Academic journal article Genetics

The Innate Immune Response Transcription Factor Relish Is Necessary for Neurodegeneration in a Drosophila Model of Ataxia-Telangiectasia

Academic journal article Genetics

The Innate Immune Response Transcription Factor Relish Is Necessary for Neurodegeneration in a Drosophila Model of Ataxia-Telangiectasia

Article excerpt

ABSTRACT Neurodegeneration is a hallmark of the human disease ataxia-telangiectasia (A-T) that is caused by mutation of the A-T mutated (ATM) gene. We have analyzed Drosophila melanogaster ATM mutants to determine the molecular mechanisms underlying neurodegeneration in A-T. Previously, we found that ATM mutants upregulate the expression of innate immune response (IIR) genes and undergo neurodegeneration in the central nervous system. Here, we present evidence that activation of the IIR is a cause of neurodegeneration in ATM mutants. Three lines of evidence indicate that ATM mutations cause neurodegeneration by activating the Nuclear Factor-κB (NF-κB) transcription factor Relish, a key regulator of the Immune deficiency (Imd) IIR signaling pathway. First, the level of upregulation of IIR genes, including Relish target genes, was directly correlated with the level of neurodegeneration in ATM mutants. Second, Relish mutations inhibited upregulation of IIR genes and neurodegeneration in ATM mutants. Third, overexpression of constitutively active Relish in glial cells activated the IIR and caused neurodegeneration. In contrast, we found that Imd and Dif mutations did not affect neurodegeneration in ATM mutants. Imd encodes an activator of Relish in the response to gram-negative bacteria, and Dif encodes an immune responsive NF-κB transcription factor in the Toll signaling pathway. These data indicate that the signal that causes neurodegeneration in ATM mutants activates a specific NF-κB protein and does so through an unknown activator. In summary, these findings suggest that neurodegeneration in human A-T is caused by activation of a specific NF-κB protein in glial cells.

ATAXIA-TELANGIECTASIA (A-T) is a rare human disease characterized by progressive degeneration of Purkinje and granule neurons in the cerebellum (Sedgwick and Boder 1960; Bundey 1994; McKinnon 2004). A-T is caused by recessive mutation of the A-T mutated (ATM) gene, which encodes a protein kinase (Savitsky et al. 1995). The molecular mechanisms underlying neurodegeneration in A-T are not well understood but probably involve events that go awry in neurons and glial cells due to inadequate phosphorylation of ATM substrate proteins. ATM phosphorylates hundreds of proteins in response to DNA damage, most notably proteins that initiate cell cycle checkpoints, DNA damage repair mechanisms, and apoptotic pathways (Bakkenist and Kastan 2003; Matsuoka et al. 2007; Derheimer and Kastan 2010; Bhatti et al. 2011).

To understand the molecular mechanisms underlying neurodegeneration in A-T, we have used Drosophila melanogaster as an experimental system. Since ATM is essential in flies, we have used a temperature-sensitive ATM allele (ATM8) and ATM RNA interference (RNAi) to conditionally inactivate ATM (Silva et al. 2004; Rimkus et al. 2008; Pedersen et al. 2010; Petersen et al. 2012). ATM8 homozygous flies cultured at 25° are pupal lethal but when cultured at 18° they are viable (Silva et al. 2004). ATM8 homozygous flies cultured at 18° and shifted to 25° as adults have negligible ATM kinase activity, as assayed by phosphorylation of serine 137 (pS137) in histone H2Av in response to ionizing radiation (IR)-induced DNA damage (Petersen et al. 2012). ATM8/+ heterozygous flies cultured at 18° and shifted to 25° as adults have an intermediate level of ATM kinase activity, relative to wild-type and ATM8 homozygous flies. Repo-ATMi flies have partially reduced ATM expression in glial cells due to ATM knockdown by RNAi. Repo-ATMi flies contain two transgenes, a target transgene that expresses an ATM short hairpin RNA (shRNA) under transcriptional control of GAL4 UAS sequences (ATMi) and a Repo-GAL4 driver transgene that expresses the GAL4 transcription factor only in glial cells and drives expression of the target transgene (Brand and Perrimon 1993; Xiong et al. 1994; Sepp et al. 2001; Rimkus et al. 2008). Based on phenotype, the level of ATM in glial cells of Repo-ATMi flies is comparable to the level in ATM heterozygous null mutant flies (Supporting Information, Figure S1). …

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