Protein misfolding and oxidative stress promote glial-mediated neurodegeneration in an Alexander disease model

Abstract

Although alterations in glial structure and function commonly accompany death of neurons in neurodegenerative diseases, the role glia play in modulating neuronal loss is poorly understood. We have created a model of Alexander disease in Drosophila by expressing disease-linked mutant versions of glial fibrillary acidic protein (GFAP) in fly glia. We find aggregation of mutant human GFAP into inclusions bearing the hallmarks of authentic Rosenthal fibers. We also observe significant toxicity of mutant human GFAP to glia, which is mediated by protein aggregation and oxidative stress. Both protein aggregation and oxidative stress contribute to activation of a robust autophagic response in glia. Toxicity of mutant GFAP to glial cells induces a non-cell-autonomous stress response and subsequent apoptosis in neurons, which is dependent on glial glutamate transport. Our findings thus establish a simple genetic model of Alexander disease and further identify cellular pathways critical for glial-induced neurodegeneration.

Figure 1.

Expression of human GFAP in Drosophila produces cell death and seizures. A, Western blot showing equivalent levels of expression of GFAP^WT and GFAP^R79H (top). The blot was reprobed with an antibody to actin to demonstrate equivalent protein loading (bottom). Flies are 1 d old. B, The number of TUNEL-positive cells increases with age and expression of mutant GFAP in glial cells. n = 6 for each genotype and time point. Serial frontal sections of the entire brain (central body and medulla) were assayed. *p < 0.01 versus control. C, There is a trend toward decreasing numbers of glial cells with advancing age (p = 0.012, control vs GFAP^R79H at 20 d). n = 6 for each genotype. A 20 μm region of lamina was assayed. D, The number of neurons decreases with age and expression of mutant GFAP in glial cells. n = 6 for each genotype. A 20 μm region of lamina was assayed. *p < 0.01 versus control. E, The number of GluRIIB neurons decreases in aged GFAP transgenic flies. At day 20, there is a significant loss of GFP-labeled GluRIIB neurons in both GFAP^WT and GFAP^R79H flies. GFAP^R79H has more severe loss of GluRIIB neurons than GFAP^WT. n = 6 for each genotype. A 20 μm region of lamina was assayed. *p < 0.01 versus control. F, There is increased seizure frequency in GFAP transgenic flies as demonstrated by the percentage of flies exhibiting seizures after mechanical stimulation. Flies are 1 d old. n = 90 for each genotype. *p < 0.01 versus control. Genotypes are as follows: control: repo–GAL4/+; GFAP^WT: repo–GAL4, UAS–GFAP^WT/+; GFAP^R79H: repo–GAL4, UAS–GFAP^R79H/+.

Figure 2.

Rosenthal fiber-like inclusion formation in GFAP^R79H transgenic flies. A, B, H&E staining shows normal neuropil in control flies (A) and numerous eosinophilic Rosenthal fibers in GFAP^R79H transgenic animals (B, arrows). Flies are 20 d old. Scale bar, 5 μm. C, Electron microscopy reveals dense osmiophilic inclusions (arrow) in a GFAP^R79H transgenic fly at 20 d of age. Scale bar, 1 μm. D–F, GFAP immunostaining shows diffuse staining of glial processes in GFAP^WT flies (E, arrowheads) and inclusions in GFAP^R79H flies (F, arrows). The medulla is shown in A, B, and D–F. Flies are 1 d old. Scale bar, 5 μm. G–J, Immunostaining reveals colocalization of endogenous Cryab (αB-crystallin) (G) with GFAP (H) in inclusions of GFAP^R79H transgenic flies (J, arrow). Nuclei are counterstained with 4′,6′-diamidino-2-phenylindole (DAPI) (I). The central body is shown. Flies are 1 d old. Scale bar, 2 μm. K, The number of inclusions increases with age and is markedly enhanced in flies expressing GFAP^R79H compared with GFAP^WT. n = 6 for each genotype and time point. The mushroom body cortex (Kenyon cells) was assayed. *p < 0.01 versus control. L, Biochemical fractionation reveals more insoluble, and less soluble, GFAP in flies expressing GFAP^R79H compared with GFAP^WT (top). The blot was reprobed with an antibody to actin to demonstrate equivalent protein loading (soluble fractions; bottom). Flies are 1 d old. Genotypes are as follows: control: repo–GAL4/+; GFAP^WT: repo–GAL4, UAS–GFAP^WT/+; GFAP^R79H: repo–GAL4, UAS–GFAP^R79H/+.

