Skip to main content
NCBI home page
Genetics logoLink to Genetics
. 2013 May;194(1):133–142. doi: 10.1534/genetics.113.150854

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

Andrew J Petersen *,†, Rebeccah J Katzenberger †, David A Wassarman *,†,1
PMCID: PMC3632461  PMID: 23502677

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.

Keywords: ATM, countercurrent, brain, glial cell, NF-κB

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).

Our analyses of ATM8/+, ATM8, and Repo-ATMi flies indicate that ATM mutation in glial cells causes neurodegeneration (Petersen et al. 2012). These flies have phenotypes that typify neurodegeneration: reduced longevity, reduced climbing ability, increased death of neurons and glial cells in the brain, and vacuolar-like holes in the brain (Lessing and Bonini 2009; Petersen et al. 2012). Furthermore, gene expression analysis of adult heads indicates that these flies activate the innate immune response (IIR) in glial cells (Petersen et al. 2012). Activation of the IIR causes photoreceptor cell neurodegeneration in fly models of human retinal degeneration and Alzheimer’s disease (Tan et al. 2008; Chinchore et al. 2012; Petersen and Wassarman 2012). Moreover, activation of a glial cell IIR occurs in Alzheimer’s disease and Parkinson’s disease and is thought to exacerbate the process of neurodegeneration (Amor et al. 2010). These findings suggest a causative link between activation of the IIR and neurodegeneration in A-T.

The IIR is primarily known to combat pathogens. In Drosophila, there are two major IIR pathways, the Toll pathway and the Imd (Immune deficiency) pathway (Lemaitre and Hoffmann 2007). These pathways activate different Nuclear Factor-κB (NF-κB) transcription factors to promote the expression of IIR genes. The Toll pathway responds to eukaryotic pathogens and gram-positive bacteria. Extracellular pathogens are recognized by peptidoglycan recognition proteins (PGRPs) and facilitate cleavage of the cytokine Spätzle, which binds to and activates the Toll receptor. Toll activation stimulates degradation of the Inhibitor of κB (IκB) protein Cactus in the cell cytoplasm, releasing the NF-κB transcription factor Dif to allow it to enter the nucleus. In the nucleus, Dif promotes the transcription of IIR genes, including antimicrobial peptide (AMP) genes that encode secreted proteins. The Imd pathway, which is homologous to the mammalian tumor necrosis factor-α (TNFα) pathway, recognizes gram-negative bacteria through membrane-bound PGRP proteins. PGRP activation signals through the Imd protein to activate the NF-κB protein Relish. Relish contains an N-terminal NF-κB motif and a C-terminal IκB motif. Activation of the Imd pathway causes Relish cleavage, releasing the NF-κB motif from its inhibitory IκB motif. Activated Relish translocates to the nucleus and promotes the transcription of IIR genes, including AMP genes. In support of this mechanism, overexpression of the Relish NF-κB motif in the fat body, a major immune-responsive tissue, is sufficient to stimulate Relish target gene expression (DiAngelo et al. 2009; Wiklund et al. 2009). Finally, NF-κB-independent pathways activate portions of the IIR, but these pathways are primarily involved in tissue-specific constitutive expression of IIR genes rather than inducible pathogen-mediated expression (Han et al. 2004; Ryu et al. 2004; Peng et al. 2005; Senger et al. 2006; Tzou et al. 2010).

Here, we present studies that tested whether activation of the IIR is necessary for neurodegeneration in the brain of ATM mutant flies. Specifically, we tested whether the level of IIR gene expression correlates with neurodegeneration, whether components of canonical IIR pathways are required for activation of IIR gene expression and neurodegeneration, and whether activation of the IIR in glial cells is sufficient to cause neurodegeneration.

Materials and Methods

Drosophila stocks and crosses

Flies were maintained on standard molasses medium at 25° unless otherwise stated. All experiments involving ATM8 flies were performed on flies raised at 18° throughout development and transferred to 25° at 0–3 days of adulthood. The pWiz-ATM RNAi construct is described by Rimkus et al. (2008). Repo-ATMi flies contained a recombined chromosome with both the Repo-GAL4 and the pWiz-ATMT4 transgenes. For ATM8 experiments with the RelE20 and RelE38 mutations, recombination was used to combine the mutations onto a single chromosome. ATM3, ATM8, RelE20, RelE38, ImdEY08573, Repo-GAL4, and UAS-Relish flies were obtained from the Bloomington Stock Center (Bloomington, IN). Dif1 and UAS-Rel-49 flies were provided by Barry Ganetzky (University of Wisconsin, Madison), ImdSDK and Imd10191 flies were provided by Mimi Shirasu-Hiza (Columbia University, New York), UAS-RelD flies were provided by Sara Cherry (University of Pennsylvania, Philadelphia), and UAS-GFP flies were provided by Morris Birnbaum (University of Pennsylvania).

