An ALS-linked mutant SOD1 produces a locomotor defect associated with aggregation and synaptic dysfunction when expressed in neurons of Caenorhabditis elegans

Abstract

The nature of toxic effects exerted on neurons by misfolded proteins, occurring in a number of neurodegenerative diseases, is poorly understood. One approach to this problem is to measure effects when such proteins are expressed in heterologous neurons. We report on effects of an ALS-associated, misfolding-prone mutant human SOD1, G85R, when expressed in the neurons of Caenorhabditis elegans. Stable mutant transgenic animals, but not wild-type human SOD1 transgenics, exhibited a strong locomotor defect associated with the presence, specifically in mutant animals, of both soluble oligomers and insoluble aggregates of G85R protein. A whole-genome RNAi screen identified chaperones and other components whose deficiency increased aggregation and further diminished locomotion. The nature of the locomotor defect was investigated. Mutant animals were resistant to paralysis by the cholinesterase inhibitor aldicarb, while exhibiting normal sensitivity to the cholinergic agonist levamisole and normal muscle morphology. When fluorescently labeled presynaptic components were examined in the dorsal nerve cord, decreased numbers of puncta corresponding to neuromuscular junctions were observed in mutant animals and brightness was also diminished. At the EM level, mutant animals exhibited a reduced number of synaptic vesicles. Neurotoxicity in this system thus appears to be mediated by misfolded SOD1 and is exerted on synaptic vesicle biogenesis and/or trafficking.

Figure 1. Locomotor defects in G85R and G85R-YFP transgenic C. elegans.

Rates of forward movement were measured as net distance traveled during 30 sec, divided by body length, normalized against non-transgenic L4 animals. The assay was carried out immediately after transfer to a fresh bacterial plate and measured by videomicroscopy. N = 20 for each time point; error bars = SEM. A, unfused constructs. B, YFP fusions. Transgenic lines employed were WTSOD (line 7), G85R (line 10), WTSOD-YFP (line 51), G85R-YFP (line 18) and H46R/H48Q-YFP (line 7).

Figure 2. Aggregation in G85R and G85R-YFP neurons.

A, B, Fluorescence analyses of G85R-YFP and WTSOD-YFP transgenic animals at stage L4, comparing cytoplasmic fluorescence in cell bodies of ventral nerve cord. WTSOD-YFP (panel B) exhibits a diffuse pattern in cell bodies (with noticeable nuclear exclusion) while G85R-YFP (panel A) exhibits a more discrete pattern in brighter, well defined, zones. C–F, EM analyses. Aggregate (Agg) in the perinuclear region of a ventral cord cell body of a G85R-YFP day 4 adult animal (panel C), and normal appearance of cell body of a WTSOD-YFP day 4 adult (panel D); Nu: nucleus. G85R (nonfused) shows fibrillar-appearing aggregate in a large nerve ring process (panels E,F). Panels C, D from chemical immersion fixed preparation and E, F from high pressure freezing preparation.

Figure 3. Biochemical analysis of SOD solubility and assembly state in transgenic animals.

A, Fractionation of worm extracts into soluble and insoluble fractions under native conditions and Western blotting. Extract was prepared by sonication and cuticle debris removed, followed by centrifugation at 120,000×g×15 min to produce soluble (S) and insoluble pellet (P) fractions. G85R and G85R-YFP exhibit substantial insoluble material whereas none is detected in wild-type. B, Western blot analysis of fractions from gel filtration chromatography of soluble fraction, showing soluble G85R-YFP oligomers extending from monomer-size up to the void volume. WTSOD-YFP, by contrast, shows only lower molecular weight species. Numbers above the blot panels indicate the size in kDa of standard proteins chromatographed on the same column; V_o = void volume, corresponding to ∼5 MDa size.

Figure 4. Distinct neuronal pattern of aggregation in G85R-YFP animals.

A, Involvement of selected lateral neurons. Mid-L4 G85R-YFP animals were scored; N = 21. Note that neuronal function and birth time do not appear to correlate with aggregate formation. B, PVDR generally presents with a well demarcated fluorescent inclusion whereas PDER, lying next to it, is usually unaffected in G85R-YFP animals. Arrows point to cytosolic aggregates.

Figure 5. Ventral nerve cord is affected in 4 day old adult G85R animals – fewer and smaller diameter processes and lack of organelles.

A, Number of neuronal processes is mildly reduced. Transverse sections of transgenic animals were prepared and examined by EM, and the number of neuronal processes in the main bundle of the ventral nerve cord was determined. N = 9. (Error bars indicate SEM.) B–E, Representative cross-sections of WTSOD and G85R animals at two levels of magnification, with white dashed lines in B, C denoting the boundaries of the main bundle of processes. The diameter of processes was reduced, and the number of organelles including mitochondria (m) and synaptic vesicles (arrows) was greatly reduced in G85R.

Figure 6. Dorsal nerve cord of G85R at L4 stage exhibits diminished numbers and brightness of puncta of fluorescent presynaptic markers and diminished fluorescence recovery of GFP-synaptobrevin after photobleaching.

A, Three different fluorescent protein-tagged synaptic proteins, synaptobrevin (SNB-1), RAB-3, and synapsin-1 (SNN-1), and one tagged presynaptic active zone protein, Rim1, were examined in WTSOD and G85R transgenic worms. Anterior portion of the dorsal cord is shown. In all cases there was a reduced number of puncta in the mutant animals. This was quantitated in panel B for GFP-SNB-1 animals, examining 10 of each genotype at L4 stage. Puncta were counted along a 50 micron distance and normalized to total body length. The number was reduced in the mutant animals, p = 0.0004. Error bars = SEM. Panel C, fluorescent recovery after photobleaching (FRAP) of GFP-synpatobrevin. Left, example of photobleaching of dorsal cord. Right, recovery of fluorescence plotted as function of time. Mid-L4 animals were subject to photobleaching, covering an area of 1 µm surrounding the dorsal punctum of interest, and fluorescence was recorded thereafter. Average intensity was normalized to the pre-bleaching intensity. G85R showed significantly slower recovery. N = 8 for each genotype. Error bars are SEM.

Figure 7. Aldicarb paralysis assay of SOD transgenic strains.

G85R and G85R-YFP transgenic animals of L4 stage are relatively resistant to aldicarb. A, Comparison of WTSOD and G85R transgenic animals at various times after exposure to aldicarb, measuring percentage that fail to exhibit movement upon physical prodding in the head region. B, Comparison of WTSOD-YFP and G85R-YFP animals for percent exhibiting aldicarb paralysis. Error bars are SEM.