ATM kinase inhibition in glial cells activates the innate immune response and causes neurodegeneration in Drosophila

Abstract

To investigate the mechanistic basis for central nervous system (CNS) neurodegeneration in the disease ataxia–telangiectasia (A-T), we analyzed flies mutant for the causative gene A-T mutated (ATM). ATM encodes a protein kinase that functions to monitor the genomic integrity of cells and control cell cycle, DNA repair, and apoptosis programs. Mutation of the C-terminal amino acid in Drosophila ATM inhibited the kinase activity and caused neuron and glial cell death in the adult brain and a reduction in mobility and longevity. These data indicate that reduced ATM kinase activity is sufficient to cause neurodegeneration in A-T. ATM kinase mutant flies also had elevated expression of innate immune response genes in glial cells. ATM knockdown in glial cells, but not neurons, was sufficient to cause neuron and glial cell death, a reduction in mobility and longevity, and elevated expression of innate immune response genes in glial cells, indicating that a non–cell-autonomous mechanism contributes to neurodegeneration in A-T. Taken together, these data suggest that early-onset CNS neurodegeneration in A-T is similar to late-onset CNS neurodegeneration in diseases such as Alzheimer's in which uncontrolled inflammatory response mediated by glial cells drives neurodegeneration.

Ataxia–telangiectasia (A-T) is a multisystem disease characterized by neurodegeneration in the central nervous system (CNS) (1–3). Progressive neurodegeneration occurs during childhood and mainly affects Purkinje and granule cells in the cerebellum. A-T is caused by mutation of the A-T mutated (ATM) gene, which encodes a serine/threonine protein kinase of the PI3K-related kinase (PIKK) family (4). ATM functions to ensure genome integrity by controlling the cell cycle, DNA repair, and apoptosis programs in response to DNA damage (5, 6). ATM is recruited to sites of DNA damage, where autophosphorylation activates the kinase activity to allow phosphorylation of many downstream targets, including histone H2AX (7–9).

Insights into why ATM loss causes neurodegeneration have come from analyses of ATM-deficient animals. Analyses of postmortem human A-T brains, as well as mouse and fly models of A-T, indicate that ATM protects postmitotic neurons from degeneration by suppressing cell cycle reentry (10, 11). ATM-deficient neurons reenter the cell cycle and may die rather than arrest or divide because of excessive DNA damage. Many aspects of neurodegeneration are not understood, including whether it is solely caused by ATM loss in neurons (12). In neurodegenerative diseases such as Alzheimer's and Parkinson disease, prolonged activation of microglia—the resident innate immune cells in the CNS—is thought to cause neurotoxicity (13–15). In addition, in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), dysfunction of astrocytes, which serve to maintain neuron metabolism and neurotransmitter release, drives neurodegeneration after onset (16). In the case of A-T, abnormal microglial cell morphology is observed in ATM knockout (KO) mice, and cultured astrocytes from ATM KO mice show markers of oxidative and endoplasmic reticulum stress and exhibit a senescence-like growth defect (17, 18). Furthermore, several lines of evidence suggest that systemic inflammation contributes to pathogenesis in patients with A-T (19, 20). Thus, ATM loss in glial cells may contribute to neurodegeneration in A-T. To investigate this possibility, we analyzed Drosophila melanogaster brains in which ATM kinase activity was reduced in all cells or ATM expression was specifically reduced in glial cells or neurons.

Results

ATM Kinase Activity Is Temperature-Sensitive in ATM⁸ Flies.

Because ATM is essential for fly viability to the adult stage, a temperature-sensitive allele of ATM (ATM⁸) was used to investigate the role of ATM in adult flies (21). ATM⁸/ATM⁸ flies (hereafter referred to as ATM⁸) developed to the adult stage when raised at 18 °C but not at 25 °C. Thus, to characterize adult phenotypes, ATM⁸ flies were raised at 18 °C and shifted to 25 °C shortly after eclosion.

ATM⁸ contains a leucine to phenylalanine mutation of the C-terminal amino acid, which is predicted to affect kinase activity but not protein stability (21, 22). Western blot analysis for histone H2Av phosphorylation (H2Av-pS137) revealed that ATM kinase activity in ATM⁸ adult fly heads was temperature-sensitive (Fig. 1). H2Av is the Drosophila homologue of mammalian H2AX, an ATM substrate in response to ionizing radiation (IR)-induced DNA damage (23). At 18 °C, IR-induced H2Av phosphorylation was not affected in ATM⁸/+ flies but was highly reduced in ATM⁸ flies relative to wildtype (WT) flies. At 25 °C, IR-induced H2Av phosphorylation was slightly reduced in ATM⁸/+ flies and was below the limit of detection in ATM⁸ flies. These data indicate that ATM⁸ kinase activity is temperature-sensitive and that ATM kinase activity in adult heads is correlated with ATM⁸ allele dose.

Fig. 1.

