Atm expression patterns suggest a contribution from the peripheral nervous system to the phenotype of ataxia–telangiectasia

doi:10.1016/S0306-4522(98)00117-1

Neuroscience

Volume 86, Issue 4, 18 June 1998, Pages 1045-1054

https://doi.org/10.1016/S0306-4522(98)00117-1 Get rights and content

Abstract

Ataxia–telangiectasia is a human autosomal recessive disease characterized by neurodegeneration, cancer predisposition and sensitivity to ionizing radiation. One of the earliest features of this disease is ataxia, which is thought to be attributable to a progressive cerebellar degeneration associated with a disruption of Purkinje cell cytoarchitecture and positioning. To investigate the neuropathology of ataxia–telangiectasia, we used in situ hybridization to map Atm (the gene mutated in ataxia–telangiectasia) expression during mouse development.

Atm expression was highest in the embryonic mouse nervous system, where it was predominantly associated with regions undergoing mitosis. During the period of Purkinje cell neurogenesis, Atm was highly expressed in the area containing Purkinje cell precursors (the ventricular zone of the fourth ventricle). However, in the postnatal cerebellum, Atm expression in Purkinje cells was very low, while expression in proliferating granule neurons was high. The only region of the adult nervous system that exhibited elevated Atm expression were the postmitotic sensory neurons of the dorsal root ganglia.

The data suggest an early developmental requirement for Atm in the cerebellum, and other regions of the central nervous system, and a potential contribution of the dorsal root ganglia/sensory input pathway to the ataxic phenotype of ataxia–telangiectasia.

Section snippets

Isolation of the mouse Atm gene

A random primed first strand brain cDNA library was made using 3 μg polyA⁺ RNA, from either mouse or human brain RNA with the Superscript cDNA system according to the manufacturers directions (Life Technologies, Gaithersburg, MD). Final reaction mix (50 μl) was diluted with water to give a volume of 150 μl. This cDNA library (2 μl) was used as a template for polymerase chain reaction with specific primers, to isolate a region of the Atm gene corresponding to nucleotides 8345–8939 from the published

Atm expression in the adult brain

The loss of cerebellar Purkinje cells is considered to be the basis of the ataxia component of A–T. Since it is unclear why the Purkinje cells should be selectively vulnerable in A–T, we examined different brain regions to assess whether Atm expression correlated with the cerebellar pathology. Northern blot analysis of polyA⁺ RNA isolated from a number of different human brain regions showed a ∼12-kb band that had a relatively uniform distribution (Fig. 1). While Atm levels in most brain

Discussion

A–T is a syndrome that affects many tissues. However, it is the ataxia that is the predominant early feature of this disease, and future treatment for A–T will require an understanding of the molecular basis of this aspect of its neuropathophysiology. The identification of Atm[29]now allows elucidation of the molecular basis of the A–T phenotype.

A number of points relevant to understanding the consequences of Atm expression in the CNS emerge from this study: (i) Atm is ubiquitously expressed in

Conclusions

The present study suggests that peripheral pathology could well be an important factor in A–T-related ataxia, and sensory neurons may play as significant a role in A–T neuropathology as cerebellar neurons. Therefore, a productive aspect of A–T research may be to evaluate the role of the sensory PNS in the phenotype of this disorder.

Acknowledgements

These studies were supported by the A–T Children's Project (P.J.M.), a Cancer Center CORE grant NIH P30 CA 21765-19 (P.J.M.) and by the American Lebanese and Syrian Associated Charities (ALSAC) of St Jude Children's Research Hospital.

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The genetic disease ataxia telangiectasia (AT) results from mutations in the ataxia telangiectasia mutated (ATM) gene. AT patients develop a progressive degeneration of cerebellar Purkinje neurons. Surprisingly, while ATM plays a criticial role in the cellular reponse to DNA damage, previous studies have localized ATM to the cytoplasm of rodent and human Purkinje neurons. Here we show that ATM is primarily localized to the nucleus in cerebellar Purkinje neurons in postmortem human brain tissue samples, although some light cytoplasmic ATM staining was also observed. No ATM staining was observed in brain tissue samples from AT patients, verifying the specificity of the antibody. We also found that antibodies against components of the Mre11/Rad50/Nbs1 (MRN) complex showed strong staining in Purkinje cell nuclei. However, while ATM is present in both the nucleoplasm and nucleolus, MRN proteins are excluded from the nucleolus. We also observed very high levels of topoisomerase 1 (TOP1) in the nucleus, and specifically the nucleolus, of human Purkinje neurons. Our results have direct implications for understanding the mechanisms of neurodegeneration in AT and AT-like disorder.
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Immunofluorescence analysis is sensitive to experimental conditions and is strictly dependent on the specificity of the antibody for the target protein. Furthermore, the low level of ATM in mature neurons (11, 26, 27) probably requires a highly specific antibody with very high affinity for ATM for such analysis. We demonstrated the specificity of our two antibodies using ATM-deficient cells (Fig. 4), and we used Wilson and Bianchi's (1999) (24) protocol, which had been adapted for immunostaining of nuclear mammalian proteins and enabled the detection of previously undetectable or poorly detectable proteins.
The protein kinase ATM (ataxia-telangiectasia mutated) activates the cellular response to double strand breaks (DSBs), a highly cytotoxic DNA lesion. ATM is activated by DSBs and in turn phosphorylates key players in numerous damage response pathways. ATM is missing or inactivated in the autosomal recessive disorder ataxia-telangiectasia (A-T), which is characterized by neuronal degeneration, immunodeficiency, genomic instability, radiation sensitivity, and cancer predisposition. The predominant symptom of A-T is a progressive loss of movement coordination due to ongoing degeneration of the cerebellar cortex and peripheral neuropathy. A major deficiency in understanding A-T is the lack of information on the role of ATM in neurons. It is unclear whether the ATM-mediated DSB response operates in these cells similarly to proliferating cells. Furthermore, ATM was reported to be cytoplasmic in neurons and suggested to function in these cells in capacities other than the DNA damage response. Recently we obtained genetic molecular evidence that the neuronal degeneration in A-T does result from defective DNA damage response. We therefore undertook to investigate this response in a model system of human neuron-like cells (NLCs) obtained by neuronal differentiation in culture. ATM was largely nuclear in NLCs, and their ATM-mediated responses to DSBs were similar to those of proliferating cells. Knocking down ATM did not interfere with neuronal differentiation but abolished ATM-mediated damage responses in NLCs. We concluded that nuclear ATM mediates the DSB response in NLCs similarly to in proliferating cells. Attempts to understand the neurodegeneration in A-T should be directed to investigating the DSB response in the nervous system.
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Ataxia-telangiectasia mutated (ATM) is the gene product mutated in ataxia-telangiectasia (A-T), which is an autosomal recessive disorder with symptoms including neurodegeneration, cancer predisposition and premature aging. ATM is thought to play a pivotal role in signal transduction in response to genotoxic DNA damage. To study the physiological and developmental functions of ATM using the zebrafish model system, we cloned the zebrafish homolog cDNA of human ATM (hATM), zebrafish ATM (zATM), analyzed the expression pattern of zATM during early development, and further developed the system to study loss of zATM function in zebrafish embryos. Employing information available from the zebrafish genomic database, we utilized a PCR-based approach to isolate zATM cDNA clones. Sequence analysis of zATM showed a high level homology in the functional domains of hATM. The putative FAT, phosphoinositide 3-kinase-like, and FATC domains of zATM, which regulate ATM kinase activity and functions, were the most highly conserved regions, exhibiting 64–94% amino acid identity to the corresponding domains in hATM, while exhibiting approximately 50% amino acid identity outside these domains. The zATM gene is expected to consist of 62 coding exons, and we have identified at least 55 exons encompassing more than 100 kb of nucleotide sequence, which encodes about 9 kb of cDNA. By in situ hybridization, zATM mRNA was detected ubiquitously with a dramatic increase at the 18-somite stage, then more specifically in the eye, brain, trunk, and tail at later stages. To inhibit zATM expression and function, we designed and synthesized splice-blocking antisense-morpholino oligonucleotides targeting the phosphoinositide 3-kinase-like domain. We demonstrated that this knockdown of zATM caused abnormal development upon ionizing radiation-induced DNA damage. Our data suggest that the ATM gene is structurally and functionally conserved in vertebrates from zebrafish to human.

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Atm expression patterns suggest a contribution from the peripheral nervous system to the phenotype of ataxia–telangiectasia

Abstract

Section snippets

Isolation of the mouse Atm gene

Atm expression in the adult brain

Discussion

Conclusions

Acknowledgements

Cell

Devl Biol.

Cell

Trends biochem. Sci.

Cell

Cell

Curr. Biol.

Physiol. Behav.

Trends biochem. Sci.

Trends Genet.

Neuroscience

Cell

Cell

Cell–cell interactions influence survival and differentiation of purified Purkinje cells in vitro

Neuron

Is the sensory neuropathy in ataxia–telangiectasia distinguishable from that in Friedreich's Ataxia?

Acta neuropath.

The mouse dystonia musculorum gene is a neural isoform of bullous pemphigoid antigen 1

Nature Genet.

Abnormalities in sensory and mixed evoked potentials in atasiatelangiectasia

J. Neurol. Neurosurg. Pyschiat.

Pleiotropic defects in ataxia–telangiectasia protein-deficient mice

Proc. natn. Acad. Sci. U.S.A.