Countercurrent climbing assay

The countercurrent apparatus is diagrammed in Figure 1 and was based on an apparatus developed by Benzer (1967). The apparatus consisted of two sets of four fly vials that were taped together. One hundred flies were loaded into the first vial of the bottom set, with the other set inverted and staggered on top of this vial. Flies were tapped to the bottom of the vial and allowed to climb for 1 min, after which time the top set of vials was shifted over one vial. Flies that were able to climb into the top vial were tapped to the bottom, resulting in flies in the first and second bottom vials. This process was repeated seven times. Vials that were not engaged with another vial were plugged with cotton to prevent flies from escaping. Flies in vials 1, 2–4, and 5–8 were designated poor, moderate, and good climbers, respectively.

Figure 1.

Figure 1

Open in a new tab

Schematic diagram of the countercurrent climbing apparatus. A description of the countercurrent assay is provided in Materials and Methods. Shaded circles indicate flies that began in vial 1. Indicated are the numbered vials that contained poor, moderate, and good climbers after the seven steps of the assay.

Western blot analysis

ATM8 and w1118 adult flies were collected at 18° and shifted to 25° for 3 days. After 3 days, ATM8 flies were subjected to countercurrent separation. w1118 flies were not separated because very few were poor or moderate climbers. IR exposure and Western blot analysis were performed as described in Petersen et al. (2012).

Neurodegeneration assays

The longevity, cell death, and brain morphology assays were performed as described in Petersen et al. (2012). Statistical analysis for the longevity assay was either log-rank analysis via the chi-square statistic (Figure 2, A and B) or one-way ANOVA with Bonferroni posttest (Figure 4). Statistical analyses of the cell death data were performed using one-way ANOVA with Bonferroni posttest, except for Figure 2C, which was analyzed using Student’s t-test. Statistical analyses were performed using Prism software (Graphpad).

Figure 2.

Figure 2

Open in a new tab

Longevity correlated with climbing ability in ATM8 and Repo-ATMi flies, and cell death in the brain correlated with climbing ability in ATM8 flies. (A and B) Graphed is the longevity of (A) ATM8 and (B) Repo-ATMi flies that were countercurrent separated into poor, moderate, and good climber groups. For ATM8 flies, the analysis was performed on 409 poor climbers, 178 moderate climbers, and 122 good climbers. For Repo-ATMi flies, the analysis was performed on 625 poor climbers, 231 moderate climbers, and 197 good climbers. Error bars indicate standard errors of the mean. Dashed lines indicate the median survival for each group. The difference between ATM8 poor and good climbers is significant (P < 0.01), and the difference between Repo-ATMi poor, moderate, and good climbers is significant for all comparisons (P < 0.01). (C) Graphed is the number of CaspAct-positive cells counted in the brains of ATM8 flies that were countercurrent separated into poor or good climbers. Flies were assayed either 0 or 4 days after countercurrent separation. Each circle represents a single brain. Horizontal lines indicate the average. *P < 0.05 based on one-way ANOVA analysis.

Figure 4.

Figure 4

Open in a new tab

Relish mutations suppressed the longevity defect of ATM8 flies. (A and B) Graphed is the median survival in days of 200 flies of the indicated genotype in (A) a wild-type or (B) an ATM8 background. Note that the scale is different for A and B. Complete longevity graphs are presented in Figure S2. For statistical analysis of A, mutant flies were compared to control w1118 flies, and for statistical analysis of B, mutant flies were compared to control ATM8 flies. **P < 0.01, based on one-way ANOVA analysis.

RNA isolation and qPCR

RNA isolation and qPCR was performed according to Petersen et al. (2012). Sequences of primer sets for Cactus, Relish, and Dif are provided in Table S1. Sequences of other primer sets are provided in Petersen et al. (2012). Statistical analyses of the qPCR data were performed using one-way ANOVA with Bonferroni posttest.

Results

Climbing ability correlates with neurodegeneration in ATM mutant flies

Previously, we found that ATM8 and Repo-ATMi flies are heterogeneous in their ability to climb (Petersen et al. 2012). When tapped to the bottom of a vial, flies normally respond by climbing to the top, a behavior called negative geotaxis. About 50% of ATM8 or Repo-ATMi flies have impaired climbing ability and are unable to climb beyond the bottom quarter of a vial within 30 sec of being tapped to the bottom. In contrast, ∼15% of ATM8 or Repo-ATMi flies have normal climbing ability and are able to climb to the top quarter of a vial within 30 sec of being tapped to the bottom. We reasoned that if climbing ability is a reflection of neurodegeneration, then the variability in climbing ability among ATM8 and Repo-ATMi flies provides an opportunity to identify the molecular causes of neurodegeneration.

To determine whether climbing ability is a reflection of neurodegeneration, we used a countercurrent assay to separate flies based on climbing ability (Benzer 1967). Flies were separated into three groups: poor climbers were unable to climb beyond the top of a vial in any of four 1-min trials (vial 1 in Figure 1), moderate climbers were able to climb beyond the top of a vial between one and three times in four to seven 1-min trials (vials 2–4), and good climbers were able to climb beyond the top of a vial four times in seven 1-min trials (vials 5–8). For ATM8 and Repo-ATMi flies, 55% and 70% were poor climbers, 30% and 16% were moderate climbers, and 15% and 14% were good climbers, respectively. In contrast, for control w1118 flies, 13% were either poor or moderate climbers and 87% were good climbers. Since the percentage of flies in the poor, moderate, and good climber groups was similar to the percentage of flies in the bottom, middle, and top of the vial in the 30-sec climbing assay, respectively, we concluded that the countercurrent method accurately separates flies based on climbing ability (Petersen et al. 2012).