ATM⁸ kinase activity is temperature-sensitive. Shown is Western blot analysis of H2Av-pS137 in adult head extracts from flies of the indicated genotypes exposed (+) or not exposed (−) to IR. Analyses of WT, ATM⁸/+, and ATM⁸ flies at 18 °C (lanes 1–6) and 25 °C (lanes 7–12) are shown. The Top panels show a short exposure of the Middle panels. The Bottom panels were probed for α-tubulin, which served as a loading control.

Reduced ATM Kinase Activity Reduces Mobility and Longevity.

As previously reported by Pedersen et al., ATM⁸ flies had mobility defects (21). They were less able to walk, fly, and right themselves when turned onto their backs. A climbing assay was used to quantify mobility. When tapped to the bottom of a vial, flies naturally respond by climbing to the top, referred to as negative geotaxis. Ten seconds after being tapped to the bottom of a vial, 65% of WT flies had climbed to the top quarter of the vial; however, only 19% of ATM⁸/+ and 6% of ATM⁸ flies had climbed to the top quarter of the vial (Fig. 2A). Similar effects were observed if climbing was assessed at different heights in the vial or if climbing was assessed after longer recovery times (20 or 30 s), suggesting that the climbing defect was not a result of incapacitation from the tapping action. The climbing defect was probably caused by reduced ATM kinase activity, as the severity of the defect correlated with the level of ATM kinase activity; less severe defects were observed for ATM⁸/+ flies than ATM⁸ flies. Many fly neurodegeneration mutants have decreased ability to climb, and turtle (tutl) mutants have decreased ability to right themselves when turned onto their backs (24, 25). The tutl gene is specifically expressed in the CNS and regulates the development of neuron dendrites (25). These data suggest that reduced ATM kinase activity causes neurological problems.

Fig. 2.

Reduced ATM kinase activity causes reduced mobility and longevity. (A) Graphed is the average percent of WT, ATM⁸/+, and ATM⁸ flies that climbed more than 75% (green), 50% to 75% (blue), 25% to 50% (gray), or less than 25% (red) of the vial height in the indicated time. Unlabeled bars had values of less than 5%. Statistical analysis by one-way ANOVA indicated a significant difference at all time points between ATM⁸ and WT flies (P < 0.01). (B) Graphed is the average percent survival at the indicated number of days after eclosion with error bars (SEM) for three independent trials of WT, ATM⁸/+, and ATM⁸ flies. Dotted lines indicate the 50% survival point for each genotype.

Adult fly longevity was used to further assess the physiological requirements for ATM kinase activity. In the longevity assay, flies were raised at 18 °C and shifted to 25 °C shortly after eclosion, and the number of surviving flies was determined each day. This assay revealed that ATM⁸/+ and ATM⁸ flies had significantly shorter lifespans than WT flies (P < 1 × 10⁻⁴; Fig. 2B). The 50% survival point for ATM⁸ flies was 18 d, compared with 27 d and 50 d for ATM⁸/+ and WT flies, respectively. Many fly neurodegeneration mutants have reduced longevity (24). Thus, neurodegeneration may cause reduced mobility and longevity of flies with reduced ATM kinase activity, but other factors, such as muscle degeneration, are also plausible.

Reduced ATM Kinase Activity Causes Neuron and Glial Cell Death.

Light microscopic analysis of paraffin sections of adult heads was used to assess the extent to which abnormal brain morphology accompanied the mobility and longevity defects of ATM⁸ flies. Neurons in the adult Drosophila brain have cell bodies at the periphery and neurites that extend internally to form the synaptic neuropil (26, 27). After 7 d at 25 °C, scattered, small holes were observed in the neuropil of ATM⁸/+ and ATM⁸ flies but not WT flies (Fig. 3 A–C and Fig. S1 A–C). After 17 d at 25 °C, the abundance and size of the holes was increased in ATM⁸/+ and ATM⁸ flies, which typifies progressive neurodegeneration in flies (Fig. 3 D–F and Fig. S1 D–F) (24).

Fig. 3.

Reduced ATM kinase activity causes neuron and glial cell death in the adult brain. Shown are representative paraffin sections of WT, ATM⁸/+, and ATM⁸ brains after 7 d (A–C) or 17 d (D–F) at 25 °C. Holes are indicated by arrows. Images are shown of the same region of the brain and at the same magnification, with anterior at the top. Full brain sections are shown in Fig. S1. Shown are representative immunofluorescence images of ATM⁸ brains stained for Repo and Casp^Act (G–I) or Elav and Casp^Act (J–L).

To directly assess cell death, an antibody to activated caspase 3 (α-Casp^Act) was used to detect cells undergoing apoptotic death (28). In apoptotic cells, cytoplasmic caspase 3 is activated by cleavage and translocated to the nucleus, so nuclear α-Casp^Act staining was used to identify dying cells (29). After 7 or 17 d at 25 °C, ATM⁸ flies had significantly more Casp^Act-positive cells than WT or ATM⁸/+ flies (P < 0.05; Fig. 3 H and K and Table 1). This finding suggests that continuous cell death underlies the progressive formation of holes in the brain and the reduced mobility and longevity of ATM⁸ flies.

Table 1.