Figure 3.

Cryab overexpression rescues GFAP toxicity and aggregation. A, Decreased numbers of TUNEL-positive cells are present when GFAP^R79H is coexpressed with Drosophila Cryab. Flies are 20 d old. n = 6 for each genotype. *p < 0.01 versus GFAP^R79H/lacZ. B, Overexpression of Cryab in GFAP^R79H flies reduces the percentage of flies with seizures. Flies are 1 d old. n = 90 (GFAP^R79H) and n = 144 (GFAP^R79H/Cryab). *p < 0.01. C, Decreased numbers of GFAP-immunoreactive inclusions are present when GFAP^R79H is coexpressed with Drosophila Cryab. Flies are 20 d old. n = 6 for each genotype. *p < 0.01 versus GFAP^R79H/lacZ. D, Overexpression of Cryab in glia of GFAP transgenic flies increases GFAP solubility compared with GFAP^R79H alone (GFAP^R79H/lacZ), without changing the total GFAP level (top). Flies are 1 d old. The blot was reprobed with an antibody to actin to demonstrate equivalent protein loading (soluble fractions; bottom). Genotypes are as follows: control: repo–GAL4/+; GFAP^R79H: repo–GAL4, UAS–GFAP^R79H/+; GFAP^R79H/lacZ: repo–GAL4, UAS–GFAP^R79H/+; UAS–lacZ/+; GFAP^R79H/Cryab: repo–GAL4, UAS–GFAP^R79H/+; UAS–Cryab/+.

Figure 4.

Overexpression of other chaperones also rescues GFAP toxicity and aggregation. A, Decreased numbers of TUNEL-positive cells are present when GFAP^R79H is coexpressed with Hsp26, Hsp27, or human Hsp70. Flies are 20 d old. n = 6 for each genotype. *p < 0.01 versus controls (GFAP^R79H and GFAP^R79H/lacZ). B, Expression of human Hsp70 in glia of GFAP^R79H flies decreases seizures. Flies are 1 d old. n = 90 (GFAP^R79H) and n = 137 (GFAP^R79H/hHsp70). *p < 0.01. C, Decreased numbers of GFAP-immunoreactive inclusions are present when GFAP^R79H is coexpressed with Hsp26, Hsp27, or human Hsp70. Flies are 20 d old. n = 6 for each genotype. *p < 0.01 versus controls (GFAP^R79H and GFAP^R79H/lacZ). D, Overexpression of Hsp26, Hsp27, or human Hsp70 in glia of GFAP transgenic flies increases GFAP solubility compared with GFAP^R79H alone (GFAP^R79H/lacZ) without changing the total GFAP levels (top). Flies are 1 d old. The blot was reprobed with an antibody to actin to demonstrate equivalent protein loading (soluble fractions; bottom). Genotypes are as follows: control: repo–GAL4/+; GFAP^R79H: repo–GAL4, UAS–GFAP^R79H/+; GFAP^R79H/lacZ: repo–GAL4, UAS–GFAP^R79H/+; UAS–lacZ/+; GFAP^R79H/Hsp26: repo–GAL4, UAS–GFAP^R79H/+; UAS–Hsp26/+; GFAP^R79H/Hsp27: repo--GAL4, UAS–GFAP^R79H/+; UAS–Hsp27/+; GFAP^R79H/hHsp70: repo–GAL4, UAS–GFAP^R79H/+; UAS–hHsp70/+.

Figure 5.

Levels of antioxidant proteins influence GFAP toxicity but do not change GFAP solubility. A, Increasing Cat levels or expressing human Sod1 reduces the number of TUNEL-positive cells, whereas reducing levels of Cat or Sod using heterozygous null alleles of Cat and Sod increases TUNEL-positive cells. Flies are 20 d old. n = 6 for each genotype. *p < 0.01 versus controls (GFAP^R79H and GFAP^R79H/lacZ). B, Overexpression of Cat in glia of GFAP^R79H flies (GFAP^R79H/Cat) reduces the percentage of flies with seizures, whereas reducing Cat using heterozygous null allele of Cat increases seizures. Flies are 1 d old. n = 90 (control and GFAP^R79H), n = 212 (GFAP^R79H/Cat), and n = 135 (GFAP^R79H/Cat^+/−). *p < 0.01. C, Oxidative stress modifiers do not influence inclusion number. Flies are 20 d old. n = 6 for each genotype. D, Overexpression of Cat or human Sod1, or reducing Cat or Sod using heterozygous null alleles of Cat and Sod in glia of GFAP transgenic flies does not change GFAP solubility compared with GFAP^R79H alone (top). Flies are 1 d old. The blot was reprobed with an antibody to actin to demonstrate equivalent protein loading (soluble fractions; bottom). Genotypes are as follows: control: repo–GAL4/+; GFAP^R79H: repo–GAL4, UAS–GFAP^R79H/+; GFAP^R79H/lacZ: repo–GAL4, UAS–GFAP^R79H/+; UAS–lacZ/+; GFAP^R79H/Cat: repo–GAL, UAS–GFAP^R79H/+; UAS–Cat/+; GFAP^R79H/hSod1: repo–GAL4, UAS–GFAP^R79H/+; UAS–hSod1/+; GFAP^R79H/Cat^+/−: repo–GAL4, UAS–GFAP^R79H/+; Cat^+/−; GFAP^R79H/Sod^+/−: repo–GAL4, UAS–GFAP^R79H/+; Sod^+/−.

Figure 6.

Overexpression of glial glutamate transporter rescues GFAP toxicity but does not change GFAP solubility. A, Decreased numbers of TUNEL-positive cells are present when GFAP^R79H is coexpressed with the Drosophila glial glutamate transporter Eaat1. Flies are 20 d old. n = 6 for each genotype. *p < 0.01 versus controls (GFAP^R79H and GFAP^R79H/lacZ). B, Overexpression of the glutamate transporter in glia of GFAP^R79H flies (GFAP^R79H/Eaat1) reduces the percentage of flies with seizures. Flies are 1 d old. n = 90 (GFAP^R79H) and n = 148 (GFAP^R79H/Eaat1). *p < 0.01. C, Expression of Eaat1 does not influence inclusion number. n = 6 for each genotype. Flies are 20 d old. D, Overexpression of the glutamate transporter does not change GFAP solubility compared with GFAP^R79H alone (top). Flies are 1 d old. The blot was reprobed with an antibody to actin to demonstrate equivalent protein loading (soluble fractions; bottom). Genotypes are as follows: control: repo–GAL4/+; GFAP^R79H: repo–GAL4, UAS–GFAP^R79H/+; GFAP^R79H/lacZ: repo–GAL4, UAS–GFAP^R79H/+; UAS–lacZ/+; GFAP^R79H/Eaat1: repo–GAL, UAS–GFAP^R79H/+; UAS–Eaat1/+.

Figure 7.

Pharmacological modulation of heat shock proteins and antioxidant defense suppresses GFAP toxicity. A, Administration of 17-AAG for 20 d after eclosion suppresses GFAP toxicity in vivo. Flies fed with 10 μg/ml 17-AAG show significantly reduced toxicity compared with flies fed with solvent (DMSO) alone. n = 6 for each data point. *p < 0.01. B, Administration of vitamin E for 20 d after eclosion suppresses GFAP toxicity in vivo in a dose-dependent manner. Flies fed with 0.5 mm vitamin E display a trend (p = 0.02) toward suppression of toxicity, whereas flies fed with 1.5 mm vitamin E show significant toxicity suppression compared with flies fed with solvent (soybean oil) alone. n = 6 for each data point. *p < 0.01. Genotypes are as follows: control: repo–GAL4/+; GFAP^R79H: repo–GAL4, UAS–GFAP^R79H/+.

Figure 8.

JNK pathway activation in GFAP transgenic flies. A, JNK activation is time and genotype dependent as measured using a puc–lacZ reporter and immunostaining in tissue sections. n = 6 for each genotype. Serial frontal sections of the entire brains (central body and medulla) were assayed. *p < 0.01 versus control. Genotypes are as follows: control: repo–GAL4/puc–lacZ; GFAP^WT/puc–lacZ: repo–GAL4, UAS–GFAP^WT/puc–lacZ; GFAP^R79H/puc–lacZ: repo--GAL4, UAS–GFAP^R79H/puc–lacZ. B–I, JNK activation as reported by expression of puc-driven β-galactosidase (β-gal; B, F) is detected in both glia and neurons of GFAP transgenic flies. REPO marks glial cells (C) and ELAV shows neurons (G). Nuclei are counterstained with DAPI (D, H). Colocalization of β-galactosidase with REPO or ELAV is shown in E and I (arrows). The central body is shown. Flies are 20 d old. Scale bar, 2 μm. J, Quantification of β-galactosidase-positive cells in GFAP transgenic flies shows more JNK activation in neurons at day 20 compared with day 10. Serial frontal sections of the entire brains (central body and medulla) were assayed. n = 6 for each data point. *p < 0.01 (Student's t test).

Figure 9.

Autophagy in GFAP transgenic flies. A, B, Induction of autophagy as measured with a GFP–LC3 transgene depends on age and genotype (top). The blot was reprobed with an antibody to actin to demonstrate equivalent protein loading (bottom). Quantification of GFP levels in B is normalized to actin levels. *p < 0.01. Genotypes are as follows: control: repo–GAL4/UAS–GFP–LC3; GFAP^WT: repo–GAL4, UAS–GFAP^WT/UAS–GFP–LC3; GFAP^R79H: repo--GAL4, UAS–GFAP^R79H/UAS–GFP–LC3. C–F, Autophagic vacuoles identified with GFP–LC3 (C) are present in glia (F, arrow). The glia cell is marked with REPO (D). Nuclei are counterstained with DAPI (E). The central body is shown. Flies are 10 d old. Scale bar, 2 μm. G, Overexpression of heat shock proteins Cryab and human Hsp70 in GFAP^R79H flies reduces autophagy. H, Decreasing levels of Cat and Sod using heterozygous null alleles of Cat and Sod in GFAP^R79H flies increases activation of autophagy. I, Overexpression of glial glutamate transporter in GFAP^R79H flies does not strongly influence autophagy activation. Flies are 1 d old. Blots were reprobed with an antibody recognizing actin as a loading control (bottom). J, Quantification of GFP levels in figure G–I is normalized to actin levels. *p < 0.05, **p < 0.01 compared with GFAP^R79H/LacZ/GFPLC3. Genotypes are as follows: control: repo–GAL4/UAS–GFP–LC3; GFAP^R79H/lacZ/GFP–LC3: repo–GAL4, UAS–GFAP^R79H, UAS–GFP–LC3/+; UAS–lacZ/+; GFAP^R79H/Cryab/GFP–LC3: repo–GAL4, UAS–GFAP^R79H, UAS–GFP–LC3/+; UAS–Cryab/+; GFAP^R79H/hHsp70/GFP–LC3: repo–GAL4, UAS–GFAP^R79H, UAS–GFP–LC3/+; UAS–hHsp70/+; GFAP^R79H/Cat^+/−/GFP–LC3: repo–GAL4, UAS–GFAP^R79H, UAS–GFP–LC3/+; Cat^+/−; GFAP^R79H/Sod^+/−/GFP–LC3: repo–GAL4, UAS–GFAP^R79H, UAS–GFP–LC3/+; Sod^+/−; GFAP^R79H/Eaat1/GFP–LC3: repo–GAL4, UAS–GFAP^R79H, UAS–GFP–LC3/+; UAS–Eaat1/+.