Neurodegenerative Ataxia Research Articles

A SEL1L Mutation Links a Canine Progressive Early-Onset Cerebellar Ataxia to the Endoplasmic Reticulum–Associated Protein Degradation (ERAD) Machinery

Inherited ataxias are characterized by degeneration of the cerebellar structures, which results in progressive motor incoordination. Hereditary ataxias occur in many species, including humans and dogs. Several mutations have been found in humans, but the genetic background has remained elusive in dogs. The Finnish Hound suffers from an early-onset progressive cerebellar ataxia. We have performed clinical, pathological, and genetic studies to describe the disease phenotype and to identify its genetic cause. Neurological examinations on ten affected dogs revealed rapidly progressing generalized cerebellar ataxia, tremors, and failure to thrive. Clinical signs were present by the age of 3 months, and cerebellar shrinkage was detectable through MRI. Pathological and histological examinations indicated cerebellum-restricted neurodegeneration. Marked loss of Purkinje cells was detected in the cerebellar cortex with secondary changes in other cortical layers. A genome-wide association study in a cohort of 31 dogs mapped the ataxia gene to a 1.5 Mb locus on canine chromosome 8 (praw = 1.1×10−7, pgenome = 7.5×10−4). Sequencing of a functional candidate gene, sel-1 suppressor of lin-12-like (SEL1L), revealed a homozygous missense mutation, c.1972T>C; p.Ser658Pro, in a highly conserved protein domain. The mutation segregated fully in the recessive pedigree, and a 10% carrier frequency was indicated in a population cohort. SEL1L is a component of the endoplasmic reticulum (ER)–associated protein degradation (ERAD) machinery and has not been previously associated to inherited ataxias. Dysfunctional protein degradation is known to cause ER stress, and we found a significant increase in expression of nine ER stress responsive genes in the cerebellar cortex of affected dogs, supporting the pathogenicity of the mutation. Our study describes the first early-onset neurodegenerative ataxia mutation in dogs, establishes an ERAD–mediated neurodegenerative disease model, and proposes SEL1L as a new candidate gene in progressive childhood ataxias. Furthermore, our results have enabled the development of a genetic test for breeders.

Neurogenin 2 regulates progenitor cell-cycle progression and Purkinje cell dendritogenesis in cerebellar development

By serving as the sole output of the cerebellar cortex, integrating a myriad of afferent stimuli, Purkinje cells (PCs) constitute the principal neuron in cerebellar circuits. Several neurodegenerative cerebellar ataxias feature a selective cell-autonomous loss of PCs, warranting the development of regenerative strategies. To date, very little is known as to the regulatory cascades controlling PC development. During central nervous system development, the proneural gene neurogenin 2 (Neurog2) contributes to many distinct neuronal types by specifying their fate and/or dictating development of their morphological features. By analyzing a mouse knock-in line expressing Cre recombinase under the control of Neurog2 cis-acting sequences we show that, in the cerebellar primordium, Neurog2 is expressed by cycling progenitors cell-autonomously fated to become PCs, even when transplanted heterochronically. During cerebellar development, Neurog2 is expressed in G1 phase by progenitors poised to exit the cell cycle. We demonstrate that, in the absence of Neurog2, both cell-cycle progression and neuronal output are significantly affected, leading to an overall reduction of the mature cerebellar volume. Although PC fate identity is correctly specified, the maturation of their dendritic arbor is severely affected in the absence of Neurog2, as null PCs develop stunted and poorly branched dendrites, a defect evident from the early stages of dendritogenesis. Thus, Neurog2 represents a key regulator of PC development and maturation.