Cell death in adult brains

	CaspAct cells per brain (brains)		Percent of Repo-positive CaspAct cells (CaspAct cells)
Genotype	7 d	17 d	7 d	17 d
WT	4.9 ± 1.5 (n = 14)	2.5 ± 0.5 (n = 17)	0 (n = 81)	5 (n = 43)
ATM8/+	12.4 ± 4.1 (n = 14)	7.3 ± 1.9 (n = 9)	77 (n = 77)	73 (n = 56)
ATM8	45.9 ± 8.1 (n = 14)*	21.4 ± 5.8 (n = 14)†	69 (n = 191)	58 (n = 218)
Repo-GAL4	1.8 ± 0.4 (n = 18)	2.2 ± 0.4 (n = 18)	0 (n = 32)	8 (n = 40)
Repo-ATMi	14.5 ± 3.7 (n = 16)‡	5.1 ± 1.3 (n = 18)‡	1 (n = 199)	29 (n = 131)
Elav-GAL4	2.3 ± 0.8 (n = 9)	2.1 ± 0.4 (n = 12)	10 (n = 21)	4 (n = 25)
Elav-ATMi	3.7 ± 0.7 (n = 9)	2.7 ± 0.4 (n = 12)	9 (n = 33)	3 (n = 32)
ElavC155-GAL4	2.0 ± 0.4 (n = 12)	1.3 ± 0.3 (n = 12)	0 (n = 24)	0 (n = 16)
ElavC155-ATMi	2.3 ± 0.6 (n = 12)	1.4 ± 0.3 (n = 13)	0 (n = 29)	0 (n = 18)

Values refer to mean ± SEM.

*P < 0.01 vs. WT and ATM⁸/+.

^†P < 0.05 vs. WT and ATM⁸/+.

^‡P < 0.05 vs. Repo-GAL4.

To determine the identity of the dying cells, brains were costained with antibodies to Casp^Act and to the glial cell-specific protein reversed polarity (Repo) or the neuron-specific protein embryonic lethal abnormal vision (Elav) (30, 31). This analysis revealed that neurons and glial cells were Casp^Act-positive in ATM⁸/+ and ATM⁸ flies (Fig. 3 G–L). Quantitation of α-Repo–stained brains further revealed that 58% to 77% of the dying cells were glial cells (Table 1). Thus, reduced ATM kinase activity causes neuron and glial cell death in the adult fly brain.

Reduced ATM Kinase Activity Causes Increased Expression of Innate Immune Response Genes.

Gene expression microarray analysis was used to identify molecular and biological events caused by reduced ATM kinase activity. With the goal of identifying events that are causative for neurodegeneration, adult heads were examined after 3 to 4 d at 25 °C, a time before the appearance of holes in the brain. Total RNA isolated from heads of ATM⁸/+ and ATM⁸ flies was converted to fluorescently labeled cDNA and used to probe NimbleGen oligonucleotide arrays representing approximately 15,000 Drosophila genes. The resulting data were quantile-normalized via robust multichip average and ATM⁸ was compared with ATM⁸/+ for fold change and statistical significance. Analysis of two independent biological replicates revealed that 163 genes met the criteria of a greater than twofold change with a moderated t test P value lower than 0.05 and a false discovery rate (FDR) P value lower than 0.10; 117 genes were up-regulated and 46 genes were down-regulated in ATM⁸ flies relative to ATM⁸/+ flies.

Gene Ontology analysis of the up-regulated genes identified the innate immune response as the most significantly changed biological event (32). Drosophila have an innate immune system for immune defense (33). The fat body is an immune-responsive tissue. In response to pathogens, the fat body expresses pathogen recognition proteins, such as peptidoglycan recognition proteins (PGRPs) and gram-negative binding proteins, that act upstream of the Toll and immune deficiency (Imd) signaling pathways to recognize pathogens (34–37). These pathways function through NF-κB transcription factors to express antimicrobial peptides (AMPs) and other proteins that combat pathogens (38, 39).

Twenty innate immune response categories were identified in the gene expression microarrays, each with a P value lower than 1 × 10⁻⁵. Table S1 provides a complete list of genes that are included in these categories and is based on comparisons to published microarray analyses of the Drosophila innate immune response (40–42). Table 2 provides a partial list that focuses on genes that encode pathogen recognition proteins and AMPs. The expression of PGRP genes, as well as members of six of seven classes of AMP genes, was up-regulated. The exception was Drosocin, but validation studies indicated that it was also up-regulated (Fig. S2C). Collectively, these data indicate that reduced ATM kinase activity activates the innate immune response in the adult head.

Table 2.

