Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Apr 9;3(4):695-708.
doi: 10.1534/g3.113.005850.

Transcriptome Profiling Following Neuronal and Glial Expression of ALS-Linked SOD1 in Drosophila

Emily L Kumimoto  1 , Taylor R Fore  1 , Bing Zhang  2
Affiliations

Transcriptome Profiling Following Neuronal and Glial Expression of ALS-Linked SOD1 in Drosophila

Emily L Kumimoto et al. G3 (Bethesda). .

Abstract

Amyotrophic lateral sclerosis (ALS) generally is a late-onset neurodegenerative disease. Mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene account for approximately 20% of familial ALS and 2% of all ALS cases. Although a number of hypotheses have been proposed to explain mutant SOD1 toxicity, the molecular mechanisms of the disease remain unclear. SOD1-linked ALS is thought to function in a non-cell-autonomous manner such that motoneurons are critical for the onset, and glia contribute to progression of the disease. Recently, it has been shown in Drosophila melanogaster that expression of human SOD1 in a subset of neuronal cells causes synaptic transmission defects, modified motor function, and altered sensitivity to compounds that induce oxidative stress. Here we used the Gal4-UAS (Upstream Activation Sequence) system to further characterize flies expressing wild-type Drosophila SOD1 (dSOD1) and the mutant human SOD1G85R (G85R) allele in motoneurons and glia. Cell-specific expression of both dSOD1 and G85R was found to influence lifespan, affect sensitivity to hydrogen peroxide, and alter lipid peroxidation levels. To better understand the genetic consequences of G85R expression in motoneurons and glia, we conducted microarray analysis of both young flies (5 days old) and old flies (45 days old) expressing G85R selectively in motoneurons or glia and concurrently in motoneurons and glia. Results from this microarray experiment identified candidate genes for further investigation and may help elucidate the individual and combined contributions of motoneurons and glia in ALS.