To determine whether differences in climbing ability are due to differences in the severity of neurodegeneration, we subjected countercurrent-separated flies to two assays for neurodegeneration. First, flies were assayed for longevity. Commonly, neurodegeneration shortens the longevity of flies (Lessing and Bonini 2009). For both ATM8 and Repo-ATMi flies, poor climbers had significantly shorter longevity than good climbers (P < 0.01) and moderate climbers had intermediate longevity (Figure 2, A and B). Second, ATM8 flies were assayed for cell death in the brain. To identify cells undergoing apoptosis, brains were stained with an antibody to the activated form of Caspase-3 (CaspAct) and analyzed by immunofluorescence microscopy (Kamada et al. 2005; Fan and Bergmann 2010; Petersen et al. 2012). When ATM8 flies were assayed immediately after countercurrent separation, poor climbers relative to good climbers exhibited a significantly higher average number of CaspAct-positive cells (24.0 ± 5.0 and 12.6 ± 2.6 cells per brain, respectively (P = 0.04)) (Figure 2C). However, when ATM8 flies were assayed 4 days after countercurrent separation, the number of CaspAct-positive cells was not significantly different between poor and good climbers (P = 0.67). Thus, immediately after separation, climbing ability directly correlated with cell death, but this difference was lost over time. In summary, the longevity and cell death data indicate that climbing ability reflects the severity of neurodegeneration: poor-climbing flies have more neurodegeneration than good-climbing flies.

Climbing ability correlates with the expression of IIR genes in ATM mutant flies

The variability in neurodegeneration among ATM8 and Repo-ATMi flies could be due to differences in ATM kinase activity. To test this possibility, we analyzed the ability of flies to carry out ATM-mediated phosphorylation of H2Av in response to IR (Petersen et al. 2012). ATM8 flies that were separated by climbing ability and control w1118 flies that were not separated were exposed to 50 Gy of IR and Western blot analysis was used to detect H2Av-pS137 in head extracts. As expected, no H2Av-pS137 was detected in flies not exposed to IR, and a high level of H2Av-pS137 was detected in w1118 flies exposed to IR (Figure 3A). In contrast, irrespective of climbing ability, H2Av-pS137 was not detected in ATM8 flies exposed to IR. These data indicate that the variability in neurodegeneration is not due to differences in ATM kinase activity.

Figure 3.

Figure 3

Open in a new tab

The expression of IIR genes correlated with climbing ability in ATM8 and Repo-ATMi flies. (A) Western blot analysis for H2Av-pS137 in adult head extracts from control w1118 (lanes 1 and 2) and ATM8 (lanes 3–8) flies exposed (+) or not exposed (−) to IR. w1118 flies were not separated by the countercurrent assay, and ATM8 flies were separated by the countercurrent assay into poor, moderate, and good climbers. As a loading control, the same membrane was probed for α-tubulin (bottom). (B–E) Graphed is qPCR analysis depicting the fold change in expression of the indicated genes in (B and C) ATM8 flies relative to unseparated control w1118 flies or in (D and E) Repo-ATMi flies relative to unseparated control Repo-GAL4 flies. Note that the scale is different for each panel. Error bars indicate standard errors of the mean. Asterisks above a bar indicate a significant difference relative to the control and asterisks above a line that spans poor and good climber bars indicate a significant difference between poor and good climber groups. *P < 0.05, **P < 0.01 based on one-way ANOVA analysis.

Alternatively, our prior studies indicated that the variability in neurodegeneration among ATM8 and Repo-ATMi flies could be due to differences in the expression of IIR genes. To test this possibility, we used quantitative real-time PCR (qPCR) to determine the level of IIR gene expression in the heads of countercurrent-separated flies. Genes that were examined included AMPs [Attacin C (AttC), Cecropin A1 (CecA1), Diptericin B (DiptB), and Metchnikowin (Mtk)] and PGRP-SC2 that are targets of both the Imd and the Toll pathways, Relish that is specific to the Imd pathway, and Cactus and Dif that are specific to the Toll pathway (Ten et al. 1992; Sun et al. 1993; Lemaitre and Hoffmann 2007). For ATM8 flies, AttC, CecA1, DiptB, Mtk, PGRP-SC2, and Relish were upregulated in all of the separated groups (except for good-climbing flies in the case of PGRP-SC2), but Cactus and Dif were not upregulated in any of the separated groups (Figure 3, B and C). These data suggest that loss of ATM kinase activity upregulates the Imd pathway but not the Toll pathway. Furthermore, poor climbers had significantly higher AMP gene expression than good climbers and moderate climbers had an intermediate level of expression (Figure 3B). Thus, the level of AMP gene expression directly correlates with the severity of neurodegeneration in ATM8 flies. Similar trends were observed for Repo-ATMi flies, but only CecA1, DiptB, and Relish had significantly higher expression in poor climbers relative to good climbers (Figure 3, D and E). Taken together, these data indicate that the variability in neurodegeneration could be due to differences in the expression of Imd IIR pathway genes in glial cells.

