A drosophila model for amyotrophic lateral sclerosis reveals motor neuron damage by human SOD1

Abstract

Amyotrophic lateral sclerosis (ALS) is a motor neuron disease that leads to loss of motor function and early death. About 5% of cases are inherited, with the majority of identified linkages in the gene encoding copper, zinc-superoxide dismutase (SOD1). Strong evidence indicates that the SOD1 mutations confer dominant toxicity on the protein. To provide new insight into mechanisms of ALS, we have generated and characterized a model for familial ALS in Drosophila with transgenic expression of human SOD1. Expression of wild type or disease-linked (A4V, G85R) mutants of human SOD1 selectively in motor neurons induced progressive climbing deficits. These effects were accompanied by defective neural circuit electrophysiology, focal accumulation of human SOD1 protein in motor neurons, and a stress response in surrounding glia. However, toxicity was not associated with oligomerization of SOD1 and did not lead to neuronal loss. These studies uncover cell-autonomous injury by SOD1 to motor neurons in vivo, as well as non-autonomous effects on glia, and provide the foundation for new insight into injury and protection of motor neurons in ALS.

FIGURE 1.

Drosophila model of SOD-linked fALS employs motor neuron-specific expression of wild type hSOD1, A4V, and G85R mutant proteins. A, comparison of hSOD1 and dSOD1. Green bars identify residues that are mutated in SOD-linked fALS. Identities are in black, and similarities are in gray (BLOSUM 62 matrix). B, expression of transgenes in young flies with D42 motor neuron driver. Western blots were probed for an internal reference tubulin (tub, E7 ascites) and with antibodies that detect both hSOD1 and dSOD1 (top: FL154) or that detect only hSOD1 (bottom: NCL-SOD). C, relative expression of hSOD1 transgenes in young (1 day), middle-aged (28 days), and old (49 days) flies with D42 motor neuron driver, from Western blots also probed for tubulin (E7 ascites), GFP, and an hSOD1 antibody that cross-reacts to fly dSOD1 (NCL-SOD or FL154). Data were expressed as a relative ratio of immunoblot reactivity of the antibody staining of antibodies to hSOD1 (or antibodies to GFP) to antibodies to tubulin and normalized to the signal for 1-day GFP from the same Western blot.

FIGURE 2.

Lifespan of flies expressing WT hSOD1, A4V, or G85R hSOD1. A, survival curves, and B, details of lifespan analysis for each genotype, from multiple experimental points. The effect of hSOD1 on lifespan is known to differ between males and females and is dependent on genetic background (64). The hSOD1 transgenes used here were generated in a similar genetic background, and we do not see an extension of lifespan with hSOD1. CI, confidence interval.

FIGURE 3.

Motor neuron expression of hSOD1 induces a reduction in climbing activity without gross loss of motor neuron nuclei. A, climbing activity was compromised in flies expressing mutant or WT hSOD1 relative to flies expressing dSOD1 (blue bars). G85R showed a deficit from 14 days onwards (green bars), WT showed a deficit from 21 days (red bars), and A4V showed a deficit at 28 days (purple bars). Bars represent climbing indices for genotypes normalized to the 1-day climbing index of dSOD1 controls, ± S.E. from at least three experiments. B, the number of motor neurons was determined by counting nuclei in the T1/T2 border (rectangular selection) in confocal stacks of whole-mounted thoracic ganglia. Shown is a thoracic ganglion of genotype D42/UAS-GFP. Ab, abdominal ganglion. the number of labeled nuclei detected in the T1/T2 border was not different at any time point when compared with controls (gold bars) or between time points for flies expressing dSOD1, WT, A4V, or G85R hSOD1 (blue, red, purple, and green bars, respectively) in motor neurons (analysis of variance, p > 0.05). The cell number is normalized to 1-day controls within each experiment; average ± S.E. from at least two experiments (5-10 flies each) is presented.

FIGURE 4.

G85R accumulates in high salt-resistant foci in retinal cells but does not cause degeneration. A, solubility assay for ionic fractionation of hSOD1 in homogenates of flies expressing WT or G85R under control of the retinal promoter gmr-GAL4. Proteins were separated in high salt buffer to break ionic bonds and then centrifuged at high speed to isolate soluble and insoluble species. B, total (T), supernatant (S), and pellet (P) fractions were separated by SDS-PAGE and probed for hSOD1. At 28 days, G85R but not wild type hSOD1 formed high molecular weight complexes that were found in both soluble and insoluble fractions. Both WT and G85R monomer were found in the pellet. dpe, days post eclosion of adult. C and D, cryosections of 28-day eyes from flies expressing WT or G85R expressed by the gmr-gal4 driver. Red fluorescence decorates hSOD1, and blue fluorescence labels nuclear DNA. G85R, but not WT, forms large foci recognized by hSOD1 antibody in the distal retina, just internal to the lens. E-J, nuclear arrays (E and F), external eye (G and H), and internal ommatidial structure (I and J) appear normal in eyes of 45-day flies expressing WT or G85R.