Keywords: ALS; Drosophila; SOD1; glia; motoneuron.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Cell specificity of Gal4 drivers and transgene expression of dSOD1 and G85R. Cell-specific expression was confirmed by crossing Gal4 drivers with a green fluorescent protein (GFP) reporter (UAS-mCD8GFP). Protein expression was confirmed by Western blot analysis. (A, B, C) Maximum intensity projections of motoneurons (D42) and glia-specific (M1B) and combined D42+M1B Gal4 drivers expressing membrane-bound GFP (A′, B′, and C′). Neuronal and glia nuclei (in red) are anti-Elav and anti-Repo markers, respectively. (A,” B,” C”) Selected z slices from the T1 segment of the ventral nerve cord (VNC). (A”) Arrowheads show the cell body of motoneurons. (B”) GFP is extensively expressed in all glial processes, with select glia nuclei. (C”) In the combined driver, motor neuron cell bodies are morphologically distinct among the glia processes. (D) Western blot analysis showing expression of the G85R transgene under the D42, M1B, and D42+M1B Gal4 drivers. Extracts from 1-day-old flies were probed with antibodies that detect dSOD1 (FL154), hSOD1 (NCL-SOD1), and tubulin (serving as a loading control).
Figure 2
Figure 2
Lifespan of flies with cell-specific Gal4 drivers crossed with wild-type (CS), dSOD1, and G85R flies. (A) Lifespan of flies with the D42 motoneuron Gal4 driver (D42::CS, n = 125; D42::dSOD1, n = 113; and D42::G85R, n = 142). D42::dSOD1 flies had a lifespan similar to that of D42::CS flies. D42::G85R flies had a reduced lifespan compared to that of D42::dSOD1 (P < 0.0001) and D42::CS flies (P < 0.0001). (B) Lifespan of flies with the M1B glia Gal4 driver (M1B::CS, n = 172; M1B::dSOD1, n = 165; and M1B::G85R, n = 138). M1B::dSOD1 flies had a reduced lifespan compared to that of M1B::CS flies (P < 0.0001). M1B::G85R flies had a reduced lifespan compared to that of M1B::CS flies (P < 0.0001) and a slightly increased lifespan compared to that of M1B::dSOD1 flies (P < 0.01). (C) Lifespan of flies with D42+M1B Gal4 driver (D42+M1B::CS, n = 102; D42+M1B::dSOD1, n = 143; and D42+M1B::G85R, n = 148). D42+M1B::dSOD1 flies had a reduced lifespan compared to that of D42+M1B::CS flies (P < 0.0001). D42+M1B::G85R flies had an increased lifespan compared to those of both D42+M1B::CS flies (P < 0.0001) and D42+M1B::dSOD1 flies (P < 0.0001).
Figure 3
Figure 3
Older flies expressing G85R were more sensitive to low levels of hydrogen peroxide. All flies were fed on 0.5% H2O2 in 3% sucrose. (A) Survivorship graphs of 5-day-old flies with motoneuron D42 Gal4 driver (D42::CS, n = 116; D42::dSOD1, n = 76; and D42::G85R, n = 113). D42::G85R flies were slightly more sensitive to H2O2 than D42::CS (P < 0.0001) and D42::dSOD1 (P < 0.0001) flies. (B) Survivorship graphs of 45-day-old flies with D42 Gal4 driver (D42::CS, n = 79; D42::dSOD1, n = 65; and D42::G85R, n = 79). D42::dSOD1 flies had increased resistance to H2O2 compared to D42::CS flies (P < 0.0001). D42::G85R flies had increased sensitivity to H2O2 compared to D42::CS (P < 0.0001) and D42::dSOD1 (P < 0.0001) flies. (C) Survivorship graphs of 5-day-old flies with glia M1B Gal4 driver (M1B::CS, n = 120; M1B::dSOD1, n = 114; M1B::G85R). M1B::dSOD1 flies were slightly more resistant to H2O2 than M1B::CS (P < 0.0001) and M1B::G85R (P < 0.0001) flies. (D) Survivorship graphs of 45-day-old flies with M1B driver (M1B::CS, n = 172; M1B::dSOD1, n = 41; M1B::G85R, n = 87). M1B::dSOD1 flies had increased resistance to H2O2 compared to M1B::CS flies (P < 0.01) flies. M1B::G85R flies had increased sensitivity to H2O2 compared to M1B::CS (P < 0.0001) and M1B::dSOD1 (P < 0.0001) flies. (E) Survivorship graphs of 5-day-old flies with D42+M1B Gal4 driver (D42+M1B::CS, n = 114, D42+M1B::dSOD1, n = 94; D42+M1B::G85R, n = 118). D42+M1B::G85R flies were slightly more sensitive to H2O2 than D42+M1B::CS (P < 0.01) and D42+M1B::dSOD1 (P < 0.0001) flies. (F) Survivorship graphs of 45-day-old flies with D42+M1B Gal4 driver (D42+M1B::CS, n = 111; D42+M1B::dSOD1, n = 63; D42+M1B::G85R, n = 94). D42+M1B::dSOD1 flies had a sensitivity similar to that of H2O2 as D42+M1B::CS flies. D42+M1B::G85R flies were more sensitive to H2O2 than D42+M1B::CS (P < 0.0001) and D42+M1B::dSOD1 (P < 0.0001) flies.
Figure 4
Figure 4
Lipid peroxidation levels of 5- and 45-day-old flies with cell-specific Gal4 drivers crossed with wild-type (CS), dSOD1, and G85R flies, as measured by malondialdehyde (MDA) concentration (nmol/mg). (A) MDA for flies with the D42 motoneuron Gal4 driver. (B) [MDA] for flies with the M1B glia Gal4 driver. (C) [MDA] for flies with the D42+M1B motoneuron and glial Gal4 drivers. (D) [MDA] for 5-day-old wild-type (CS) flies treated with 10 mM paraquat in 3% sucrose solution or the sucrose solution. *P < 0.05; **P < 0.001; ***P < 0.0001.
Figure 5
Figure 5
Venn diagrams showing overlap in differentially expressed genes with at least a two-fold change when G85R was expressed in motoneurons, glia, or together in both cell types. (A) Up-regulated genes in 5-day-old flies. (B) Down-regulated genes in 5-day-old flies. (C) Up-regulated genes in 45-day-old flies. (D) Down-regulated genes in 45-day-old flies.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Afifi A. K., Aleu F. P., Goodgold J., Mackay B., 1966. Ultrastructure of atrophic muscle in amyotrophic lateral sclerosis. Neurology 16: 475–481 - PubMed
    1. Alexianu M. E., Kozovska M., Appel S. H., 2001. Immune reactivity in a mouse model of familial ALS correlates with disease progression. Neurology 57: 1282–1289 - PubMed
    1. Barrett T., Troup D. B., Wilhite S. E., Ledoux P., Evangelista C., et al. , 2011. NCBI GEO: archive for functional genomics data sets–10 years on. Nucleic Acids Res. 39: D1005–D1010 - PMC - PubMed
    1. Bier E., Jan L. Y., Jan Y. N., 1990. rhomboid, a gene required for dorsoventral axis establishment and peripheral nervous system development in Drosophila melanogaster. Genes Dev. 4: 190–203 - PubMed
    1. Boillee S., Vande Velde C., Cleveland D. W., 2006a ALS: a disease of motor neurons and their nonneuronal neighbors. Neuron 52: 39–59 - PubMed

Publication types