Relish upregulates the expression of IIR genes in ATM8 and ATM8/+ flies

To determine whether the Imd pathway is necessary for activation of the IIR, we examined the expression of IIR genes in ATM8/+ and ATM8 flies that carried mutations in Relish (Rel) or Imd. The RelE20 and RelE38 alleles result from imprecise excision of a P element (Hedengren et al. 1999). Both alleles eliminate the Relish translation start codon, suggesting that they are null alleles. The ImdEY08573 allele contains a P-element insertion within the 5′-UTR, and the Imd10191 and ImdSDK alleles have unknown lesions. However, all three Imd alleles inhibit activation of the IIR by gram-negative bacteria (Taylor and Kimbrell 2007; Costa et al. 2009). To determine whether effects on the expression of IIR genes are specific to the Imd pathway, we examined ATM8/+ and ATM8 flies that carried a mutation in the Toll pathway gene Dif. The Dif1 allele contains a missense mutation that is thought to inhibit the ability of Dif to interact with DNA (Rutschmann et al. 2000).

qPCR analysis of RNA isolated from fly heads revealed that, relative to control w1118 flies, both ATM8/+ and ATM8 flies had significantly higher expression of IIR genes, with ATM8/+ flies having an intermediate level of expression (Table 1). This is consistent with the intermediate level of ATM kinase activity in ATM8/+ flies relative to w1118 and ATM8 flies (Petersen et al. 2012). The Dif1 mutation significantly increased the expression of IIR genes in ATM8/+ flies but had no effect in ATM8 flies, and the ImdEY08573, ImdSDK, and Imd10191 mutations significantly reduced the expression of IIR genes in ATM8/+ flies but had no effect in ATM8 flies. At this point, it is unclear why Dif and Imd mutations affected the expression of IIR genes in the context of intermediate but not negligible ATM kinase activity. In contrast, Relish mutations significantly reduced the expression of IIR genes in both ATM8/+ and ATM8 flies. For almost all of the IIR genes tested, the RelE20 and RelE38 mutations reduced expression to near normal levels. These data indicate that Relish is necessary for transcription upregulation of IIR genes in ATM mutants.

Table 1. Effects of IIR gene mutations on gene expression.

w1118 RelE20 RelE38 Dif1 ImdEY08573 ImdSDK Imd10191
WT
AttC 1.0 ± 0.2 0.7 ± 0.1 40.0 ± 12.0* 4.5 ± 1.2 0.3 ± 0.4 2.8 ± 0.4 0.7 ± 0.2
CecA1 1.0 ± 0.2 0.5 ± 0.2 0.4 ± 0.4 1.3 ± 0.6 0.5 ± 0.2 1.1 ± 0.2 0.2 ± 0.01**
DiptB 1.0 ± 0.4 0.4 ± 0.1 1.1 ± 0.3 2.0 ± 0.3* 0.6 ± 0.1 1.4 ± 0.1 0.1 ± 0.02**
Mtk 1.0 ± 0.2 0.9 ± 0.4 12.8 ± 2.8* 1.4 ± 0.4 1.1 ± 0.2 2.8 ± 0.5 0.8 ± 0.1
PGRP-SC2 1.0 ± 0.3 0.8 ± 0.2 0.5 ± 0.1 0.5 ± 0.1 0.7 ± 0.1 0.4 ± 0.1** 0.2 ± 0.1**
ATM8/+
AttC 39.4 ± 13.1 1.7 ± 0.6** 3.6 ± 0.9** 223.1 ± 43.6* 3.0 ± 0.9** 7.4 ± 1.5** 12.0 ± 4.2**
CecA1 36.0 ± 13.5 0.8 ± 0.3** 0.1 ± 0.1** 158.3 ± 38.0* 1.46 ± 0.4** 2.3 ± 0.7** 8.7 ± 3.0**
DiptB 11.5 ± 2.7 1.0 ± 0.2** 1.5 ± 0.5** 54.7 ± 10.3* 1.2 ± 0.3** 2.5 ± 0.9** 6.8 ± 1.8
Mtk 50.9 ± 10.1 1.1 ± 0.4** 9.7 ± 1.7** 88.0 ± 26.1* 10.1 ± 2.1** 4.1 ± 1.3** 24.7 ± 5.0**
PGRP-SC2 1.3 ± 0.4 1.0 ± 0.2 0.4 ± 0.1 8.6 ± 4.3* 0.7 ± 0.3 0.4 ± 0.04 0.6 ± 0.2
ATM8
AttC 156.5 ± 48.0 0.6 ± 0.2** 4.8 ± 1.7** 161.5 ± 59.7 149.7 ± 11.0 115.8 ± 29.7 189.5 ± 37.9
CecA1 65.4 ± 19.4 0.2 ± 0.1** 0.1 ± 0.01** 109.5 ± 29.4 141.3 ± 30.1 127.2 ± 58.6 45.0 ± 5.6
DiptB 45.5 ± 13.1 0.7 ± 0.3** 0.5 ± 0.1** 29.1 ± 8.7 41.8 ± 8.9 39.8 ± 7.4 51.8 ± 11.3
Mtk 134.9 ± 31.7 0.8 ± 0.4** 11.1 ± 3.2** 60.6 ± 11.6 132.3 ± 27.0 53.6 ± 6.9 134.2 ± 20.4
PGRP-SC2 5.7 ± 2.1 0.5 ± 0.1** 0.4 ± 0.04** 7.4 ± 3.0 20.0 ± 4.6 8.0 ± 3.4 4.1 ± 1.6
Open in a new tab
*

P < 0.05, significant increase in expression relative to the control; **P < 0.05, significant reduction in expression relative to the control. All values were normalized to the expression level in w1118 flies.