Structure of an aprataxin–DNA complex with insights into AOA1 neurodegenerative disease

DNA ligases finalize DNA replication and repair through DNA nick-sealing reactions that can abort to generate cytotoxic 5′-adenylation DNA damage (5′-AMP). Aprataxin (Aptx) catalyses direct reversal of 5′-AMP adducts to protect genome integrity. Here, the structure of an Aptx-DNA-AMP-Zn complex reveals active site and DNA interaction clefts formed by fusing a HIT (histidine triad) nucleotide hydrolase with an unprecedented DNA minor groove binding C2HE Zn-finger (Znf). An Aptx helical wedge interrogates the base stack for DNA end/nick sensing. HIT-Znf, the wedge, and an "[F/Y]PK" pivot motif cooperate to distort terminal DNA base-pairing and direct 5′-AMP into the active site pocket. Structural and mutational data support a wedge-pivot-cut HIT-Znf catalytic mechanism for 5′-AMP adduct recognition and removal, and suggest mutations impacting protein folding, the active site pocket, and the pivot underlie Aptx dysfunction in the neurodegenerative disorder Ataxia Oculomotor Apraxia 1 (AOA1).

Distinct Neurochemical Profiles of Spinocerebellar Ataxias 1, 2, 6, and Cerebellar Multiple System Atrophy

Hereditary and sporadic neurodegenerative ataxias are movement disorders that affect the cerebellum. Robust and objective biomarkers are critical for treatment trials of ataxias. In addition, such biomarkers may help discriminate between ataxia subtypes because these diseases display substantial overlap in clinical presentation and conventional MRI. Profiles of 10-13 neurochemical concentrations obtained in vivo by high field proton magnetic resonance spectroscopy ((1)H MRS) can potentially provide ataxia-type specific biomarkers. We compared cerebellar and brainstem neurochemical profiles measured at 4T from 26 patients with spinocerebellar ataxias (SCA1, N = 9; SCA2, N = 7; SCA6, N = 5) or cerebellar multiple system atrophy (MSA-C, N = 5) and 15 age-matched healthy controls. The Scale for the Assessment and Rating of Ataxia (SARA) was used to assess disease severity. The patterns of neurochemical alterations relative to controls differed between ataxia types. Myo-inositol levels in the vermis, myo-inositol, total N-acetylaspartate, total creatine, glutamate, glutamine in the cerebellar hemispheres and myo-inositol, total N-acetylaspartate, glutamate in the pons were significantly different between patient groups (Bonferroni corrected p < 0.05). The best MRS predictors were selected by a tree classification procedure and lead to 89% accurate classification of all subjects while the SARA scores overlapped considerably between patient groups. Therefore, this study demonstrated multiple neurochemical alterations in SCAs and MSA-C relative to controls and the potential for these neurochemical levels to differentiate ataxia types. Studies with higher numbers of patients and other ataxias are warranted to further investigate the clinical utility of neurochemical levels as measured by high-field MRS as ataxia biomarkers.

Genetic interactions between HNT3/Aprataxin and RAD27/FEN1 suggest parallel pathways for 5′ end processing during base excision repair

Mutations in Aprataxin cause the neurodegenerative syndrome ataxia oculomotor apraxia type 1. Aprataxin catalyzes removal of adenosine monophosphate (AMP) from the 5′ end of a DNA strand, which results from an aborted attempt to ligate a strand break containing a damaged end. To gain insight into which DNA lesions are substrates for Aprataxin action in vivo, we deleted the Saccharomyces cerevisiae HNT3 gene, which encodes the Aprataxin homolog, in combination with known DNA repair genes. While hnt3Δ single mutants were not sensitive to DNA damaging agents, loss of HNT3 caused synergistic sensitivity to H 2O 2 in backgrounds that accumulate strand breaks with blocked termini, including apn1Δ apn2Δ tpp1Δ and ntg1Δ ntg2Δ ogg1Δ. Loss of HNT3 in rad27Δ cells, which are deficient in long-patch base excision repair (LP-BER), resulted in synergistic sensitivity to H 2O 2 and MMS, indicating that Hnt3 and LP-BER provide parallel pathways for processing 5′ AMPs. Loss of HNT3 also increased the sister chromatid exchange frequency. Surprisingly, HNT3 deletion partially rescued H 2O 2 sensitivity in recombination-deficient rad51Δ and rad52Δ cells, suggesting that Hnt3 promotes formation of a repair intermediate that is resolved by recombination.