Up-regulated innate immunity genes

CG no.	Gene	ATM8	Repo-ATMi
Pathogen recognition genes
CG13422	GNBP-like	2.05	2.06
CG11709	PGRP-SA	2.24	—
CG9681	PGRP-SB1	2.65	3.47
CG14745	PGRP-SC2	6.45	5.22
CG7496	PGRP-SD	2.25	2.81
AMP genes
CG10146	Attacin-A (AttA)	4.16	3.11
CG18372	Attacin-B (AttB)	4.56	2.47
CG4740	Attacin-C (AttC)	3.37	2.94
CG7629	Attacin-D (AttD)	7.91	—
CG1365	Cecropin A1 (CecA1)	5.07	3.85
CG1367	Cecropin A2 (CecA2)	2.35	—
CG1878	Cecropin B (CecB)	8.55	—
CG1373	Cecropin C (CecC)	7.04	2.30
CG1385	Defensin (Def)	3.33	—
CG12763	Diptericin (Dipt)	2.29*	—
CG10794	Diptericin B (DiptB)	2.99*	—
CG10810	Drosomycin (Drs)	5.11	—
CG32282	drosomycin-4 (dro4)	2.13	—
CG8175	Metchnikowin (Mtk)	3.01	2.26

*FDR P < 0.14.

Quantitative PCR (qPCR) was used to independently examine innate immune response gene expression. Comparison of WT and ATM⁸ flies that had been shifted to 25 °C for 0, 3, or 8 d confirmed the microarray finding that reduced ATM kinase activity caused up-regulation of PGRP and AMP gene expression (Fig. 4A). Furthermore, because WT flies rather than ATM⁸/+ flies were used for the comparison, a higher level of innate immune response gene expression was observed relative to the microarray analysis. Finally, up-regulation of PGRP and AMP gene expression was detected immediately after eclosion and, in some cases, persisted through the first 8 d of adulthood. These data indicate that reduced ATM kinase activity throughout development induces a prolonged innate immune response in the head of young adult flies.

Fig. 4.

Reduced ATM kinase activity causes up-regulation of AMP gene expression in glial cells. (A) Shown is qPCR analysis of mRNA expression in ATM⁸ heads after 0, 3, or 8 d at 25 °C that was normalized to age-matched WT heads. Asterisks represent P < 0.05 based on Student t test analysis. Shown are representative immunofluorescence images of WT (B–D) or ATM⁸/+ (E–G) brains stained for Repo and detecting the AttA::GFP reporter gene. Other AMP::GFP reporter genes are shown in Fig. S2.

Reduced ATM Kinase Activity Causes AMP Gene Expression in Glial Cells.

To further characterize the innate immune response, transgenic reporter flies were used to determine the location of AMP gene expression in the head. Flies expressing green fluorescent protein (GFP) under control of AMP gene transcriptional regulatory sequences were crossed to WT or ATM⁸/+ flies at 25 °C, the progeny were cultured for 3 to 5 d, and GFP expression was examined in dissected head fat body cells and brain whole mounts (43). For the Attacin A (AttA)::GFP reporter, GFP expression was not detected in head fat body cells of ATM⁸/+ or WT flies. In contrast, GFP expression was detected in the brain of ATM⁸/+ but not WT flies (Fig. 4 C and F). GFP expression was most prominent in the outer area of the optic lobe and the region where the optic lobe juxtaposes the central brain neuropil, regions that are highly populated by neuron cell bodies (26, 27). Similar results were observed for other AMP transgenic reporter lines (Cecropin A1::GFP, Defensin::GFP, Drosomycin::GFP, Metchnikowin::GFP, and Drosocin::GFP; Fig. S2) (43). These data indicate that reduced ATM kinase activity causes transcriptional up-regulation of AMP genes in the brain.

To identify the AMP-expressing cell type in ATM⁸/+ flies, brains were stained with antibodies to Repo or Elav (30, 31). GFP was found to exclusively colocalize with Repo-positive cells, indicating that reduced ATM kinase activity causes transcriptional up-regulation of AMP genes in glial cells (Fig. 4 B–G).

ATM Knockdown in Glial Cells Causes Neurodegeneration.

The studies of ATM⁸ flies indicated that reduced ATM kinase activity in glial cells is responsible for activation of the innate immune response and neuron and glial cell death. To test this hypothesis, RNA interference (RNAi) was used to knock down ATM only in glial cells. A Repo-GAL4 transgene was used to drive expression of a pWIZ-ATM^T4 transgene that expresses an ATM short hairpin RNA (shRNA) under transcriptional control of UAS sequences (11, 44, 45). Our prior studies demonstrated that GAL4-driven expression of pWIZ-ATM^T4 specifically knocks down ATM (11). Control Repo-GAL4 flies and experimental Repo-GAL4/pWIZ-ATM^T4 (Repo-ATMi) flies were assayed as described for ATM⁸ flies.

Repo-ATMi flies had similar phenotypes to ATM⁸ flies. Repo-ATMi flies had impaired climbing ability; only 6% to 17% of Repo-ATMi flies compared with 82% to 84% of Repo-GAL4 flies climbed to the top quarter of the vial within 10, 20, or 30 s after being tapped to the bottom (Fig. 5A). Repo-ATMi flies also had significantly reduced longevity (P < 1 × 10⁻⁴; Fig. 5B). The 50% survival point for Repo-ATMi flies was 16 d, compared with 25 d and 43 d for Repo-GAL4 and WT flies, respectively. Repo-ATMi flies also had holes in the adult lamina and neuropil. At 1 d posteclosion, large holes were present in the lamina and small holes in the neuropil, and at 7 d posteclosion, numerous small and large holes were present in the neuropil (Fig. 6 A–C and Fig. S1 G–J). Finally, at 7 d and 17 d posteclosion, Repo-ATMi flies had significantly more Casp^Act-positive cells than Repo-GAL4 flies (P < 0.05; Table 1). Costaining for Casp^Act and Elav or Repo revealed that cell death at 7 d was limited to neurons, but at 17 d also included glial cells (Fig. 6 G–L and Table 1). Taken together, these data indicate that ATM is required in glial cells for the survival of neurons.