Relish activation causes neurodegeneration in ATM8/+ and ATM8 flies

To determine whether Relish is necessary for neurodegeneration in ATM mutant flies, we examined the effect of Relish mutations on longevity and cell death in the brain. In the longevity assay, the number of days that it took for 50% of the flies to die (i.e., median survival) was used as a measure of longevity. The longevity of ATM8 flies was not affected by Dif or Imd mutations, but it was significantly increased from 16.1 to 24.3 or 24.9 days by the RelE20 and RelE38 mutations, respectively (Figure 4). The complete longevity graphs revealed that Relish mutations delayed the onset of death in ATM8 flies, suggesting that Relish functions to initiate neurodegeneration (Figure S2). These data indicate that Relish is necessary for neurodegeneration in ATM8 flies.

In the cell death assay, the number of CaspAct-positive cells per adult brain was used as a measure of cell death. Rel, Dif, and Imd mutant flies had a similar level of cell death to that of control w1118 flies (Figure 5A). However, ATM8/+ and ATM8 flies had a significantly higher level of cell death than control flies, with ATM8/+ flies having an intermediate level (Figure 5, B and C). The Dif1, ImdEY08573, ImdSDK, and Imd10191 mutations did not affect the level of cell death in ATM8/+ and ATM8 flies. In contrast, the RelE20 and RelE38 mutations significantly reduced the level of cell death in ATM8/+ and ATM8 flies (P < 0.01). These data indicate that Relish is necessary for neurodegeneration in ATM8/+ and ATM8 flies.

Figure 5.

Figure 5

Open in a new tab

Relish mutation reduced cell death in the brain of ATM8 flies. (A–C) Graphed is the number of CaspAct-positive cells in the brains of flies of the indicated genotype in a (A) wild-type, (B) ATM8/+, or (C) ATM8 background. Note that the scale is different for each panel. Each circle represents a single brain. Horizontal lines indicate the average. For statistical analysis of A, mutant flies were compared to control w1118 flies. For statistical analysis of B, mutant flies were compared to control ATM8/+ flies. For statistical analysis of C, mutant flies were compared to control ATM8 flies. **P < 0.01, based on one-way ANOVA analysis.

Expression of constitutively active Relish in glial cells is sufficient to activate the IIR and to cause neurodegeneration

The data presented thus far indicate that Relish activity in glial cells is required to activate the IIR and cause neurodegeneration in ATM mutant flies. To determine whether Relish activation in glial cells is sufficient to produce these outcomes, we characterized Repo-GAL4,UAS-RelD flies that overexpressed the constitutively active Relish NF-κB motif only in glial cells (Sepp et al. 2001; DiAngelo et al. 2009). To control for effects due to protein overexpression, we characterized Repo-GAL4,UAS-GFP (Green fluorescent protein) flies. To control for effects due to Relish expression that are independent of Relish activation, we characterized Repo-GAL4,UAS-Relish and Repo-GAL4,UAS-Rel-49 flies, which express full-length Relish and the Relish IκB domain, respectively, and do not activate the transcription of Relish target genes (Hedengren et al. 1999; Wiklund et al. 2009). To control for the cell type specificity of the effects, we used Elav-GAL4 to drive overexpression of these UAS transgenes only in neurons (Luo et al. 1994; Yao and White 1994).

qPCR was used to determine the level of expression of IIR genes in the heads of flies that overexpressed Relish proteins. This analysis revealed that overexpression of constitutively active Relish, RelD, in glial cells was sufficient to activate the expression of IIR genes (Table 2). In contrast, expression of full-length Relish or the Relish IκB domain in glial cells had no effect on the expression of IIR genes. Furthermore, expression of RelD in neurons had no effect on the expression of IIR genes, indicating that glial cells are specifically competent to respond to Relish activation. These data indicate that Relish activation in glial cells is sufficient to upregulate the expression of IIR genes.

Table 2. Effects of Relish overexpression in glial cells or neurons on gene expression.

Repo-GAL4
 Elav-GAL4
UAS-GFP UAS-Relish UAS-Rel-49 UAS-RelD UAS-GFP UAS-RelD
AttC 1.0 ± 0.4 1.1 ± 0.5 0.5 ± 0.2 84.1 ± 32.3* 1.0 ± 0.3 1.1 ± 0.5
CecA1 1.0 ± 0.3 0.6 ± 0.1 1.0 ± 0.3 442.4 ± 70.7* 1.0 ± 0.5 0.6 ± 0.3
DiptB 1.0 ± 0.3 0.2 ± 0.1 0.2 ± 0.1 31.5 ± 7.9* 1.0 ± 0.3 0.4 ± 0.1
Mtk 1.0 ± 0.3 0.7 ± 0.3 0.7 ± 0.1 70.1 ± 19.1* 1.0 ± 0.2 1.9 ± 0.6
Rel 1.0 ± 0.1 8.2 ± 1.7** 0.9 ± 0.2 20.8 ± 4.8** 1.0 ± 0.1 4.5 ± 1.8**
Open in a new tab

Values were normalized to UAS-GFP expression for each GAL4 construct. *P < 0.01, significant increase compared to Repo-GAL4,UAS-GFP and Elav-GAL4,UAS-RelD; **P < 0.05, significant increase compared to the corresponding UAS-GFP control.