FIGURE 5.

hSOD1 induces age-dependent electrophysiological defects in the giant fiber neural circuit. A, schematic illustration of the giant fiber pathway responsible for jump-flight escape behavior. The giant fiber neuron (GFn) is located in the brain and descends to the thoracic ganglion, where it excites the motor neuron (TTMn) that innervates the TTM via an electrical synapse (marked with a lightning bolt). GFn also excites the peripherally synapsing interneuron (PSI) via an electrical synapse, which in turn excites five motor neurons (DLMn) innervating DLMs. Both DLM and TTM motor neurons synapse with their respective muscles via glutamatergic synapses. For illustrational purposes only, the DLMn is shown outside the thoracic ganglion. B, histograms of the average cutoff frequency in DLM (left panel) and TTM (right panel) in 55-day flies. DLM in control flies (D42/+ and dSOD1) was able to follow a train of 10 stimuli up to ∼140-Hz stimulation of the GFn, whereas DLM in flies expressing WT or G85R was only capable of following up to 80-90 Hz. Although TTM in the control flies followed up to 300 Hz, the ability of the TTM to follow high frequency stimulation was compromised in flies expressing WT. (^**, p < 0.05) and slightly reduced in G85R. n ≥ 5 independent flies for each genotype. C, representative responses of DLM following 140-Hz stimulation of the GFn in control flies (top panels), flies expressing WT hSOD1 (second panels), flies expressing G85R (third panels), and those expressing dSOD1 (bottom panels). The muscle responded normally to each stimulus at 10 days but failed to follow each stimulus when aged (55 days) in the experimental flies. The arrows indicate failed responses. D, representative responses of TTM following 300-Hz stimulation of the GFn in control flies (top panels), flies expressing WT (second panels), flies expressing G85R (third panels), and those expressing dSOD1 (bottom panels). The muscle responded normally to each stimulus at 10 days, but experimental flies showed minor failures to a train of 10 stimuli at older time points (55 days). Arrows indicate failed responses. For each genotype and age group presented, n = 5.

FIGURE 6.

hSOD1 accumulates in foci with age in motor neurons. The accumulation of hSOD1 into foci in the thoracic ganglion of animals expressing G85R with the D42 driver at young (0-1 days) and old (28 days) ages, when compared with animals expressing GFP only (left) is shown. Green, GFP or hSOD1 immunostaining; red, the neuronal nuclear marker Elav, in whole-mount thoracic ganglia. B-D, details of C showing homogenous GFP fluorescence in a 28-day fly. F-H, details of G showing homogenous immunoreactivity for G85R hSOD1 at 0-1 days. J-L, details of K showing striking focal accumulation of G85R at 28 days, with many foci in a single cell (arrows).

FIGURE 7.

Quantitative analysis of hSOD1 foci accumulation in motor neurons. Top, analysis of WT, A4V, and G85R hSOD1 accumulation with time. WT and mutant forms of hSOD1 accumulated into foci progressively with age (chi square p < 0.001). Bottom, whole-mount thoracic ganglia immunolabeled for hSOD1 to illustrate classification of focal protein accumulation. Arrows denote SOD-positive foci in motor neurons and neuropil. Only the T1-T2 border is shown here, but immunofluorescence in the entire thoracic ganglia was used to make the determination. Absent, SOD immunofluorescence was uniform and smooth. Mild, SOD immunofluorescence was mostly smooth and uniform, a few cells exhibited focal accumulations, and not more than one focus was observed per cell. Moderate, some smooth immunofluorescence was visible, and many cells contained at least one focal accumulation. Severe, the vast majority of visible immunofluorescence was present in foci, and most cells contained large numbers of accumulations.

FIGURE 8.

Expression of SOD1 in motor neurons is associated with a stress response in glia. A-D, confocal images of a thoracic ganglion from a fly expressing G85R in motor neurons, stained for Hsc/Hsp70 (blue), hSOD1 (green), and Elav (neurons, red). Hsc/hsp70 immunoreactivity was often seen near, but not overlapping with, hSOD1 and Elav (arrows). E, WT hSOD1 induced mild to strong expression of hsc/hsp70 protein at 49 days, whereas both A4V and G85R induced immunostaining at 28 days, which was increased at 49 days. Differences when compared with control at each time point and differences due to age within genotype are statistically significant (p < 0.0001). F-H, the chaperone signal was in glia, not motor neurons. Hsp70 signal (blue) overlapped with the glial cell marker Repo (yellow). Arrowheads highlight examples of cells that immunostain strongly for both Hsc/Hsp70 and Repo.

FIGURE 9.

Expression of Hsp70 in motor neurons is associated with a neuronal stress response upon polyglutamine protein expression. Shown are confocal images of thoracic ganglia from flies demonstrating expression of SCA3tr-Q78 in motor neurons, stained for Hsc/Hsp70 (red) and polyglutamine protein (A and C, green) or glia with Repo (D and F, green). Neuronal expression induces robust Hsc/Hsp70 immunoreativity in neurons (here and in Refs. and 37), with a minimal response in Repo-positive cells. HA, hemagglutinin.