Fig. 5.

ATM knockdown in glial cells, but not neurons, causes reduced mobility and longevity. Graphed is the average percent of Repo-GAL4 and Repo-ATMi flies (A) or Elav-GAL4 and Elav-ATMi flies (C) that climbed more than 75% (green), 50% to 75% (blue), 25% to 50% (gray), or less than 25% (red) of the vial height in the indicated time. Unlabeled bars had values of less than 5%. Statistical analysis by one-way ANOVA indicated a significant difference at all time points between Repo-ATMi and Repo-GAL4 flies (P < 0.01) but not between Elav-ATMi and Elav-GAL4 flies (P > 0.05). Fig. 2A shows WT fly climbing data. Graphed is the average percent survival at the indicated number of days posteclosion with error bars (SEM) for three independent trials of WT, Repo-GAL4, and Repo-ATMi flies (B) or WT, Elav-GAL4, and Elav-ATMi flies (D). Dotted lines indicate the 50% survival point for each genotype.

Fig. 6.

ATM knockdown in glial cells, but not neurons, causes neuron and glial cell death in the adult brain. Shown are representative paraffin sections of Repo-GAL4 and Repo-ATMi brains (A–C) or Elav-GAL4 and Elav-ATMi brains (D–F) at the indicated age in days. Holes are indicated by arrows. Images are shown of the same region of the brain and at the same magnification, with anterior at the top. Full brain sections are shown in Fig. S1. Shown are representative immunofluorescence images of Repo-ATMi brains stained for Repo and Casp^Act (G–I) or Elav and Casp^Act (J–L).

ATM Knockdown in Glial Cells Causes Increased Expression of Innate Immune Response Genes.

Gene expression microarray analysis was used to identify molecular and biological events caused by ATM knockdown in glial cells. Using parameters described for the ATM⁸ microarray analysis, two independent biological replicates of 3- to 5-d-old Repo-GAL4 and Repo-ATMi flies were analyzed. This analysis revealed that 246 genes were up-regulated and 106 genes were down-regulated in Repo-ATMi flies relative to Repo-GAL4 flies.

Gene Ontology analysis of the up-regulated genes identified the innate immune response as the most significantly changed biological event (32). Twelve innate immune response categories were identified, each with a P value lower than 1 × 10⁻⁵. As expected from this result, comparison of the genes up-regulated in ATM⁸ flies and Repo-ATMi flies showed a significant overlap (P < 0.01), with 30 overlapping genes that were primarily innate immune response genes. Table S1 provides a complete list of genes that were included in these categories, and Table 2 provides a partial list that focuses on PGRP and AMP genes. qPCR analysis confirmed that PGRP and AMP genes were up-regulated in Repo-ATMi flies (Fig. 7A). Analyses at 0, 3, and 8 d posteclosion revealed prolonged AMP gene expression. Finally, the AMP::GFP transgenic reporter assay revealed AMP gene expression only in glial cells of Repo-ATMi flies (Fig. 7 B–G and Fig. S2). Taken together, these data suggest that ATM knockdown in glial cells causes cell autonomous and prolonged activation of the innate immune response.

Fig. 7.

ATM knockdown in glial cells causes up-regulation of AMP gene expression in glial cells. (A) Shown is qPCR analysis of mRNA expression in Repo-ATMi heads after 0, 3, or 8 d that was normalized to age-matched Repo-GAL4 heads. Asterisks represent P < 0.05 based on Student t test analysis. Shown are representative immunofluorescence images of Repo-GAL4 (B–D) or Repo-ATMi (E–G) brains stained for Repo and detecting the AttA::GFP reporter gene. Other AMP::GFP reporter genes are shown in Fig. S2.

ATM Knockdown in Neurons Does Not Cause Neurodegeneration or Increased Expression of Innate Immune Response Genes.

To determine the extent to which reduced ATM kinase activity in neurons plays a role in ATM⁸ phenotypes, Elav-GAL4 and pWIZ-ATM^T4 transgenes were used to knock down ATM only in neurons. Two Elav-GAL4 driver flies were used: Elav-GAL4 flies, which have a transgene on the second chromosome that expresses GAL4 under control of the Elav promoter, and Elav^C155-GAL4 flies, which have a GAL4-containing transgene insertion downstream of the endogenous Elav promoter on the X chromosome (46, 47). Control Elav-GAL4 and Elav^C155-GAL4 flies and experimental Elav-GAL4/pWIZ-ATM^T4 (Elav-ATMi) and Elav^C155-GAL4/pWIZ-ATM^T4 (Elav^C155-ATMi) flies were assayed as described for ATM⁸ flies.