The CaspAct cell death assay was used to determine the level of neurodegeneration in the heads of flies that overexpressed Relish proteins. This analysis revealed that only overexpression of constitutively active Relish, RelD, in glial cells caused a significant increase in the level of neurodegeneration (Figure 6A). In addition, RelD expression in glial cells, but not in neurons, caused vacuolar-like holes in the lamina (Figure 6, B and C). A similar phenotype was observed in Repo-ATMi flies (Figure 6D). Vacuolar-like holes in the adult brain also occur in ATM8 flies and are a common feature of neurodegeneration in flies (Lessing and Bonini 2009; Petersen et al. 2012). Thus, Relish activation in glial cells is sufficient to cause neurodegeneration.

Figure 6.

Figure 6

Open in a new tab

Overexpression of a constitutively active form of Relish, RelD, in glial cells but not in neurons caused neurodegeneration. (A) Graphed is the number of CaspAct-positive cells in the brains of flies of the indicated genotype. (B–D) Images of paraffin sections of the optic lobe and retina of flies of the indicated genotypes. In B, the optic lobe (OL), lamina (LA), and retina (RE) are labeled. In C and D, open arrowheads indicate vacuolar-like holes in the lamina.

Discussion

Insights into why neurodegeneration occurs in A-T

Our data indicate that a glial cell IIR promotes neurodegeneration in ATM mutant flies. Furthermore, our data indicate that the level of activation of the IIR affects the severity of neurodegeneration. Flies with the same ATM mutation activated the IIR to different extents and had correspondingly different severities of neurodegeneration (Figures 1–3). This suggests that there are environmental or genetic factors that modulate the IIR in ATM mutant flies. Analogous factors may underlie variability in the severity of neurodegeneration in A-T patients. For example, suppressing factors may explain why some individuals with undetectable levels of ATM have mild clinical phenotypes (Lavin et al. 2006).

Relish activation in the process of neurodegeneration

Our data indicate that the NF-κB transcription factor Relish is necessary for activation of the IIR and neurodegeneration in ATM mutant flies. Likewise, Chinchore et al. (2012) have also shown that Relish is necessary for activation of the IIR and neurodegeneration in a fly model of human retinal degeneration. However, in neither case is it known what factor substitutes for the pathogen to initiate the IIR or what pathway of events is required to trigger Relish activation. We found that mutation of Imd, a canonical activator of Relish in response to pathogens, had variable effects on the expression of IIR genes and no effect on neurodegeneration (Figures 4 and 5 and Table 1). Likewise, Chinchore et al. (2012) found that mutation of Imd had no effect on neurodegeneration. This suggests that a noncanonical pathway activates Relish to cause neurodegeneration. The unknown pathway may be specific to Relish since mutation of another NF-κB protein Dif did not affect neurodegeneration in ATM mutant flies (Figures 4 and 5). This does not mean that Dif activation cannot cause neurodegeneration. In fact, Dif has been implicated in causing neurodegeneration in a fly model of Alzheimer’s disease (Tan et al. 2008). Therefore, future efforts will focus on determining the mechanisms that regulate Relish activation in ATM mutant cells.

Relish transcriptional targets that cause neurodegeneration

Our data indicate that proteins encoded by transcriptional targets of Relish cause neurodegeneration in ATM mutant flies. Indeed, constitutive activation of Relish in glial cells was sufficient to upregulate the expression of IIR genes and to cause neurodegeneration (Table 2 and Figure 6). Proteins encoded by AMP genes are obvious candidates because their expression was highly increased in ATM mutant flies and correlated with the severity of neurodegeneration (Figure 3 and Table 1). AMPs are small secreted peptides that contribute to the elimination of pathogens through mechanisms that remain largely unknown. However, it is generally believed that positively charged AMPs bind negatively charged membranes of pathogens, leading to increased membrane permeability and death (Zaiou 2007). Thus, neurodegeneration may be caused by AMP-mediated disruption of neuron membranes. Arguing against a role for AMPs in neurodegeneration is our finding that neurodegeneration was not affected in ATM8/+ flies that have a Dif or Imd mutation, despite the fact that the level of AMP gene expression was significantly affected (Figure 5B and Table 1). Therefore, knowledge of the genome-wide transcriptional changes caused by Rel mutations in ATM mutant flies may help identify gene targets that are relevant to neurodegeneration.

