findings at structural and functional brain imaging, and the pathological picture of the sural nerve biopsy. The report emphasized the genetical and phenotypical heterogeneity of AOA1 by demonstrating atypical features such as absence of oculomotor apraxia and signs of pyramidal involvement. Expression studies by Western blotting on fibroblasts demonstrated that the homozygous Val230Gly mutation was associated with decreased levels of APTX indicating a loss-of-function mechanism.

Senataxin, defective in ataxia oculomotor apraxia type 2, is involved in the defense against oxidative DNA damage

Adefective response to DNA damage is observed in several human autosomal recessive ataxias with oculomotor apraxia, including ataxia-telangiectasia. We report that senataxin, defective in ataxia oculomotor apraxia (AOA) type 2, is a nuclear protein involved in the DNA damage response. AOA2 cells are sensitive to H2O2, camptothecin, and mitomycin C, but not to ionizing radiation, and sensitivity was rescued with full-length SETX cDNA. AOA2 cells exhibited constitutive oxidative DNA damage and enhanced chromosomal instability in response to H2O2. Rejoining of H2O2-induced DNA double-strand breaks (DSBs) was significantly reduced in AOA2 cells compared to controls, and there was no evidence for a defect in DNA single-strand break repair. This defect in DSB repair was corrected by full-length SETX cDNA. These results provide evidence that an additional member of the autosomal recessive AOA is also characterized by a defective response to DNA damage, which may contribute to the neurodegeneration seen in this syndrome.

Short half-lives of ataxia-associated aprataxin proteins in neuronal cells

Early-onset ataxia with ocular motor apraxia and hypoalbuminemia (EAOH)/ataxia with oculomotor apraxia type 1 (AOA1) is caused by mutations in the gene encoding aprataxin (APTX). Although several in vitro findings proposed that impaired enzymatic activities of APTX are responsible for EAOH/AOA1, potential instability of mutant proteins has also been suggested as the pathogenesis based on in vivo finding that mutant proteins are almost undetectable in EAOH/AOA1 tissues or cells. The present study aimed to experimentally prove instability of mutant proteins in neuronal cells, the cell type preferentially affected by this disease. Results of pulse-chase experiments demonstrated that all of the disease-associated mutants had extremely shorter half-lives than the WT. We further found that mutants were targeted for rapid proteasome-mediated degradation. These results help establish pathogenic and physiological protein characteristics of APTX in neuronal cells.

Actions of Aprataxin in Multiple DNA Repair Pathways

Mutations in the Aptx gene lead to a neurological disorder known as ataxia oculomotor apraxia-1. The product of Aptx is Aprataxin (Aptx), a DNA-binding protein that resolves abortive DNA ligation intermediates. Aprataxin catalyzes the nucleophilic release of adenylate groups covalently linked to 5' phosphate termini, resulting in termini that can again serve as substrates for DNA ligases. Here we show that Aprataxin acts preferentially on adenylated nicks and double-strand breaks rather than on single-stranded DNA. Moreover, we show that whereas the catalytic activity of Aptx resides within the HIT domain, the C-terminal zinc finger domain provides stabilizing contacts that lock the enzyme onto its high affinity AMP-DNA target site. Both domains are therefore required for efficient AMP-DNA hydrolase activity. Additionally, we find a role for Aprataxin in base excision repair, specifically in the removal of adenylates that arise from abortive ligation reactions that take place at incised abasic sites in DNA. We suggest that Aprataxin may have a general proofreading function in DNA repair, removing DNA adenylates as they arise during single-strand break repair, double-strand break repair, and in base excision repair.

DNA single‐strand break repair is impaired in aprataxin‐related ataxia

Early-onset ataxia with ocular motor apraxia and hypoalbuminemia (EAOH)/ataxia with oculomotor apraxia type 1 (AOA1) is an autosomal recessive form of cerebellar ataxia. The causative protein for EAOH/AOA1, aprataxin (APTX), interacts with X-ray repair cross-complementing 1 (XRCC1), a scaffold DNA repair protein for single-strand breaks (SSBs). The goal of this study was to prove the functional involvement of APTX in SSB repair (SSBR). We visualized the SSBR process with a recently developed laser irradiation system that allows real-time observation of SSBR proteins and with a local ultraviolet-irradiation system using a XPA-UVDE cell line that repairs DNA lesions exclusively via SSBR. APTX was knocked down using small interference RNA in the cells. Oxidative stress-induced DNA damage and cell death were assessed in EAOH fibroblasts and cerebellum. Our systems showed the XRCC1-dependent recruitment of APTX to SSBs. SSBR was impaired in APTX-knocked-down cells. Oxidative stress in EAOH fibroblasts readily induced SSBs and cell death, which were blocked by antioxidants. Accumulated oxidative DNA damage was confirmed in EAOH cerebellum. This study provides the first direct evidence for the functional involvement of APTX in SSBR and in vivo DNA damage in EAOH/AOA1, and suggests a benefit of antioxidant treatment.

Muscle coenzyme Q10 deficiencies in ataxia with oculomotor apraxia 1

APTX gene mutations responsible for ataxia-oculomotor apraxia 1 (AOA1) were identified in a family previously reported with ataxia and coenzyme Q10 (CoQ10) deficiency. We measured muscle CoQ10 levels in six patients with AOA1 and found decreased levels in five. Patients homozygous for the W279X mutation had lower values (p = 0.003). A therapeutic trial of CoQ10 may be warranted in patients with AOA1.

Senataxin, defective in ataxia oculomotor apraxia type 2, is involved in the defense against oxidative DNA damage

Senataxin, defective in ataxia oculomotor apraxia type 2, is involved in the defense against oxidative DNA damage

The appearance of aprataxin in rat cultured neuron and glia

The appearance of aprataxin in rat cultured neuron and glia

In cis autosomal dominant mutation of Senataxin associated with tremor/ataxia syndrome

Senataxin mutations are the molecular basis of two distinct syndromes: (1) ataxia oculomotor apraxia type 2 (AOA2) and (2) juvenile amyotrophic lateral sclerosis 4 (ALS4). The authors describe clinical and molecular genetic studies of mother and daughter who display symptoms of cerebellar ataxia/atrophy, oculomotor defects, and tremor. Both patients share Senataxin mutations N603D and Q653K in cis (N603D-Q653K), adjacent to an N-terminal domain thought to function in protein-protein interaction. The N-terminal and helicase domains appear to harbor missense mutation clusters associated with AOA2 and ALS4. Working synergistically, the N603D-Q653K mutations may confer a partial dominant negative effect, acting on the senataxin N-terminal, further expanding the phenotypic spectrum associated with Senataxin mutations.

Aprataxin ( APTX) gene mutations resembling multiple system atrophy

Mutations of the aprataxin ( APTX) gene cause early-onset ataxia with ocular motor apraxia and hypoalbuminemia (EAOH), also called ataxia with oculomotor apraxia type 1. Recent studies showed clinical heterogeneity in patients with EAOH. We describe 2 patients whose clinical features resembled those of multiple system atrophy of the cerebellar subtype (MSA-C) but without ocular motor apraxia and hypoalbuminemia. Each had a different nucleotide transition in the APTX gene (725G→A and 457A→G). These variants on the APTX gene exhibit phenotypic variability.