Mobility, longevity, brain morphology, and Casp^Act expression were not adversely affected in Elav-ATMi flies (Figs. 5 C and D and 6 D–F, Table 1, and Fig. S1 K–N). In fact, Elav-ATMi flies had significantly increased longevity relative to Elav-GAL4 flies (P < 0.001). This contrasts the reduced longevity in A-T and may be a result of ATM knockdown exclusively in neurons as opposed to all cells. Longevity and Casp^Act expression were also not noticeably affected in Elav^C155-ATMi flies (Table 1 and Fig. S3B). However, mobility and brain morphology were affected in Elav^C155-ATMi flies. Elav^C155-ATMi flies had slightly reduced climbing ability immediately (10 s) after being tapped to the bottom of the vial, but climbing ability was not affected at the 20-s and 30-s time points (Fig. S3A). This contrasts the sustained and more severe reduction in climbing ability observed for ATM⁸ and Repo-ATMi flies (Figs. 2A and 5A). Elav^C155-ATMi brains contained several 2- to 4-μm round structures that were distinct in location and morphology from holes that are characteristic of neurodegeneration (Fig. S1 O–R and S3 C–E) (24). The round structures occurred within the optic lobes, but not the central body, and had a clear zone around the periphery and a darkly stained interior core. This contrasts the holes in ATM⁸ and Repo-ATMi flies that occurred in the optic lobes and the central body and had an empty core (Figs. 3C and 6C). Taken together, these data indicate that ATM knockdown in neurons is not sufficient to cause neurodegeneration in the brain.

Gene expression microarray analysis was used to identify molecular and biological events caused by ATM knockdown in neurons. Using parameters described for the ATM⁸ microarray analysis, at least two independent biological replicates of 3- to 5-d-old flies were analyzed. This analysis revealed that 124 genes were up-regulated and 36 genes were down-regulated in Elav-ATMi flies relative to Elav-GAL4 flies and that 81 genes were up-regulated and 39 genes were down-regulated in Elav^C155-ATMi flies relative to Elav^C155-GAL4 flies. Gene Ontology analysis revealed that the innate immune response was not significantly affected in Elav-ATMi or Elav^C155-ATMi flies, and qPCR analysis confirmed that the expression of PGRP and AMP genes was not affected in Elav-ATMi or Elav^C155-ATMi flies (Fig. S4) (32). The genes that were affected will be reported elsewhere. These data indicate that ATM knockdown in neurons is not sufficient to activate the innate immune response in the brain.

Discussion

ATM Kinase Activity Is Required for Neuron Survival.

This study indicates that the kinase activity of ATM⁸ is temperature-sensitive. ATM⁸ was identified in a screen for ATM alleles and was found to contain a leucine-to-phenylalanine mutation of the final amino acid (21). This amino acid falls within the FATC (FRAP, ATM, TRRAP C-terminal) domain, which is located directly downstream of the kinase domain in PIKK family members (48). Functional and structural analyses indicate that the FATC domain regulates PIKK kinase activity (22). Mutation of one or two amino acids in the FATC domain of several PIKKs reduces kinase activity. Structural studies demonstrate that the FATC domain is physically close to the activation loop of the kinase domain, suggesting that temperature sensitivity of ATM⁸ kinase activity is caused by disruption of FATC domain contacts with the kinase domain at high temperature (25 °C) that are weak but functionally sufficient at low temperature (18 °C) (49). Taken together with the finding that a single amino acid mutation in the FATC domain was found to naturally occur in a patient with A-T, evidence of neurodegeneration in ATM⁸ flies suggests that ATM kinase activity is required for neuron survival in humans (50).

Reduced ATM Kinase Activity in Glial Cells Causes Neurodegeneration.

This study indicates that neuron survival requires ATM kinase activity in glial cells. Phenotypes caused by ATM knockdown only in glial cells (Repo-ATMi flies) were similar to those caused by reduced ATM kinase activity in both neurons and glial cells (ATM⁸ flies). These phenotypes include reduced mobility, reduced longevity, holes in the brain, and neuron and glial cell death. Collectively, these data indicate that reduced ATM kinase activity in glial cells causes neurodegeneration and imply that interactions between glial cells and neurons are important for neuron survival. Reduced ATM kinase activity in glial cells may inhibit the production of protective signals or allow the production of toxic signals that result in neurodegeneration.

ATM knockdown only in neurons (Elav-ATMi and Elav^C155-ATMi flies) did not cause neurodegeneration in the adult brain. This does not mean that ATM loss in neurons does not contribute to neurodegeneration; rather, it means that ATM loss in neurons is not sufficient to cause neurodegeneration in particular cellular contexts. In fact, we previously found that ATM knockdown in neurons (Elav^C155-ATMi flies) causes neurodegeneration in another cellular context, the eye (11). The different results observed in the brain and the eye may result from different levels of ATM knockdown. Alternatively, the different results may result from unique properties of neurons, glial cells, or other cells in the brain or eye. This alternative explanation is attractive because, in A-T and other neurodegenerative diseases, neuron types differ in their susceptibility to degeneration, with Purkinje and granule neurons being particularly susceptible in A-T (1, 2).