Supplementary Material

Supporting Information
supp_194_1_133__index.html (1.4KB, html)

Acknowledgments

We thank Nicole Bertram, Grace Boekhoff-Falk, Barry Ganetzky and members of his laboratory, and Randy Tibbetts for their insights throughout the course of the research. We also thank Satoshi Kinoshita for paraffin sectioning. Flies were kindly provided by Mimi Shirasu-Hiza, Sara Cherry, Morris Birnbaum, and Barry Ganetzky. This work was supported by a grant from the National Institutes of Health (NIH) (R01 NS059001 to D.A.W.) and a predoctoral fellowship from NIH training grant T32 GM08688 (to A.J.P.).

Footnotes

Communicating editor: I. K. Hariharan

Literature Cited

  1. Amor S., Puentes F., Baker D., van der Valk P., 2010.  Inflammation in neurodegenerative disease. Immunology 129: 154–169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bakkenist C. J., Kastan M. B., 2003.  DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 421: 499–506. [DOI] [PubMed] [Google Scholar]
  3. Benzer S., 1967.  Behavioral mutants of Drosophila isolated by countercurrent distribution. Proc. Natl. Acad. Sci. USA 58: 1112–1119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bhatti S., Kozlov S., Farooqi A. A., Naqi A., Lavin M., et al. , 2011.  ATM protein kinase: the linchpin of cellular defenses to stress. Cell. Mol. Life Sci. 68: 2977–3006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brand A. H., Perrimon N., 1993.  Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118: 401–415. [DOI] [PubMed] [Google Scholar]
  6. Bundey S., 1994.  Clinical and genetic features of ataxia-telangiectasia. Int. J. Radiat. Biol. 66: S23–S29. [PubMed] [Google Scholar]
  7. Chinchore Y., Gerber G. F., Dolph P. J., 2012.  Alternative pathway of cell death in Drosophila mediated by NF-κB transcription factor Relish. Proc. Natl. Acad. Sci. USA 109: E605–E612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Costa A., Jan E., Sarnow P., Schneider D., 2009.  The Imd pathway is involved in antiviral immune responses in Drosophila. PLoS ONE 4: e7436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Derheimer F. A., Kastan M. B., 2010.  Multiple roles of ATM in monitoring and maintaining DNA integrity. FEBS Lett. 584: 3675–3681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. DiAngelo J. R., Bland M. L., Bambina S., Cherry S., Birnbaum M. J., 2009.  The immune response attenuates growth and nutrient storage in Drosophila by reducing insulin signaling. Proc. Natl. Acad. Sci. USA 106: 20853–20858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fan Y., Bergmann A., 2010.  The cleaved-Caspase-3 antibody is a marker of Caspase-9-like DRONC activity in Drosophila. Cell Death Differ. 17: 534–539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Han S. H., Ryu J. H., Oh C. T., Nam K. B., Nam H. J., et al. , 2004.  The moleskin gene product is essential for Caudal-mediated constitutive antifungal Drosomycin gene expression in Drosophila epithelia. Insect Mol. Biol. 13: 323–327. [DOI] [PubMed] [Google Scholar]
  13. Hedengren M., Asling B., Dushay M. S., Ando I., Ekengren S., et al. , 1999.  Relish, a central factor in the control of humoral but not cellular immunity in Drosophila. Mol. Cell 4: 827–837. [DOI] [PubMed] [Google Scholar]
  14. Kamada S., Kikkawa U., Tsujimoto Y., Hunter T., 2005.  Nuclear translocation of caspase-3 is dependent on its proteolytic activation and recognition of a substrate-like protein(s). J. Biol. Chem. 280: 857–860. [DOI] [PubMed] [Google Scholar]
  15. Lavin M. F., Delia D., Chessa L., 2006.  ATM and the DNA damage response. Workshop on ataxia-telangiectasia and related syndromes. EMBO Rep. 7: 154–160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lemaitre B., Hoffmann J., 2007.  The host defense of Drosophila melanogaster. Annu. Rev. Immunol. 25: 697–743. [DOI] [PubMed] [Google Scholar]
  17. Lessing D., Bonini N. M., 2009.  Maintaining the brain: insight into human neurodegeneration from Drosophila melanogaster mutants. Nat. Rev. Genet. 10: 359–370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Luo L., Liao Y. J., Jan L. Y., Jan Y. N., 1994.  Distinct morphogenetic functions of similar small GTPases: Drosophila Drac1 is involved in axonal outgrowth and myoblast fusion. Genes Dev. 8: 1787–1802. [DOI] [PubMed] [Google Scholar]
  19. Matsuoka S., Ballif B. A., Smogorzewska A., McDonald E. R., Hurov K. E., et al. , 2007.  ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. Science 316: 1160–1166. [DOI] [PubMed] [Google Scholar]
  20. McKinnon P. J., 2004.  ATM and ataxia telangiectasia. EMBO Rep. 5: 772–776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pedersen M., Tiong S., Campbell S. D., 2010.  Molecular genetic characterization of Drosophila ATM conserved functional domains. Genome 53: 778–786. [DOI] [PubMed] [Google Scholar]
