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Neurodegenarative comes from the prefix "neuro" which means nerve cells (neuron) and "degeneration" which refers to a process of losing structure and function of any tissues or organ (Przedborski et al., 2003). Neurodegenerative diseases are a heterogeneous group of disorders characterized by gradually progressive, selective loss of anatomically or physiologically related neuronal system (Przedborski et al., 2003). Some examples of the most common inherited neurodegenerative diseases are Alzheimer disease (AD), Parkinson disease (PD), Huntington disease (HD) and Amyotrophic Lateral Sclerosis (ALS) (Bossy-Wetzel et al., 2004). Less common inherited neurodegenerative diseases include Ataxia-telangiectasia (A-T), Ataxia-telangiectasia like disorder (A-TLD), Ataxia oculomotor apraxia type 1 (AOA1), Ataxia oculomotor apraxia type 2 (AOA2) and the recently described, Ataxia oculomotor apraxia type 3 (AOA3) (Gueven et al., 2007, Suraweera et al., 2007).
Based on the phenotypic features, neurodegenerative diseases can be divided into two categories which involve problems in movements or ataxia and conditions triggering memory loss such as dementia. Ataxia implies dysfunction of central nervous system (CNS) controlling muscle movement and can be further classified according to affected brain regions eg.the cerebral cortex, the basal ganglia, the brainstem and cerebellum or the spinal cord. Dementia is defined as progressive decline in cognitive function due to damage or disease in the brain beyond that expected from normal ageing processes. Memory, attention, language and problem solving are normally affected. Dementia can be divided into two groups depending on the area of the affected brain. First, is the cortical dementia which may lead to AD, Vascular dementia, or Dementia with Lewy Bodies (DLB). Subcortical dementia includes Hungtinton disease, Hypothyroidism, Parkinson disease, and deficiency in Vitamin B1, B2 and folate. Aspects of dementia can be observed in at least 50 different diseases (Tomlinson, 1977).
The number of neurodegenerative diseases is estimated to be several hundred and many of these show clinical and pathological overlapping (Burn & Jaros, 2001). Furthermore, these diseases often involve multisystem atrophy where several areas of the brain are affected, combinations of lesions giving rise to different clinical pictures and rendering the classification of neurodegenerative diseases challenging and early diagnosis difficult.
Ataxias are a subgroup of neurodegenerative diseases defined by unsteady and clumsy motion of the limbs and torso (http://neuromuscular.wustl.edu). The clinical manifestation of ataxia is caused by dysfunction of the cerebellum, a part of the brain which is important for movement coordination. Ataxias can be divided into hereditary and acquired ataxia (http://neuromuscular.wustl.edu). Hereditary ataxia includes autosomal dominant, autosomal recessive, X-linked, congenital DNA repair defects, metabolic disorders and mitochondrial multisystem disorders (http://neuromuscular.wustl.edu). The most common autosomal recessive cerebellar ataxias include Autosomal Recessive Cerebellar Ataxia Type 1 (ARCA1), Friedreich's ataxia and A-T (Di Donato et al., 2001). Other forms of Autosomal Recessive Cerebellar Ataxia (ARCA) such as A-TLD, AOA1, AOA2, AOA3, Ataxia with vitamin E deficiency, Spastic ataxia of Charlvoix-Saguenay and Infantile early-onset spinocerebellar ataxia have also been described (Di Donato et al., 2001).
Autosomal Recessive Cerebellar Ataxia (ARCA)
Heterogenous ARCA are characterized by degeneration of the cerebellum and spinal cord, autosomal recessive inheritance and generally occur at an early onset (before 20 years old) (Palau & Espinos, 2006). This group includes a large number of rare diseases but amongst these, Friedreich's ataxia and A-T are the most frequent with a prevalence of 1 in 30, 000 to 50, 000 (Delatycki et al., 2000) and 1 in 100, 000 (Schulz et al., 2009) individuals respectively. Although most cases of ARCA are caused by mutations in specific genes, others result from mutations at multiple loci, therefore contributing to the genetic heterogeneity. Table 1.1 shows a classification of ARCA disorders into four categories: congenital ataxias, metabolic ataxias, DNA repair defects and degenerative ataxias.
Table 1.1 Genetic data on ARCA Disorders
- JBTS1 (cerebelloparenchymal disorder IV, CPD IV) JBST1 9q34
- JBTS2 (CORS2) JBST2 11p12-p13.3
- JBTS3 AHII 6q23
- JBTS4 NPHPI 2q13
- JBTS5 Necrocystin6 12q21.32
Cayman ataxia Cayataxin (ATCAY) 19p13.3
Ataxia with isolated vitamin E deficiency (AVED) a-TTP 8q13
Abetalipoproteinemia MTP 4q22-q24 Cerebrotendinous xanthomatosis CYP27 2q33-qter
Refsum disease PhyH 10pter-p11.2
ARCA2 ADCK3 1q42.3
DNA repair defects
Ataxia-telangiectasia (A-T) ATM 11q22.3
Ataxia-telangiectasia-like disorder (ATLD) MRE11A 11q21
Ataxia with oculomotor apraxia 1 (AOA1) APTX 9p13
Ataxia with oculomotor apraxia 2 (AOA2) SETX 9q34
Spinocerebellar ataxia with axonal neuropathy (SCAN1) TDP1 14q31
Xeroderma pigmentosum (XP)
XP of complementation group A XPA 9q22.3
XP of complementation group B XPB/ERCC3 2q21
XP of complementation group C XPC 3p25
XP of complementation group D XPB/ERCC2 19q13.2-q13.3
XP of complementation group E XPE (DDB2) 11p12-p11
XP of complementation group F XPF/ERCC4 16p13.3-p13.3
XP of complementation group G XPB/ERCC5 13q32-q33
XP variant (XPV) or XP with normal DNA repair rates POLH 6p21.1-p12
Friedreich's ataxia FRDA or FXN 9q13
Mitochondrial recessive ataxic syndrome (MIRAS) POLG
Charlevoix-Saguenay spastic ataxia SACS 13q12
Early onset cerebellar ataxia with retained tendon reflexes ? 13q11-12
Infantile onset spinocerebellar ataxia (IOSCA) CI0orf2 10q22.3-q24.1
Marinesco-Sjogren syndrome SILI 5q32
Classical MSS ?
MSS with myoglobinuria ? 18qter
Coenzyme Q10 deficiency with cerebellar ataxia ?
Posterior column ataxia with retinitis pigmentosa (PCARP) AXPCI 1q31
1.3.1 Friedreich's ataxia
Friedreich's ataxia (FRDA) which was first described by Nicolaus Friedreich, a German pathologist, is inherited as an autosomal recessive trait and was first described in nine individuals, two females and seven males of three sibships (Morrison & Harding, 1994). The frequency of FRDA is estimated to be about 1/50, 000 individuals and is characterized by cardiac failure due to impaired ATP synthesis in cardiac muscles (Lodi et al., 2006). Friedreich observed that the onset of the disease was around puberty defined by the presence of ataxia, dysarthria, sensory loss, muscle weakness, scoliosis, foot deformity, cardiac symptoms and tendon reflexes. Most patients show absence of reflexes in upper limbs, distal loss of joint position and sense in lower limbs, scoliosis, and abnormal electrocardiogram suggesting the presence of cardiomyopathy (Morrison & Harding, 1994). Other clinical signs include pes cavus, nysgtagmus, optic atrophy, deafness and diabetes mellitus (Morrison & Harding, 1994).
Mutations in the FRDA gene which mapped to chromosome 9q13 have been found to be the cause of Friedreich's ataxia (Chamberlain et al., 1988). The FRDA gene spans 80 kb and consists of seven exons which encode a 210 amino acids protein known as frataxin (Campuzano et al., 1996). In humans, frataxin mRNA is found to be abundant in the heart, spinal cord and developing brain (Trushina & McMurray, 2007). Frataxin is a soluble protein with unknown function that localizes in the mitochondrial internal membrane and mitochondrial matrix (Koutnikova et al., 1998). Studies in yeast, Saccharomyces cerevisiae, have shown that frataxin is involved in iron transport (Babcock et al., 1997), iron-sulfur cluster (ISC) biosynthesis (Rotig et al., 1997), iron storage (Adamec et al., 2000), as an antioxidant (Chantrel-Groussard et al., 2001) and in the stimulation of oxidative phosphorylation (Ristow et al., 2000).
The most frequent mutation is a GAA trinucleotiode repeat expansion (67 - 1700) which is located in the first intron of the FRDA gene. These expanded GAA repeats result in the inhibition of the FRDA gene expression (Christodoulou et al., 2001).
1.3.2 Ataxia-Telangiectasia (A-T)
A-T, a progressive neurological disorder resulting from cerebellar degeneration is inherited in an autosomal recessive manner. It is a multisystem disorder characterized by progressive cerebellar ataxia, oculomotor apraxia, oculocutaneous telangiectasia, recurrent sinopulmonary infections, a variable immunodeficiency state with involvement of cellular and humoral immunity, high risk of malignancy (especially leukemia and lymphoma) and enhanced sensitivity to ionizing radiation (Palau & Espinos, 2006). Onset starts usually at an age when the child requires to walk, with ataxia of both upper and lower limbs. By the early teenage years, most patients require a wheelchair for mobility. Patients with A-T also show thymic hypoplasia, high serum concentration of a-fetoprotein (AFP), growth retardation, and telangiectasia in various parts of the body, most noticeably in the bulbar conjunctiva (Gatti et al., 1991).
A-T is caused by mutation in the ATM gene (McKinnon, 2004) that spans about 150kb of genomic DNA, and encodes a ubiquitously expressed transcript of approximately 13kb, with 66 exons giving rise to a 350 kDa protein of 3056 amino acids (Savitsky et al., 1995). More than 200 distinct mutations have been reported associated with A-T. Almost all mutations involve the coding region and are distributed throughout the gene. Most of the patients are compound heterozygous for mutations resulting in truncated proteins, although missense mutations that alter the protein function have also been described (Becker-Catania et al., 2000).
The ATM protein has a central role in the cellular response to DNA damage (Lavin et al., 2006). ATM is a serine-threonine protein kinase that undergoes autophosphorylation after DNA damage and initiates a phosphorylation cascade of multiple substrates involved in cell cycle control including p53, breast-cancer-associated 1 (BRCA1), p53-binding protein 1 (53BP1) and the checkpoint kinase (CHK2) (Figure 1.1) (Kastan & Lim, 2000, Shiloh, 1997). The Mre11/Nbs/Rad50 (MRN) complex is required for the activation of ATM (Lee & Paull, 2004). Mutations in the NBS1 and MRE11 genes cause diseases known as Nijmegen breakage syndrome (NBS) and Ataxia-Telangiectasia like disorder (A-TLD), both of which show some overlapping features to A-T.
Figure 1.1 A model of ATM activation by DNA double strand break (DSBs) and other forms of DNA damage. DSBs and other forms of DNA damage are detected by the MRN complex (Mre11, Rad50 and Nbs1). ATM is activated via its autophosphorylation on serine 1981, resulting in the conversion of ATM from an inactive dimer to an active monomer form. ATM activation subsequently leads to the phosphorylation of downstream targets that include proteins involved in DNA repair (dark blue), in apoptosis (green), in the G1/S checkpoint (violet), in the intra S-phase checkpoint (yellow), in the G2/M checkpoint (light blue) and in gene regulation/translation (beige) [Adapted from (Kurz & Lees-Miller, 2004)].
1.3.3 Ataxia-Telangiectasia related disorders
22.214.171.124 Ataxia-Telangiectasia-Like Disorder (A-TLD)
Ataxia-Telangiectasia Like Disorder (A-TLD) is an autosomal recessive syndrome caused by mutations in the MRE11 gene (Taylor & Byrd, 2005). Given that the MRE11 gene is located near the ATM gene at chromosome 11q, it is difficult to distinguish between A-T and A-TLD (Palau & Espinos, 2006). Individuals with A-TLD are characterized by neurodegeneration, but less severe than A-T with the absence of telangiectasia and increased levels of AFP (Taylor & Byrd, 2005). The cells from A-TLD patients show increased sensitivity to ionizing radiation, chromosomal instability, defective induction of stress-activated signal transduction pathways and radioresistant DNA synthesis (Palau & Espinos, 2006). The MRE 11 gene encodes for the MRE11 protein, one of the components of the MRE11/RAD50/NBS (MRN complex), a highly conserved protein which plays a major role in DNA double strand break repair involving homologous recombinant repair (HRR) and non homologous end joining (NHEJ) (Lamarche et al., 2010). The MRN complex is also involved in telomere maintenance and in DNA replication (Kavitha et al., 2010). In the presence of DNA double stand break, the MRN complex acts as a sensor which is required for the recruitment of the ataxia telangiectasia mutated (ATM) protein to sites of DNA breaks and subsequently get activated (Lee et al., 2010). Thus, mutation in the MRE11 gene could disrupt the MRN complex, its function and subsequently the DNA repair mechanisms which would lead to disease state (Taylor & Byrd, 2005).
126.96.36.199 Nijmegen Breakage Syndrome (NBS)
Mutations in the NBN gene lead to the NBS syndrome, which is characterized by microcephaly, growth retardation, immunodeficiency, radiosensitivity and cancer predisposition (Taylor & Byrd, 2005). The NBS syndrome shows clinical overlapping with A-T particularly in radiosensitivity, immunodeficiency and increased risk of lymphoma (Taylor & Byrd, 2005). Mental retardation was also observed in NBS patients but there was no cerebellar degeneration or telangiectasia (Taylor & Byrd, 2005). NBS is associated with defective DNA damage response mechanisms since NBN is part of the MRN complex which is involved in the response to DNA double strand break breaks (DSBs) generated by both endogenous and environmental factors such as ionizing radiation (di Masi & Antoccia, 2008, Carney et al., 1998).
188.8.131.52 Ataxia Oculomotor Apraxia Type 1 (AOA1)
Aicardi and colleagues (1988) were first to describe AOA diseases in 14 patients with slowly progressive ataxia, choreoathetosis and ocular motor apraxia (Aicardi et al., 1988). The characteristics of these diseases were similar to A-T but could be differentiated by late onset symptoms and absence of multisystem involvement. AOA1 was originally reported in Portugal and Japan (Le Ber et al., 2003, Shimazaki et al., 2002). In Japan, AOA1 is the most frequent recessive ataxia while in Portugal it is ranked second after Friedreich ataxia (Barbot et al., 2001). AOA1 is characterized by early onset gait ataxia that develops between 2 and 6 years of age, dysarthria, limb dysmetria (later in the disease course), oculomotor apraxia, distal and symmetric muscle weakness, mild loss of vibration and joint position sense, and slow progression (Palau & Espinos, 2006). Dystonia, masked facies, and mental retardation also have been reported in some patients (Palau & Espinos, 2006). Magnetic resonance image (MRI) scans of AOA1 patients demonstrated, motor and sensory axonal neuropathy, mild loss of large myelinated axons and cerebellar and brainstem atrophy (Le Ber et al., 2003). Hypoalbuminemia and hypercholesterolaemia are also present in patients with AOA1 (Palau & Espinos, 2006).
AOA1 is caused by mutations in the APTX gene, located on chromosome 9p13.3, which contains seven exons and codes for aprataxin (Date et al., 2001, Moreira et al., 2001). Aprataxin is a nuclear protein composed of three domains: an N-terminal forkhead-associated domain (FHA), a histidine-triad (HIT) domain and a zinc-finger domain. Aprataxin interacts with proteins involved in the repair of DNA breaks and was shown to be involved in the repair of DNA single strand breaks by resolving abortive DNA ligations (Gueven et al., 2004, Clements et al., 2004, Lou et al., 2004, Hirano et al., 2007, Ahel et al., 2006). In addition, aprataxin was found to interact with proteins involved in the repair of DNA double strand breaks suggesting a possible role in processing double stranded DNA ends (Rass et al., 2007, Clements et al., 2004, Becherel et al., 2009).
184.108.40.206 Ataxia Oculomotor Apraxia Type 2 (AOA2)
Ataxia with oculomotor apraxia type 2 (AOA2) also referred as non-Friedreich spinocerebellar ataxia type 1 (SCAR1), is an autosomal recessive disorder that represents approximately 8% of non-Friedreich ARCA (Le Ber et al., 2003). The SETX gene which has recently been characterized and mapped to chromosome 9q34, encodes a 2 677 amino acids protein, senataxin. Mutations of the SETX gene associated with AOA2 were first reported by Moreira and colleagues (Moreira et al., 2001).
A superfamily I DNA/RNA helicase domain at the C-terminus region (residues 1931 - 2456) of senataxin was the only conserved domain in this gene (Moreira et al., 2004, Chen et al., 2004). This domain shows significant homology to the helicase domain of human proteins, Immunoglobulin Mu-Binding Protein 2 (IGHMBP2) and Regulator of Nonsense Transcripts-1 (RENT1) proteins and with 42% and 46% of homology respectively (Chen et al., 2004, Moreira et al., 2004). The presence of a helicase domain in SETX gene suggests that senataxin might play a role in nucleic acid metabolism. DNA/RNA helicases play major roles in DNA/RNA processing including: DNA repair, replication, recombination, transcription, RNA processing, transcript stability and translation initiation (Tanner & Linder, 2001). The helicase domain also shows strong homology with the budding yeast protein, splicing endonuclease 1 (Sen1p). Sen1p is involved in the processing of non-protein encoding RNAs which are important for growth (Ursic et al., 1997). A schematic diagram of the senataxin protein is illustrated in figure 1.2. The protein-protein interaction domain of senataxin protein is located at the N-terminal. Senataxin interacts with several proteins involved in RNA metabolisms including nucleolin, heterogenous ribonucleoprotein M, poly(A) binding protein 1, spliceosomal protein 155, RNA polymerase II and survival motor neuron protein (Suraweera et al., 2007). A nuclear localization signal is been located at the extreme C-terminal which contains 17 amino acid sequences (Chen et al., 2006).
Figure 1.2 A schematic map of the Senataxin protein domains. Senataxin protein contains 2677 amino acids, the N-terminal consists of 600 amino acids protein-protein interaction domain and C-terminal is indicated by the RNA/DNA superfamily 1 helicase domain. The nuclear localization signal is located at extreme of the C-terminal which consists of 17 amino acids (Chen et al., 2004).
AOA2 is characterized by spinocerebellar ataxia with an onset between 11 and 22 years of age, cerebellar atrophy, choreoathetosis, and a dystonic posturing of walking. Occasionally it is associated with oculomotor apraxia, and elevated levels of Î³-globulin, a fetoprotein, creatinine kinase (CK), as well as hypoalbuminemia and hypercholesterolaemia. A total of 15 different mutations were described in the original AOA2 report, 10 of which caused premature protein termination (Moreira et al., 2001). Mutations in the SETX gene also have been identified in the autosomal dominant form of juvenile myotrophic lateral sclerosis, known as ALS4 (Chen et al., 2004). In contrast to AOA2, ALS is a disease which involves regions of the nervous system that controls voluntary muscle movement (Morrison & Harding, 1994). The hallmark of ALS is the progressive muscle weakness resulting from the degeneration of motor neurons due to atrophy and spasticity. This is correlated with the degeneration and death of upper and lower motor neurons in the brain and spinal cord (Morrison & Harding, 1994).
220.127.116.11 Autosomal Recessive Cerebellar Ataxia Type 2 (ARCA2)
Autosomal Recessive Cerebellar Ataxia Type 2 (ARCA2) is a novel form of ARCA caused by mutations in the aarF-domain containing kinase3 gene (ADCK3) also known as chaperone activity of the bc1 (CABC1) complex gene (Lagier-Tourenne et al., 2008). ADCK3 is a mitochondrial protein homologous to COQ8 in yeast and UbiB proteins in bacteria which are important for the biosynthesis of CoQ (Lagier-Tourenne et al., 2008). A SNP-based genome-wide scan identified the locus on chromosome 1q41-q42 and a homozygous splice-site mutation was causative of ARCA2 (Lagier-Tourenne et al., 2008). The linkage study was performed within a large consanguineous Algerian family (4 individuals with childhood onset cerebellar ataxia). Using 10K SNP arrays, all affected individuals showed a unique region of homozygosity whereas the healthy sibling was heterozygous at chromosome 1q41-q42 which was confirmed by a dense set of microsatellite markers (Lagier-Tourenne et al., 2008). Four genes encoding mitochondrial proteins have been identified to cause recessive ataxia when defective (Nikali et al., 2005). Sequencing analysis revealed a homozygous donor splice-site mutation (c.1398+2T®C) in intron 11 of ADCK3 (NM_020247; aarF-domain containing kinase 3 or ADCK3 [MIM 606980]. Three splice variants were also identified from the mutant allele using RT-PCR analysis. Exon 10 was skipped resulting in a frameshift mutation (p.Asp420TrpfsX40) in the shorter variant. Other variants were identified in exon 11 resulting in insertions of 68 and 70 nucleotides with a stop codon after 21 residues (p.Ile467AlafsX22). Using homozygosity mapping at 1q41-q42 and direct sequencing of ADCK3 exons and flanking sequences, six additional mutations were identified. These includes one single-amino acid deletion, two truncating mutation, three missense mutations) in four sporadic cases of non-Friedreich ataxia.
All affected individuals with mutations in this gene had childhood onset cerebellar ataxia with slow progression, cerebellar atrophy and three of six individuals showed elevated lactate levels. Table 1.2 describes the clinical and biochemical features of ARCA2 patients, and table 1.3 highlights the coenzyme Q10 levels and respiratory chain enzyme activities in these patients.
Table 1.2 ADCK3 gene mutations and clinical features of ARCA2 patient cells (Lagier-Tourenne et al., 2008)
c.1398+2T â†’ C
c.[993Câ†’ T] + [1645G â†’ A]
Exons 8 and 14
Predicted amino acid change
p.[Lys314_Gln360 del] + [Gly549Ser]
Age onset (years)
Disease duration (years)
None to mild
Table 1.3 Coenzyme Q10 and respiratory chain enzyme activities in ARCA2 patients (Lagier-Tourenne et al., 2008)
CoQ levels in fibroblasts (ng/mg protein)
58.5 + 4.1, n=15
CoQ levels in lymphoblast
62.2 + 2.8, n=3
CoQ10 biosynthesis assay in fibroblast
(CoQ10 DPM/mg prot/day)
4108 + 52
2006 + 15
3569 + 255, n=5
CI + CIII/COX
13.86 + 1.27, n=3
CI + CIII/CS
8.03 + 0.67, n=3
CI + CIII/CS fold increase after addition of CoQ2
1.26 + 0.07, n=3
CII + CIII/CS
0.46 + 0.02, n=2
0.81 + 0.18, n=2
Abnormal values are shown in bold. Values are given as means + standard error of the mean.
Abbreviations: CI, complex I (NADH ubiquinone oxidoreductase); CII, complex II (succinate ubiquinone oxidoreductase); CIII, complex III (ubiquinol cytochrome c oxidoreductase); COX, cytochrome c oxidase; CS, citrate synthase; CoQ2, coenzyme Q2 (strong rescue by CoQ2 is indicative of CoQ deficiency); DPM, decays per min.
In another study, ADCK3 gene mutations led to ubiquinone deficiency with cerebellar ataxia and seizures (Mollet et al., 2008). Four mutations were identified including a homozygous G to A transition at nucleotide 1655 in exon 14 resulting in E551K, a compound heterozygous mutation which involved a C to T transition at nucleotide 636 (R213W) and a G to T transversion at nucleotide 815 (G272V), and finally compound heterozygotes for one missense G to A transition at nucleotide 815 (G272D) and one base pair insertion, 1812_1813insG. These mutations were identified in children with respiratory chain deficiency and correlated with reduced ubiquinone levels in muscle tissue. The clinical characteristics of these patients were similar, and included muscle weakness, abnormal exercise fatigability (between 18 months to 3 years) and cerebellar ataxia. In addition, MRI scans also indicated cerebellar atrophy. The patients displayed various degrees of deficiency in mitochondrial respiratory enzymes activities as shown in table 1.4.
Table 1.4 Respiratory-chain enzyme activities in muscle mitochondria and cultured skin fibroblasts (Mollet et al., 2008)
Activities (nmol/min/mg protein)
Malate+ glutamate oxidase
Malate + pyruvate oxidase
CI + III
CII + III
CII + III activation by DQ
80 + 30
80 + 30
80 + 30
80 + 30
80 + 30
80 + 30
59.5 + 12.2
11.9 + 2.3
73 + 15
98 + 20
1458 + 257
740 + 146
335 + 68
80 + 30
22 + 7
107 + 7
56 + 17
17 + 10
18 + 7
153 + 35
41 + 5
74 + 8
762 + 103
384 + 44
110 + 14
138 + 16
238.5 + 25.6
CII + III/CS
CIV/CII + III
CII + III/CII
80 + 30
80 + 30
80 + 30
3.2 + 0.3
2.5 + 0.2
1.7 + 0.2
0.58 + 0.3
2.0 + 0.2
2.1 + 0.2
Enzyme activities on muscle mitochondria have been determined in two different laboratories for patients 1 and 2. Abnormal values are indicated with an asterisk. P1 (E551K), P2 (R231W/G272V)
aPreviously published by(Aure et al., 2004).
The aarF-domain containing kinase3 (ADCK3), a novel gene which encodes for a mitochondrial protein, was isolated in 2002 (Figure 1.3) (Iiizumi et al., 2002). ADCK3 shows similarity in amino acids sequence with ABC1, a member of an electron-transferring membrane protein complex of lower eukaryotes such as in S. Pombe, A. Thaliana and S. Cerevisiae (Iiizumi et al., 2002). ABC1 is important for proper conformation and functioning of the bc1 complex and the neigbouring complexes (CI and CIV) in the electron transport chain (ETC) (Iiizumi et al., 2002). Similarity between ADCK3 and ABC1 proteins suggest an important role for ADCK3 in the ETC where ADCK3 could alter the electrochemical gradient through its effect on complex formation (Iiizumi et al., 2002). This may lead to changes in the ETC and mitochondrial membrane potential (MMP) which is important for apoptosis via the mitochondrial-dependant pathway.
In humans, ADCK3 plays an important role in the p53-inducible mitochondrial apoptotic pathway (Iiizumi et al., 2002). p53 is a tumor suppressor protein that plays a major role in protecting cells against malignant transformation (Levine, 1997). Almost half of human cancers are associated with functional loss of p53 (Levine, 1997). In response to cellular stresses such as DNA damaging agents, p53 is stabilized by post-translational modifications that include phosphorylations of serines residues and acetylations of lysine residues (Lowe et al., 1993). p53 is also a transcription factor that inhibits cell growth by activating transcription of multiple genes involved in cell cycle arrest such as p21Waf1 and/or apoptosis. Iiizumi showed that ADCK3 was induced after transfection with adenovirus vectors with wild type-p53 (Adp53) into p53-defective cells, U373MG (Iiizumi et al., 2002). Treatments with various genotoxic agents also induce expression of ADCK3. Overexpression of exogenous ADCK3 in two cell lines with reduced wild type p53 resulted in decreased numbers of colonies. This is in agreement with subsequent analysis, where the use of antisense ADCK3 oligonucleotides resulted in the inhibition of p53-induced apoptosis. Together these data indicate a role for ADCK3 in p53-mediated apoptosis.
Figure 1.3 AarF-domain containing kinase3 (ADCK3) gene. ADCK3 gene is located at 1q42.2. ADCK3 has been shown to encode a mitochondrial protein that is closely related to the yeast activity of bc1 complex. The ADCK3 gene contains 15 exons that give rise to a 47 kb transcript and a 647 amino acids protein (Iiizumi et al., 2002).
1.4 Ataxia Oculomotor Apraxia Type 3 (AOA3)
AOA3 is a novel form of AOA that was first described in a single Australian patient by Gueven and colleagues from the Queensland Institute of Medical Research (QIMR) (Gueven et al., 2006, Gueven et al., 2007). Briefly, cells from this patient showed hypersensitivity to several DNA damaging agents, specifically to those inducing DNA single strand breaks (SSB). Increased levels of Poly (ADP-ribose) polymerase (PARP-1) auto-poly (ADP-ribosylation) suggested the presence of residual SSBs in DNA from the patient (Gueven et al., 2007). However, normal repair of these breaks was observed (Gueven et al., 2007). Increased reactive oxygen species/reactive nitrogen species (ROS/RNS) and high levels of oxidative DNA damage were observed in these cells. There was also evidence of increased protein damage and lipid peroxidation. The mode of cell death in AOA3 cells was not by apoptosis. This was explained by a defective mitochondrial membrane depolarization after DNA damage, reduced cytochrome C release and defective translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus. These data suggest possible mitochondrial dysfunction which is supported by the reduced levels of ATP observed in these cells. The clinical, molecular and cellular characteristics of this novel AOA will be described extensively in the following sections (Gueven et al., 2006, Gueven et al., 2007).
1.4.1 Clinical features
The AOA3 patient is a boy and first child of unrelated parents from Eastern European background. There was no family history of cancer or neurological disorders. Clinical features of this patient include progressive ataxia, oculomotor apraxia and childhood onset. Head CT scan at 1 year was normal. The patient had "jerky" eye movements at the age of 8 years. There was no nystagmus but there was a bilateral finger-nose ataxia. MRI scans showed a progressive pan-cerebellar atrophy over a 4 years period. In addition, a mild brainstem atrophy was also observed.
Full blood parameters were normal. These included full blood count, creatinine, urea, electrolytes, calcium, magnesium, phosphate, liver function tests including serum albumin, cholesterol and triglycerides, a-feotoprotein, transferring isoforms, lysosomal enzyme analysis, vitamin E levels, copper, ceruloplasmin, lactate and pyruvate. A metabolic screen of urine was normal. No trinucleotide expansions were detected during the testing for spinocerebellar ataxias 1,2,3,6 and 7. At the age of 10 years, nerve conduction studies indicated normal right sural and common peroneal nerves. Electrocardiogram (ECG) and echocardiogram were normal. A comparison of the clinical and laboratory features between different forms of AOA is detailed in Table 1.5. The onset of AOA3 disease is at the first decade of life similar to that of A-T, A-TLD and AOA1 but different from that of AOA2 which usually occurs in the second decade of life. The disease progression in AOA3 is slow, comparable to A-TLD, AOA1 and AOA2, in contrast to A-T which is faster. All of these diseases share common neurological features that include ataxia, ocular motor apraxia and cerebellar atrophy. Chorea/dystonia was present in A-T, A-TLD, AOA1 and AOA2 but not in AOA3. Only A-T and several cases of AOA2 showed telangiectasia. Axonal neuropathy was present in A-T, AOA1, AOA2 but absence in A-TLD and AOA3. Immunodeficiency is associated with A-T and A-TLD, but not in any of the AOA diseases. Alpha-fetoprotein was high in A-T and AOA1, mildly elevated in AOA3 and showed normal levels in A-TLD and AOA3. AOA1 and several cases of AOA2 patients showed increased levels of cholesterol. A-T, A-TLD and AOA3 patients showed normal cholesterol levels. AOA3 patient showed normal albumin comparable to A-T and A-TLD, whereas AOA1 and AOA3 showed hypoalbuminemia.
Table 1.5 Comparison of the clinical and laboratory features between different forms of AOA (Gueven et al., 2007)
Ataxia Telangiectasia Like Disorder
Ataxia Oculomotor Apraxia type 1
Ataxia Oculomotor Apraxia type 2
Ataxia Oculomotor Apraxia type 3 (AOA3)
Ocular motor apraxia
Ocular motor apraxia +/-
Ocular motor apraxia
Cerebellar atrophy & mild brainstem atrophy
(fungal infection ?)
Reduced IgE, IgA, IgG2
Rare increase of Ig
Alpha-fetoprotein mildly elevated
1.4.2 Molecular and cellular characteristics of AOA3
Screening for germline mutations for all coding exons of the ATM and APTX genes was performed using polymerase chain reaction (PCR) and denaturing high performance liquid chromatography (DHPLC). Full-length SETX cDNA was amplified and sequenced for mutational screening. No mutations were identified in the ATM gene and there was normal ATM activation in response to ionizing radiation (Gueven et al., 2006). Levels of ATM protein and the MRN complex were also normal (Gueven et al., 2006). Sequencing of the SETX gene revealed no mutation and senataxin protein was expressed at normal levels. PCR and DHPLC of APTX gene also revealed the absence of mutation, and aprataxin protein levels were also normal when compared to control cell lines. This suggested that AOA3 did not result from mutations in ATM, SETX, APTX genes and that AOA3 is genetically distinct disorder. 18.104.22.168 Mutational Analysis
22.214.171.124 Sensitivity to DNA damaging agents and chromosomal aberrations
The phenotype of the AOA3 patient resembles that of A-T, A-TLD, AOA1 and AOA2. Thus it was possible that AOA3 cells would also be sensitive to DNA damaging agents. Treatment of AOA3 cells with DNA damaging agents such as camptothecin (CPT), etoposide, mitomycin C (MMC), methylmethanesulfonate (MMS), methylnitrosoguanidine (MNNG), hydrogen peroxide (H2O2) and ionizing radiation were employed to determine the sensitivity these cells. AOA3 cells displayed increased sensitivity to hydrogen peroxide (H2O2) and intermediate sensitivity to ionizing radiation compared to controls (Gueven et al., 2007). A normal response to CPT and MMS was also observed. As a comparison, A-T cells showed a normal response to H2O2 but extreme sensitivity to ionizing radiation. Finally, both A-T and AOA3 cells were sensitive to MMC, etoposide and MNNG.
Radiation-induced chromosome aberrations (ICA) were determined to investigate chromosomal instability after exposure to DNA damaging agents. AOA3 cells showed double the number of ICA compared to control cells but were intermediate when compared to A-T cells (Gueven et al., 2007). AOA3 cells had double the number of MMC-induced ICAs compared to controls (Gueven et al., 2007). The father, mother and one of the siblings of the AOA3 patient also displayed elevated ICAs as compared to control (Gueven et al., 2007).
126.96.36.199 ATM activation and DNA damage signaling
ATM is one of the key players of the DNA damage response, which signals DNA damage to the DNA repair and cell cycle machineries to minimize genome instability. In normal conditions, ATM is present as an inactive dimer in the nucleus. Upon exposure to damaging agents that induce DNA DSB, ATM becomes activated by autophosphorylation on Ser1981 and other sites (Kurz & Lees-Miller, 2004). Autophosphorlyation causes the dissociation of the ATM dimer to form active monomeric ATM molecules which initiate the downstream phosphorylation of multiple substrates involved in DNA repair and cell cycle control (Bakkenist & Kastan, 2003).
AOA3 cells showed normal ATM activation as measured by in vitro phosphorylation of GST-p531-44 fragments in ATM kinase assays when compared to control cells. The autophosphorylation of Ser1981 in AOA3 cells was comparable to that of control cells (Gueven et al., 2007). Phosphorylation of histone gH2AX, an early event after ATM activation showed a response in AOA3 cells similar to control cells (Burma et al., 2001, Gueven et al., 2006). After DNA damage, ATM is rapidly recruited by the MRE11-Rad50-Nbs1 (MRN) complex at sites of DNA double strand breaks which subsequently phosphorylates Nbs1 (NBN), a member of this complex, to assist downstream signaling (Cerosaletti & Concannon, 2004).
Nbs1/NBN phosphorylation after DNA damage detected as a shift in electrophoretic migration was observed in AOA3 cells and was comparable to the other family members and control cells. Chk2 another ATM substrate normally activated in response to ionizing radiation phosphorylates cdc25c. Again, AOA3 cells showed normal radiation-induced phosphorylation of cdc25c by invitro kinase assay (Gueven et al., 2006).
In summary, AOA3 showed normal ATM activation and phosphorylation of downstream targets, in agreement with normal ATM protein levels and the absence of mutation in the ATM gene.
188.8.131.52 Defective p53 stabilization in AOA3
The p53 protein plays a critical role in maintaining the integrity of the genome (Liu & Kulesz-Martin, 2001). It is an important component for cell cycle progression and apoptosis after DNA damage (Liu & Kulesz-Martin, 2001). p53 activity is tightly controlled in normal cells, however, in response to DNA damage such as ionizing radiation (Kastan et al., 1991), UV light (Maltzman & Czyzyk, 1984) and ribonucleotide depletion (Linke et al., 1996), p53 is rapidly induced and stabilized. Once induced, p53 acts as a transcription factor and regulate the expression of more than 20 genes (el-Deiry, 1998). These genes include p21 in the G1/S phase checkpoint, 14-3-3s in the G2/M checkpoint, BAX, PUMA, NOXA and p53-induced genes (PIGs) in apoptosis, GADD45 and XPE in DNA repair (Liu & Kulesz-Martin, 2001).
AOA3 cells displayed defective radiation-induced p53 stabilization (Gueven et al., 2006). Phosphorylation of Ser 15 and Ser20, and Lys 382 acetylation are early events in the process of p53 stabilization (Kastan et al., 1991, Lavin & Gueven, 2006). These cells also demonstrated reduced phosphorylation of p53-Ser15 which confirmed the defect in p53 stabilization. In addition, p53 was inefficiently stabilized in these cells as shown by a reduced signal of Ser20 phosphorylation and Lys382 acetylation.
184.108.40.206 Defective p53 effector genes in AOA3
p53 acts as a transcription factor, induces the expression of various genes important for DNA damage signaling, cell cycle control and apoptosis (Lavin & Gueven, 2006). Mdm2, initially identified in the mouse tumorigenic cell line 3T3DM, plays a key role in regulating p53 protein levels by modulating p53 (Lakin & Jackson, 1999). Interaction of Mdm2 with p53 leads to ubiquitination of p53 and its targeting for proteasome degradation (Haupt et al., 1996). The peptidyl-prolyl-isomerase (Pin1), another p53-binding protein also regulates the function and stability of p53. Interaction between Pin1 and p53 leads to conformational changes of the p53 which supports its biological function (Zacchi et al., 2002).
Induction of a number of p53 effector genes was determined to investigate whether the p53 defect observed in AOA3 cells was associated with its transcription factor activity. Induction of the p21 and Mdm2, two downstream targets of p53, were significantly reduced in AOA3 compared to control cells after exposure to ionizing radiations (Gueven et al., 2006). Since the interaction of p53 with Mdm2 is the major mechanism to control its stability and its targeting for proteosome degradation, it was hypothesized that AOA3 cells may have an abnormal p53-Mdm2 interaction. A constitutive interaction between Mdm2 and p53 was observed in control cells by co-immunoprecipitation and this association was lost after irradiation as expected. However, irradiation had only a minimal effect on the dissociation of p53-Mdm2 interaction in AOA3 cells suggesting that the mechanisms involved in releasing p53 from Mdm2 was defective in these cells. In contrast, there was a normal level of interaction between p53 and Pin1 in control and AOA3, in response to radiation damage, indicating that the post-translational modifications to p53 are intact in these cells but operating at reduced level.