Reduced ATM Kinase Activity Triggers an Innate Immune Response in Glial Cells.

This study indicates that glial cells in the Drosophila CNS produce an innate immune response. This adds glial cells in the CNS to the existing list of innate immune response-competent cells that includes surface epithelia and the fat body in adult labellar glands, midgut, malpighian tubules, trachea, and male and female reproductive tracts (43, 51). However, among the identified types of glial cells, the relevant type remains to be determined (52, 53). Additionally, the involvement of the Toll and Imd pathways in the glial cell innate immune response remains to be determined. None of the components of these pathways, including the NF-κB genes that are transcriptionally up-regulated in response to microbial infection, was found to have altered expression in the ATM⁸ or Repo-ATMi microarrays (40, 41). Activation of AMP gene expression independently of the Toll and Imd pathways is not unprecedented. FOXO and GATA transcription factors have been shown to mediate the innate immune response independently of the pathogen-responsive innate immunity pathways (54, 55). Future studies will assess the details of the glial cell innate immune response.

Increased expression of innate immune response genes may directly result from reduced ATM kinase activity or indirectly result from increased susceptibility to infection or altered physiology. Data presented here are consistent with a model in which ATM directly represses the innate immune response through protein phosphorylation. However, there are no obvious candidates for substrates. ATM-dependent phosphorylation is essential for activation of NF-κB in response to DNA damage (56). Although NF-κB proteins are also activated in the innate immune response, results presented here suggest that ATM-dependent phosphorylation inhibits NF-κB proteins, as reduced ATM kinase activity activated the innate immune response. In regard to an indirect mechanism, AMP gene expression in glial cells has not been observed in response to infection or to physiological perturbations such as starvation, altered circadian rhythm, and altered sleep pattern that up-regulate AMP gene expression in the fat body (42, 57, 58). However, AMP gene expression in glial cells may not have been adequately examined under these conditions. Finally, prolonged expression of AMP genes in ATM⁸ flies argues against a burst of pathogen growth that is fought back, but it does not rule out persistent infection. Future studies will establish the role of ATM in regulating the glial cell innate immune response.

Activation of Innate Immune Response May Be a Common Feature of Neurodegeneration.

This study revealed a correlation between neurodegeneration and the innate immune response in a fly model of the human neurodegenerative disease A-T. A link between neurodegeneration and the innate immune response may be a common phenomenon in flies. Innate immune response genes are up-regulated in a fly model of Parkinson disease, and the Toll pathway mediates neurodegeneration in a fly model of Alzheimer's disease (59, 60). Moreover, studies in mammalian systems have uncovered considerable evidence linking the innate immune response and neurodegeneration in a variety of human neurological disorders, including Alzheimer's disease, Parkinson disease, Huntington disease, multiple sclerosis, and ALS (14, 15, 61). In mammals, microglial cells are responsible for the innate immune response. They recognize pathogens through their Toll-like receptors, leading to release of proinflammatory cytokines including Tumor necrosis factor-alpha (TNF-α) (62). Current thinking is that prolonged activation of microglial cells is a causative factor for neurodegeneration. For example, in Alzheimer's disease, amyloid-β activates microglia to release neurotoxic factors such as TNF-α and reactive oxygen species, and in Parkinson disease, overactivated microglia produce reactive oxygen species in response to damaged ascending dopaminergic neurons (63, 64). The discovery of a glial cell immune response in Drosophila makes this model organism well suited for studying the effects of neuroinflammation on neuron survival.

Materials and Methods

Drosophila Genetics.

Flies were maintained on standard molasses medium at 25 °C unless otherwise stated. For all experiments, WT flies were w¹¹¹⁸. pWIZ-ATM^T4 is described by Rimkus et al. (2008) (11). ATM⁸, Repo-GAL4, and Elav-GAL4 flies were obtained from the Bloomington Stock Center; Elav^C155-GAL4 flies were obtained from Barry Ganetzky (University of Wisconsin, Madison, WI); and AMP::GFP flies were obtained from Ylva Engstrom (Stockholm University, Stockholm, Sweden) (12, 43, 44, 46, 47). Repo-ATMi flies were generated by recombining the Repo-GAL4 and the pWIZ-ATM^T4 transgenes onto the same chromosome. To generate ATM⁸ flies, ATM⁸/TM3 flies were raised at 18 °C and their progeny were screened for the absence of TM3. For ATM⁸ experiments, flies were cultured at 18 °C until 0 to 3 d posteclosion and then transferred to 25 °C for the indicated time.

Western Blot Analysis.

For analysis of ATM kinase activity, vials containing 10 flies (4–5 d old) were untreated or irradiated with 50 Gy by using a Mark 1 irradiator. Flies were allowed to recover for 30 min and heads were isolated by manual dissection and homogenized in Laemmli sample buffer (Bio-Rad). Head lysates were fractionated on polyacrylamide gels and probed with H2Av-pS137 (1:500; Rockland) and α-tubulin (1:10,000, Sigma) antibodies. The secondary antibody was α-rabbit IgG horseradish peroxidase (1:5,000; GE Healthcare/Amersham).

Mobility Assay.

A climbing assay was used to quantify mobility. Groups of 20 adult flies of the indicated genotype were aged for 3 d and were placed in 2.5-cm by 9.5-cm (diameter by height) plastic vials with graduation marks at 25%, 50%, and 75% of the vial height. Vials were sealed with Parafilm. Flies were tapped to the bottom of the vial and videotaped for 30 s. The tape was paused at 10, 20, and 30 s, and the number of flies in each quadrant was counted. For each genotype, four independent replicates were averaged and plotted as a percentage of flies in each quadrant at the indicated time points.

Longevity Assay.

Longevity assays were performed in triplicate with control and experimental genotypes at the same time. For each of the triplicate experiments, 100 flies were examined (five vials of 20 flies each). ATM⁸, ATM⁸/+, and w¹¹¹⁸ flies were raised at 18 °C and transferred to 25 °C at 0 to 1 d posteclosion. All other flies were collected 0 to 3 d posteclosion at 25 °C. The day of collection was designated day 1, and the number of surviving flies was counted daily until all flies had died. Flies were transferred to new vials approximately every 3 d. The number of surviving flies for each genotype was averaged, with errors representing the SEM between replicates. Statistical significance was determined by using log-rank analysis and the χ² statistic.

Paraffin Sectioning.

Fly heads were hand dissected and incubated in ethanol:chloroform:acetic acid (6:3:1) at room temperature overnight. Heads were then incubated in 70% ethanol, processed into paraffin, sectioned at 5 μm, and stained with hematoxylin (Harris modified with acetic acid; Fisher) and eosin (Eosin Y powder; Polysciences) by using standard procedures (65).

Microarray Analysis.

For each genotype, approximately 150 flies (approximately equal numbers of males and females) were collected and aged for 3 d at 25 °C. To collect head, flies were transferred to 1.5 mL tubes and frozen in liquid N₂, tubes were shaken vigorously to shear heads from bodies, and heads were isolated from other body parts by sequential use of US Standard no. 25 (710 μm) and no. 40 (425 μm) sieves. Total RNA was isolated from heads by using TRI reagent (Sigma). The resulting RNA was resuspended in 40 μL diethylpyrocarbonate-treated water and subjected to DNase treatment using the TURBO DNA-free kit (Ambion). The resulting RNA was ammonium acetate precipitated and resuspended in 20 μL of diethylpyrocarbonate-treated water. Typically, 40 μg of RNA was recovered per sample. RNA (1 mg/mL) was shipped at −80 °C to NimbleGen (Reykjavik, Iceland) for probe generation and analysis on D. melanogaster 4 × 72-plex arrays, containing a minimum of four probes per gene for 15,473 genes. The data were analyzed by using the ArrayStar program (DNAStar). Pair files were used as input, and normalization was conducted by using robust multichip average processing and quantile normalization. Two replicates were performed for all genotypes except Elav-GAL4 and Elav-ATMi flies, which had four replicates. All replicates were used to generate fold changes and statistical confidence limits. Before statistical analysis, genes with a low coefficient of variation (<0.05) were filtered. Initial statistical analysis identified genes with a P value lower than 0.05 by using a moderated t test analysis with no additional corrections. Further statistical analysis using the FDR test was used to generate the final list of genes, with the final criteria being a greater than twofold change in expression, a moderated t test P < 0.05, and an FDR P < 0.10. Raw and normalized microarray data have been deposited in the Gene Expression Omnibus database (accession no. GSE34315).

qPCR.

Flies were collected as indicated for the microarrays and aged for the stated times. The 0-d time point indicates that RNA was processed on the day of collection. Heads were isolated from approximately 25 flies per genotype, and RNA was isolated using the RNeasy Plus Mini Kit (Qiagen). cDNA was generated with an iScript cDNA Synthesis Kit (Bio-Rad). Real-time qPCR was carried out as described by Katzenberger et al. (2006) (66). Primer sequences are provided in Table S2.

Immunofluorescence Microscopy.

For the AMP::GFP experiments, brains were dissected from flies aged 4 to 6 d at 25 °C. For the Casp^Act experiments, brains were dissected, fixed for 15 min in fresh 4% formaldehyde, washed in 1× PBS solution with 0.3% Triton-X, and incubated in primary antibody overnight at 4 °C. Primary antibodies used were α-Repo (1:200; Developmental Studies Hybridoma Bank), α-Elav (1:200; Developmental Studies Hybridoma Bank), and α-Casp^Act (1:50; Millipore). Fluorescently labeled secondary antibodies used were α-mouse rhodamine (1:300; Invitrogen), α-rat rhodamine (1:200; Invitrogen), and α-rabbit Alexa Fluor 488 (1:300; Invitrogen). Brains were mounted in Vectashield (Vector Laboratories) and imaged on a Zeiss Axiovert 200M inverted microscope or a Bio-Rad MRC-1024 confocal microscope (W. M. Keck Laboratory for Biological Imaging, University of Wisconsin, Madison, WI).