CK2 phosphorylation-dependent interaction between aprataxin and MDC1 in the DNA damage response.

Aprataxin, defective in the neurodegenerative disorder ataxia oculomotor apraxia type 1, resolves abortive DNA ligation intermediates during DNA repair. Here, we demonstrate that aprataxin localizes at sites of DNA damage induced by high LET radiation and binds to mediator of DNA-damage checkpoint protein 1 (MDC1/NFBD1) through a phosphorylation-dependent interaction. This interaction is mediated via the aprataxin FHA domain and multiple casein kinase 2 di-phosphorylated S-D-T-D motifs in MDC1. X-ray structural and mutagenic analysis of aprataxin FHA domain, combined with modelling of the pSDpTD peptide interaction suggest an unusual FHA binding mechanism mediated by a cluster of basic residues at and around the canonical pT-docking site. Mutation of aprataxin FHA Arg29 prevented its interaction with MDC1 and recruitment to sites of DNA damage. These results indicate that aprataxin is involved not only in single strand break repair but also in the processing of a subset of double strand breaks presumably through its interaction with MDC1.

Cerebellar ataxias

The term 'cerebellar ataxias' encompasses the various cerebellar disorders encountered during daily practice. Patients exhibit a cerebellar syndrome and can also present with pigmentary retinopathy, extrapyramidal movement disorders, pyramidal signs, cortical symptoms (seizures, cognitive impairment/behavioural symptoms), and peripheral neuropathy. The clinical diagnosis of subtypes of ataxias is complicated by the salient overlap of the phenotypes between genetic subtypes. The identification of the causative mutations of many hereditary ataxias and the development of relevant animal models bring hope for effective therapies in neurodegenerative ataxias. We describe the current classification of cerebellar ataxias and underline the recent discoveries in molecular pathogenesis. Cerebellar disorders can be divided into sporadic forms and inherited diseases. Inherited ataxias include autosomal recessive cerebellar ataxias, autosomal dominant cerebellar ataxias/spinocerebellar ataxia) and episodic ataxias, and X-linked ataxias. From a motor control point of view, the leading theories of ataxia are based on neural representations or 'internal models' to emulate fundamental natural processes such as body motion. Recent molecular advances have direct implications for research and daily practice. We provide a framework for the diagnosis of ataxias. For the first time, the therapeutic agents under investigation are targeted to deleterious pathways.

Aprataxin, poly-ADP ribose polymerase 1 (PARP-1) and apurinic endonuclease 1 (APE1) function together to protect the genome against oxidative damage

Aprataxin, defective in the neurodegenerative disorder ataxia oculomotor apraxia type 1 (AOA1), is a DNA repair protein that processes the product of abortive ligations, 5' adenylated DNA. In addition to its interaction with the single-strand break repair protein XRCC1, aprataxin also interacts with poly-ADP ribose polymerase 1 (PARP-1), a key player in the detection of DNA single-strand breaks. Here, we reveal reduced expression of PARP-1, apurinic endonuclease 1 (APE1) and OGG1 in AOA1 cells and demonstrate a requirement for PARP-1 in the recruitment of aprataxin to sites of DNA breaks. While inhibition of PARP activity did not affect aprataxin activity in vitro, it retarded its recruitment to sites of DNA damage in vivo. We also demonstrate the presence of elevated levels of oxidative DNA damage in AOA1 cells coupled with reduced base excision and gap filling repair efficiencies indicative of a synergy between aprataxin, PARP-1, APE-1 and OGG1 in the DNA damage response. These data support both direct and indirect modulating functions for aprataxin on base excision repair.

Irreversible chromosome damage accumulates rapidly in the absence of ATM kinase activity

Mutations in the ATM kinase cause the neurodegenerative disorder ataxia telangiectasia (A-T) and affected individuals are exquisitely radiation-sensitive and cancer-prone. Cells derived from A-T individuals contain chromosome aberrations and exhibit profound cellular radiosensitivity. ATM is an apical kinase critical for the activation of cell cycle checkpoints and the induction of apoptosis in irradiated cells. However, defects in these pathways are insufficient to account for the chromosomal instability seen in A-T cells. We show here that the small molecule KU55933 can be used as a “molecular switch” to selectively and transiently inhibit ATM kinase activity in cells. We subsequently show that the cellular radiosensitization seen when ATM kinase activity is inhibited for one hour following exposure to γ-rays, accounts for over 70% of the total cellular radiosensitization seen when ATM kinase activity is inhibited for 17h. Finally, we show that inhibition of ATM kinase activity for one hour following exposure to irradiation doubles the number of chromosome aberrations occurring in late-S- and G2-, but not M-phase, cells. These observations are unexpected and suggest that irreversible chromosome damage accumulates very rapidly when ATM kinase activity is transiently inhibited following irradiation. We propose that we have revealed an essential, yet previously undescribed, role for ATM kinase in suppressing chromosomal instability

Genetic Interactions of the Aspergillus nidulans atmAATM Homolog With Different Components of the DNA Damage Response Pathway

Ataxia telangiectasia mutated (ATM) is a phosphatidyl-3-kinase-related protein kinase that functions as a central regulator of the DNA damage response in eukaryotic cells. In humans, mutations in ATM cause the devastating neurodegenerative disease ataxia telangiectasia. Previously, we characterized the homolog of ATM (AtmA) in the filamentous fungus Aspergillus nidulans. In addition to its expected role in the DNA damage response, we found that AtmA is also required for polarized hyphal growth. Here, we extended these studies by investigating which components of the DNA damage response pathway are interacting with AtmA. The AtmA(ATM) loss of function caused synthetic lethality when combined with mutation in UvsB(ATR). Our results suggest that AtmA and UvsB are interacting and they are probably partially redundant in terms of DNA damage sensing and/or repairing and polar growth. We identified and inactivated A. nidulans chkA(CHK1) and chkB(CHK2) genes. These genes are also redundantly involved in A. nidulans DNA damage response. We constructed several combinations of double mutants for DeltaatmA, DeltauvsB, DeltachkA, and DeltachkB. We observed a complex genetic relationship with these mutations during the DNA replication checkpoint and DNA damage response. Finally, we observed epistatic and synergistic interactions between AtmA, and bimE(APC1), ankA(WEE1) and the cdc2-related kinase npkA, at S-phase checkpoint and in response to DNA-damaging agents.

Chronic Pain in Machado-Joseph Disease

Machado-Joseph disease (MJD) is one of the most common forms of neurodegenerative ataxia characterized by remarkable phenotypic heterogeneity. Although patients frequently report pain, systematic evaluation of this clinical feature is lacking. To compare the frequency of chronic pain among patients with genetically confirmed MJD, an age- and sex-matched healthy control group, and a disease control group of patients with amyotrophic lateral sclerosis (ALS). We included 70 patients with MJD, 20 patients with ALS, and 70 control subjects from 2 clinical centers. All individuals underwent assessment with a standardized pain questionnaire. In addition, we used a visual analog scale to quantify pain intensity. Thirty-three patients with MJD (47%), 3 patients with ALS (15%), and 6 controls (9%) reported chronic pain. Lower back pain preceded ataxia in 6 patients with MJD. Twenty-nine patients with MJD had daily pain, which was continuous in 23. The mean visual analog scale score was 6.1 in patients with MJD. Pain was musculoskeletal in 26 patients with MJD, dystonic in 2, neuropathic in 2, and mixed in 3. Typically, pain was lumbar (n = 17) or in the lower limbs (n = 15). We did not find significant differences regarding duration of disease, sex, or severity of ataxia among patients with MJD with and without chronic pain. Expanded (CAG)(n) tandem repeats were longer in patients with MJD who experienced chronic pain (67.3 vs 65.2; P = .04). In our series, pain was significantly more frequent in patients with MJD than in controls. Chronic pain was a frequent and often disabling complaint among patients with MJD. The lower back was the most frequently reported location of pain in patients with MJD.

Protein kinase C γ mutations in the C1B domain cause caspase-3-linked apoptosis in lens epithelial cells through gap junctions

Failure to control oxidative stress is closely related to aging and to a diverse range of human diseases. We have reported that protein kinase C γ (PKCγ) acts as a primary oxidative stress sensor in the lens. PKCγ has a Zn-finger C1B stress switch domain, residues 101–150. Mutation, H101Y, in the C1B domain of PKCγ proteins causes a failure of the PKCγ oxidative stress response [Lin, D., Takemoto, D.J., 2005. Oxidative activation of protein kinase Cgamma through the C1 domain. Effects on gap junctions. J. Biol. Chem. 280, 13682-13693]. Some human neurodegenerative spinocerebellar ataxia type 14 are caused by mutations in the PKCγ C1B domain. In the current study we have investigated the effects of these mutations on lens epithelial cell responses to oxidative stress. The results demonstrate that PKCγ C1B mutants had lower basal enzyme activities and were not activated by H 2O 2. Furthermore, the PKCγ mutations caused a failure of endogenous wild type PKCγ to be activated by H 2O 2. These PKCγ mutations abolished the effect of H 2O 2 on phosphorylation of Cx43 and Cx50 by H 2O 2 activation of PKCγ. The cells with PKCγ C1B mutations had more Cx43 and/or Cx50 gap junction plaques which were not decreased by H 2O 2. Since open gap junctions could have a bystander effect this could cause apoptosis to occur. H 2O 2 (100 μM, 3 h) activated a caspase-3 apoptotic pathway in the lens epithelial cells but was more severe in cells expressing PKCγ mutations. The presence of 18α-glycyrrhetinic acid (AGA), an inhibitor of gap junctions, decreased Cx43 and Cx50 protein levels and gap junction plaque number. This reduction in gap junctions by AGA resulted in inhibition of H 2O 2-induced apoptosis. Our results demonstrate that there is a dominant negative effect of PKCγ C1B mutations on endogenous PKCγ which results in loss of control of gap junctions. Modeled structures suggest that the severity of C1B mutation effects may be related to the extent of loss of C1B structure. Mutations in the C1B domain of PKCγ result in increased apoptosis in lens epithelial cells. This can be prevented by a gap junction inhibitor. Thus, propagation of apoptosis from cell-to-cell in lens epithelial cells may be through open gap junctions. The control of gap junctions requires PKCγ.

Structure validation of the Josephin domain of ataxin-3: Conclusive evidence for an open conformation

The availability of new and fast tools in structure determination has led to a more than exponential growth of the number of structures solved per year. It is therefore increasingly essential to assess the accuracy of the new structures by reliable approaches able to assist validation. Here, we discuss a specific example in which the use of different complementary techniques, which include Bayesian methods and small angle scattering, resulted essential for validating the two currently available structures of the Josephin domain of ataxin-3, a protein involved in the ubiquitin/proteasome pathway and responsible for neurodegenerative spinocerebellar ataxia of type 3. Taken together, our results demonstrate that only one of the two structures is compatible with the experimental information. Based on the high precision of our refined structure, we show that Josephin contains an open cleft which could be directly implicated in the interaction with polyubiquitin chains and other partners.

Crystal structure of human alpha-tocopherol transfer protein bound to its ligand: implications for ataxia with vitamin E deficiency.

Human alpha-tocopherol (alpha-T) transfer protein (ATTP) plays a central role in vitamin E homeostasis, preventing degradation of alpha-T by routing this lipophilic molecule for secretion by hepatocytes. Mutations in the gene encoding ATTP have been shown to cause a severe deficiency in alpha-T, which results in a progressive neurodegenerative spinocerebellar ataxia, known as ataxia with vitamin E deficiency (AVED). We have determined the high-resolution crystal structure of human ATTP with (2R,4'R,8'R)-alpha-T in the binding pocket. Surprisingly, the ligand is sequestered deep in the hydrophobic core of the protein, implicating a large structural rearrangement for the entry and release of alpha-T. A comparison to the structure of a related protein, Sec14p, crystallized without a bona fide ligand, shows a possibly relevant open conformation for this family of proteins. Furthermore, of the known mutations that cause AVED, one mutation, L183P, is located directly in the binding pocket. Finally, three mutations associated with AVED involve arginine residues that are grouped together on the surface of ATTP. We propose that this positively charged surface may serve to orient an interacting protein, which might function to regulate the release of alpha-T through an induced change in conformation of ATTP.

Mifepristone (RU486) protects Purkinje cells from cell death in organotypic slice cultures of postnatal rat and mouse cerebellum.

Mifepristone (RU486), which binds with high affinity to both progesterone and glucocorticosteroid receptors (PR and GR), is well known for its use in the termination of unwanted pregnancy, but other activities including neuroprotection have been suggested. Cerebellar organotypic cultures from 3 to 7 postnatal day rat (P3-P7) were studied to examine the neuroprotective potential of RU486. In such cultures, Purkinje cells enter a process of apoptosis with a maximum at P3. This study shows that RU486 (20 microM) can protect Purkinje cells from this apoptotic process. The neuroprotective effect did involve neither PR nor GR, because it could not be mimicked or inhibited by other ligands of these receptors, and because it still took place in PR mutant (PR-KO) mice and in brain-specific GR mutant mice (GRNes/Cre). Potent antioxidant agents did not prevent Purkinje cells from this developmental cell death. The neuroprotective effect of RU486 could also be observed in pathological Purkinje cell death. Indeed, this steroid is able to prevent Purkinje cells from death in organotypic cultures of cerebellar slices from Purkinje cell degeneration (pcd) mutant mice, a murine model of hereditary neurodegenerative ataxia. In P0 cerebellar slices treated with RU486 for 6 days and further kept in culture up to 21 days, the synthetic steroid increased by 16.2-fold the survival of pcd/pcd Purkinje cells. Our results show that RU486 may act through a new mechanism, not yet elucidated, to protect Purkinje cells from death.

Dramatically different phenotypes in mouse models of human Tay-Sachs and Sandhoff diseases.

We have generated mouse models of human Tay-Sachs and Sandhoff diseases by targeted disruption of the Hexa (alpha subunit) or Hexb (beta subunit) genes, respectively, encoding lysosomal beta-hexosaminidase A (structure, alpha) and B (structure, beta beta). Both mutant mice accumulate GM2 ganglioside in brain, much more so in Hexb -/- mice, and the latter also accumulate glycolipid GA2. Hexa -/- mice suffer no obvious behavioral or neurological deficit, while Hexb -/- mice develop a fatal neurodegenerative disease, with spasticity, muscle weakness, rigidity, tremor and ataxia. The Hexb -/- but not the Hexa -/- mice have massive depletion of spinal cord axons as an apparent consequence of neuronal storage of GM2. We propose that Hexa -/- mice escape disease through partial catabolism of accumulated GM2 via GA2 (asialo-GM2) through the combined action of sialidase and beta-hexosaminidase B.

The Machado-Joseph disease locus is different from the spinocerebellar ataxia locus (SCA1)

Machado-Joseph disease (MJD) is an autosomal dominant neurodegenerative spinocerebellar ataxia that has been described primarily in families of Azorean or Protuguese descent. MJD and chromosome 6p-linked spinocerebellar ataxia (SCA1) are difficult to differentiate clinically, and it has been suggested that they may be allelic variants of the same disorder. We have tested MJD families for linkage to six DNA sequence polymorphisms located on chromosome 6p, including the highly informative dinucleotide repeat, D6S89. Seventeen centimorgans telomeric to and 41 cM centromeric to D6S89, a region that includes the SCA1 locus reported to be within 3 cM of D6S89, have been excluded. These data provide conclusive evidence that MJD and SCA1 are nonallelic.