  22. Peng J., Zipperlen P., Kubli E., 2005.  Drosophila sex peptide stimulates female innate immune system after mating via the Toll and Imd pathways. Curr. Biol. 15: 1690–1694. [DOI] [PubMed] [Google Scholar]
  23. Petersen A. J., Wassarman D. A., 2012.  Drosophila innate immune response pathways moonlight in neurodegeneration. Fly 6: 169–172. [DOI] [PubMed] [Google Scholar]
  24. Petersen A. J., Rimkus S. A., Wassarman D. A., 2012.  ATM kinase inhibition in glial cells activates the innate immune response and causes neurodegeneration in Drosophila. Proc. Natl. Acad. Sci. USA 109: E656–E664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rimkus S. A., Katzenberger R. J., Trinh A. T., Dodson G. E., Tibbetts R. S., et al. , 2008.  Mutations in String/CDC25 inhibit cell cycle re-entry and neurodegeneration in a Drosophila model of Ataxia telangiectasia. Genes Dev. 22: 1205–1220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rutschmann S., Jung A. C., Hetru C., Reichhart J. M., Hoffmann J. A., et al. , 2000.  The Rel protein DIF mediates the antifungal but not the antibacterial host defense in Drosophila. Immunity 12: 569–580. [DOI] [PubMed] [Google Scholar]
  27. Ryu J. H., Nam K. B., Oh C. T., Nam H. J., Kim S. H., et al. , 2004.  The homeobox gene Caudal regulates constitutive local expression of antimicrobial peptide genes in Drosophila epithelia. Mol. Cell. Biol. 24: 172–185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Savitsky K., Bar-Shira A., Gilad S., Rotman G., Ziv Y., et al. , 1995.  A single ataxia telangiectasia gene with a product similar to PI-3 kinase. Science 268: 1749–1753. [DOI] [PubMed] [Google Scholar]
  29. Sedgwick R. P., Boder E., 1960.  Progressive ataxia in childhood with particular reference to ataxia-telangiectasia. Neurology 10: 705–715. [DOI] [PubMed] [Google Scholar]
  30. Senger K., Harris K., Levine M., 2006.  GATA factors participate in tissue-specific immune responses in Drosophila larvae. Proc. Natl. Acad. Sci. USA 103: 15957–15962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sepp K. J., Schulte J., Auld V. J., 2001.  Peripheral glia direct axon guidance across the CNS/PNS transition zone. Dev. Biol. 238: 47–63. [DOI] [PubMed] [Google Scholar]
  32. Silva E., Tiong S., Pedersen M., Homola E., Royou A., et al. , 2004.  ATM is required for telomere maintenance and chromosome stability during Drosophila development. Curr. Biol. 14: 1341–1347. [DOI] [PubMed] [Google Scholar]
  33. Sun S. C., Ganchi P. A., Ballard D. W., Greene W. C., 1993.  NF-kappa B controls expression of inhibitor I kappa B alpha: evidence for an inducible autoregulatory pathway. Science 259: 1912–1915. [DOI] [PubMed] [Google Scholar]
  34. Tan L., Schedl P., Song H. J., Garza D., Konsolaki M., 2008.  The Toll→NFkappaB signaling pathway mediates the neuropathological effects of the human Alzheimer’s Abeta42 polypeptide in Drosophila. PLoS ONE 3: e3966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Taylor K., Kimbrell D. A., 2007.  Host immune response and differential survival of the sexes in Drosophila. Fly 1: 197–204. [DOI] [PubMed] [Google Scholar]
  36. Ten R. M., Paya C. V., Israël N., Le Bail O., Mattei M. G., et al. , 1992.  The characterization of the promoter of the gene encoding the p50 subunit of NF-kappa B indicates that it participates in its own regulation. EMBO J. 11: 195–203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tzou P., Ohresser S., Ferrandon D., Capovilla M., Reichhart J. M., et al. , 2010.  Tissue-specific inducible expression of antimicrobial peptide genes in Drosophila surface epithelia. Immunity 25: 1–5. [DOI] [PubMed] [Google Scholar]
  38. Wiklund M.-L., Steinert S., Junell A., Hultmark D., Stöven S., 2009.  The N-terminal half of the Drosophila Rel/NF-kappaB factor Relish, REL-68, constitutively activates transcription of specific Relish target genes. Dev. Comp. Immunol. 33: 690–696. [DOI] [PubMed] [Google Scholar]
  39. Xiong W. C., Okano H., Patel N. H., Blendy J. A., Montell C., 1994.  repo encodes a glial-specific homeodomain protein required in the Drosophila nervous system. Genes Dev. 8: 981–994. [DOI] [PubMed] [Google Scholar]
  40. Yao K. M., White K., 1994.  Neural specificity of elav expression: defining a Drosophila promoter for directing expression to the nervous system. J. Neurochem. 63: 41–51. [DOI] [PubMed] [Google Scholar]
  41. Zaiou M., 2007.  Multifunctional antimicrobial peptides: therapeutic targets in several human diseases. J. Mol. Med. 85: 317–329. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Information
supp_194_1_133__index.html (1.4KB, html)
ee293cf9b5ae6f9faf904a68477e516a_genetics.113.150854-1.pdf (118.7KB, pdf)
b7d6e2c29ceacc0b9a7b491bbd86d0d5_genetics.113.150854-3.pdf (64.6KB, pdf)
cb12eb03f85782b1a4cef585cdf8649e_genetics.113.150854-2.pdf (115.2KB, pdf)
6555122c7e82b7eba78515396e54c1df_genetics.113.150854-4.pdf (57KB, pdf)

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES