An ALS-Linked Mutant SOD1 Produces a Locomotor Defect Associated with Aggregation and Synaptic Dysfunction When Expressed in Neurons of Caenorhabditis elegans

1A number of neurodegenerative diseases have been associated with protein misfolding and aggregation, with a specific protein in each case observed to aggregate in a particular population of neurons. For example, in the case of amyotrophic lateral sclerosis (Lou Gehrig's Disease), a dominantly inherited form of this condition, accounting for ∼2% of cases, is associated with mutant forms of the abundant cytosolic homodimeric enzyme superoxide dismutase (SOD1), which accumulate in insoluble aggregates in motor neurons –. Mutational studies of SOD1-linked ALS have uncovered single residue substitutions throughout the enzyme subunit, and studies in vitro indicate that the substitutions generally destabilize the protein, disposing to misfolding and aggregation –. It remains unknown, however, exactly how apparent misfolding and aggregation of SOD1 exerts toxic effects on motor neurons. Is there a central common effect shared by the various mutant alleles that comprises a common pathway of motor neuron injury?

2Mice transgenic for a variety of mutant SOD1 alleles also develop motor neuron disease resembling that of affected humans,, enabling a variety of pathological and biochemical studies. A survey of pathology reported for various alleles implicates a variety of potential physical sites of toxicity, including mitochondria, endoplasmic reticulum, and axonal traffic. For example, abnormal-appearing mitochondria have been observed in animals transgenic for G93A and G37R SOD1 –, and several mutant SOD1's have been coisolated with spinal cord mitochondria –. Concerning ER function, an unfolded protein response (UPR) was observed in spinal cord of G93A mice, and a recent report suggests that mutant SOD1 induces this response by binding to the ER membrane component Derlin-1, blocking retrograde traffic of ER proteins to the cytosol for proteasomal degradation (ERAD) at the level of ubiquitination. Concerning axonal traffic, both anterograde and retrograde transport have been observed to be retarded in mice transgenic for mutant SOD1 –. Which, if any, of these effects is primary to mutant SOD1-induced motor neuron damage?

3One approach to resolving this question is to produce mutant human SOD1 in neurons in an invertebrate system to inspect for effects on function, with the idea that this might reveal a minimal target of the toxic effect which could ultimately be further evaluated in the mammalian system. Such an approach has been taken, for example, with other neurodegenerative disease-associated proteins, expressing them in Drosophila or C. elegans, including polyglutamine repeat proteins, and α-synuclein,. Here we have taken such an approach with SOD1 using C. elegans, programming pan-neuronal expression of a mutant version of human SOD1, G85R, that obligatorily misfolds. We observe a locomotor defect in transgenic animals expressing the mutant SOD1, associated with aggregation of the mutant SOD1 protein and with synaptic dysfunction, involving deficient numbers and possibly deficient trafficking of pre-synaptic vesicles.

4To examine the effects of an ALS-associated human mutant SOD1 on a collective of neurons in an optically accessible nervous system, we produced transgenic C. elegans expressing G85R mutant or wild-type human SOD1 (referred to hereafter as SOD). G85R SOD has been identified in human cases of ALS, and G85R SOD transgenic mice develop a similar disease. In the latter setting, the protein has been shown to behave as a misfolded monomer. That is, it fails to form the normal SOD homodimer, and it lacks the normal disulfide bond that is formed between Cys 57 and Cys 146 when the protein is properly folded. (This disulfide bond is normally formed despite localization of SOD to the relatively reducing cytosol). To express the wild-type and G85R SOD proteins in as many of the 302 neurons of a C. elegans hermaphrodite as possible, a pan-neuronal promoter, the promoter of the C. elegans synptobrevin gene (snb-1), was used to drive the respective wild-type and G85R human cDNAs encoding SOD. To allow direct observation of the expressed SOD proteins, two additional constructs were employed that join a YFP reporter sequence via a flexible peptide linker to the C-terminus of the wild-type or mutant SOD. Multiple stable transgenic C. elegans lines of both unfused and fused constructs were produced.

5We noticed immediately that G85R SOD transgenic animals exhibited minimal forward movement across the culture medium compared with normal movement of age and protein expression-matched wild-type SOD transgenic strains (Figure 1A and Video S1; see Figure S1 for immunoblot analysis and activity blot analysis). The wild-type SOD transgenic animals exhibited a forward movement speed similar to that of the parental nontransgenic C. elegans N2 strain (Figure 1A). In addition to severely reduced forward crawling of the mutant transgenic animals, their side-to-side thrashing movement in liquid was also severely affected (Figure S2 and Videos S2, S3). The rates of forward movement of the animals transgenic for the fusion proteins, WTSOD-YFP and G85R-YFP, likewise showed a large difference (Figure 1B), although the mutant now exhibited significant forward movement, and the wild-type fusion was somewhat slower than the nonfused wild-type transgenic animals (compare Figure 1B with 1A). These same effects were observed on thrashing (Figure S2). Thus the addition of the YFP moiety has effects on the mutant and wild-type SOD proteins, exerted in opposite directions. Overall, however, even with the YFP fusion protein, there was still markedly slower movement of the G85R animals as compared with the wild-type (Figure 1B).

6To assess whether the biological behavior of the G85R-YFP fusion protein has any relevance to the mammalian context where expression is associated with an identifiable clinical neuronal disorder, G85R-YFP was produced in transgenic mice from a human SOD genomic clone with YFP fused to the last coding exon. This fusion produced an ALS phenotype in mice at 3–9 months of age, with the age of onset of the motor deficit depending on copy number and expression level of the transgene (JW, GF, KF, and ALH, unpublished). By contrast, expression-matched wild-type SOD-YFP transgenic mice produced in parallel did not develop disease even at ages of 2 years and beyond. Thus the SOD-YFP fusions tested here reflect the behavior of the nonfused counterparts in the context of production of ALS-like disease in the mammalian setting.

7An additional SOD mutant transgenic C. elegans strain, H46R/H48Q-YFP, containing an SOD double mutant allele that blocks copper binding by SOD and produces ALS in transgenic mice, was also examined. Here it produced a movement defect less prominent than that seen in G85R-YFP (Figure 1B).

8Fluorescence microscopy of transgenic C. elegans at both larval and adult stages revealed fluorescence in many neurons in the nerve ring (head region), the ventral nerve cord, lateral body wall, and tail ganglia (Figure S3A). Non-neuronal fluorescence of the spermatheca and two gonadal distal tip cells was also observed. The character of fluorescence of neuronal cell bodies of G85R-YFP transgenic animals differed from that of WTSOD-YFP transgenics as exemplified by cell bodies of motor neurons along the ventral nerve cord (Figure 2A, B). [Neuronal processes, by contrast, did not exhibit altered fluorescence (Figure S3B).] Cell bodies from the wild-type animals exhibited a more diffuse cytosolic fluorescence pattern, whereas those from the G85R-YFP mutant exhibited a well-demarcated pattern, suggestive of aggregate formation. Consistent with this, a FRAP experiment (fluorescent recovery after photobleaching) showed very slow recovery of fluorescence in mutant cell bodies as compared with wild-type (see Figure S4).

9Aggregates were directly observed in the mutant by EM examination following chemical fixation (Figure 2C, D), which revealed well-delineated electron-dense inclusions in the cytosol of ventral nerve cord cell bodies of G85R-YFP animals (Figure 2C, Agg.). By contrast, there were no recognizable aggregates in WTSOD-YFP cell bodies (Figure 2D). The location of G85R-YFP aggregates in a perinuclear position is reminiscent of aggresomes or of perinuclear structures distinct from the centrosome known as JUNQ, involved in juxtranuclear quality control, but aggregates formed in the unfused G85R animals exhibited a more diffuse, “fluffy” character (Figure S5A, asterisks). When high pressure freezing/fixation was employed on the G85R animals, these regions were now observed as amorphous inclusions (e.g. Figure 2E, Agg.), which at high magnification appeared to contain loosely stacked fibrillar material (Figure 2F).

10Biochemical analysis also revealed insoluble SOD protein as well as soluble oligomers to be present specifically in mutant animals. Animals were homogenized in non-denaturing buffer, the extracts were centrifuged at 120,000×g×15 min, and the supernatant and pellet fractions were solubilized under reducing conditions and analyzed by immunoblotting with anti-SOD antibodies. As shown in Figure 3A, both G85R and G85R-YFP animals exhibited a substantial portion of SOD protein in the insoluble fraction (P) whereas all of the SOD protein in wild-type animals localized to the soluble fraction (S). The soluble fractions of G85R-YFP and WTSOD-YFP were further analyzed by gel filtration chromatography (under reducing conditions), and immunoblotting of the fractions revealed the presence of soluble oligomers specifically present in the mutant extending in size from ∼45 kDa (size of the fusion monomer) up to ∼5 MDa (void volume). Such soluble oligomers are likely to be the precursors to insoluble aggregates and have been implicated in cellular toxicity –.

11To address the distribution of neurons containing aggregates, first we counted fluorescent aggregates in the ventral nerve cord cell bodies (all belonging to motor neurons) at stage L4. Individual cell bodies could not be readily assigned due to their high density, but we observed greater than 40 aggregate-containing cell bodies out of the total of 56 cell bodies of the ventral nerve cord. The aggregates in these cell bodies were typically of large size, like those shown in Figure 2A. Next, a number of individually identifiable neurons of the lateral body wall were scored in L4 stage G85R-YFP animals for presence or absence of a visible fluorescent aggregate (Figure 4A). This revealed that some neurons, e.g. PVDR and SDQR, nearly always contained an aggregate, whereas other neurons only rarely exhibited an aggregate. Of interest in regard to such differences is the behavior of the two neighboring lateral body wall mechanosensory neurons, PVDR and PDER (Figure 4B). PVDR nearly always exhibited fluorescent aggregates, usually first observed at the L4 stage, whereas PDER was generally spared (Figure 4B; note that PDER was present in G85R-YFP animals, delineated by diffuse green fluorescence not clearly visible in this image). The nature of the difference between these two neurons that governs the different aggregation behavior is interesting to contemplate and may relate to specific neuronal activity. More generally, whether neurons which have sequestered misfolded SOD-YFP into an insoluble fraction are functionally more affected in vivo than neurons lacking aggregates, as suggested by Matsumoto et al, who observed reduced viability of SOD-YFP aggregate-containing cultured cells, or, conversely, are protected by aggregation, as suggested by primary neuron studies with Httex1-polyQ-GFP, is unknown.

12Concerning temporal development of aggregates, fluorescent aggregates in the head region and ventral cord were detectable as early as L1, while lateral body wall aggregates were not detected until L3. Aggregation was not restricted to motor neurons, as indicated in Figure 4. Concerning other sensory neurons, we note that the DiI-stainable chemosensory amphid neurons in the anterior region, as well as the stainable phasmid tail neurons, exhibited normal DiI staining, but no green fluorescence was observed in these cells in either mutant or wild-type animals, leaving uncertain whether any expression of SOD-YFP occurred in these neurons. Consistent with broad neuronal involvement, however, additional general functions of mutant animals were found to be affected, including survival, brood size, and rate of development (see Figures S6, S7).

13In further EM studies, transverse sections of the ventral nerve cord were prepared and the processes examined. In 4 day old adults, the number of processes (mostly axons) in G85R animals was slightly reduced compared with wild-type (Figure 5A). More striking, however, were morphologic abnormalities that included reduced diameter (compare Figure 5B, C), and reduced numbers of organelles, both mitochondria and vesicles, within them (compare Figure 5D, E). Some of the processes in G85R animals were also swollen by large vesicular structures (Figure 5C). In contrast with these abnormalities of the ventral nerve cord, the innervated body wall muscles appeared normal, with normal sarcomeres and subcellular organelles (not shown).

14To address whether synapses might be affected, labeling was carried out with GFP-synaptobrevin. This protein, when expressed in DA motor neurons, has been observed to produce a continuous pattern of fluorescent puncta in the dorsal nerve cord, where neuromuscular junctions are present (e.g. ref. 39). Wild-type and G85R (unfused) transgenic animals were crossed to Punc-129::GFP-synaptobrevin transgenic animals, and the double transgenic progeny were examined for the number of dorsally located green synaptic puncta that demarcate neuromuscular junctions (Figure 6A). Whereas wild-type animals exhibited a continuous band of such puncta, the mutant animals exhibited a discontinuous pattern. In addition to the reduced number of puncta (Figure 6B), their signals were also reduced. A similar decrease in number and brightness of puncta relative to wild-type was also observed when the fluorescent-tagged synaptic vesicle proteins YFP-RAB-3 and synapsin-1-YFP (SNN-1-YFP) were similarly crossed in and examined (Figure 6A). These data suggest that both synapse number and vesicle content in the dorsal cord is reduced. Consistent with decreased numbers of synapses, when the presynaptic active zone protein UNC-10 (RIM1) was examined, it exhibited a diminished number of puncta in the mutant (Figure 6A).

15A FRAP experiment was also carried out on the GFP-synaptobrevin animals, showing slower recovery of fluorescence at puncta in the dorsal region in G85R animals vs wild-type (Figure 6C). This potentially reflects abnormal dynamic behavior of synaptic vesicles in this dorsal region.

16To inspect synapses at the ultrastructural level, EM studies of the nerve ring were carried out, because this region has the greatest concentration of synapses. Thin sections were prepared from 4 day old adult G85R or wild-type animals using the high pressure freezing method. The character of synapses appeared to be affected (Figure S8). For example, in many synapses of the mutant animals (panels C,D), there appeared to be a paucity of synaptic vesicles which was more pronounced in the region closest to the presynaptic density (37 of 50 synapses examined) as compared with wild-type (panels A,B), where vesicles were densely packed in the presynaptic region including the active zone and periactive zone (44 of 50 synapses examined). In view of the paucity of vesicles, further EM analysis was carried out to identify whether there might be a corresponding accumulation of vesicles in cell bodies, as occurs for example in unc-104 kinesin-deficient animals, but this was not observed. Instead, vesicles were observed in some cases to be trapped within aggregate material.

17To assess synaptic function, the effects of the cholinesterase inhibitor alidcarb in paralyzing wild-type or G85R transgenic animals was measured,. Resistance to this inhibitor can be caused by loss of cholinergic synaptic transmission. Both G85R and G85R-YFP animals were strongly resistant to aldicarb as compared with the corresponding wild-type animals (Figure 7), indicating deficient synaptic function. To distinguish whether postsynaptic function was contributing, a second compound, levamisole, a cholinergic receptor agonist, was employed. G85R animals displayed the same sensitivity to levamisole-induced paralysis as wild-type (data not shown). Thus both morphologic and functional assays point to defective presynaptic function as a proximate cause to the locomotor defect of G85R transgenic animals.

18To identify genetic modifiers of neuronal aggregation in the G85R SOD-YFP transgenic animals, we carried out an RNAi screen. Neurons of C. elegans are relatively resistant to RNA interference, so alleles previously identified to enhance interference activity, eri-1 (mg366) and lin-15B (n744), were introduced. Strikingly, the introduction of these alleles led to a significant reduction in fluorescence from the G85R-YFP protein as compared with the parental strain (Figure S9A), associated with a decrease in the number of fluorescent inclusions observed in the ventral nerve cord (Figure S9B) and with somewhat improved movement. These results are consistent with a previous report that lin-15B could produce silencing of multicopy transgenes. We observed that either allele alone could produce substantial silencing of the G85R-YFP transgene, although lin-15B exerted a greater effect. The combination of alleles proved to sufficiently reduce fluorescence and aggregate formation of G85R-YFP to a degree that could allow for a “dynamic” range of effects of RNAi screening, i.e. both reduction and enhancement of green fluorescence patterns reflecting aggregation would be detectable. A bacterial feeding library was employed for RNAi testing. Collectives of animals at all stages were transferred onto feeding plates and visually examined by multiple observers after 3–6 days for changes in the number and intensity of fluorescent aggregates in the nerve ring and ventral nerve cord as compared with the strain fed bacteria with an empty vector.

19In the general case of observing decreased fluorescent aggregates when animals were placed on a particular interfering bacterial strain, the development of the animals was generally also slowed and viability in many cases reduced, reflecting likely effects on general health or on general gene expression, and these interfering RNAs were not studied further. There was one exception to this, involving an interfering RNA for a ubiquitin specific protease, where fluorescent aggregation was reduced and viability somewhat improved, and this gene is under further study.

20In the general case of animals with increased fluorescent aggregates on a particular interfering bacterial strain, these animals generally exhibited larger numbers of fluorescent inclusions and diminished locomotion. There were 88 such hits (Table S1). 7 of these appeared likely to suppress eri-1; lin-15B action and were not studied further (see last entries in Table S1), while the remaining 81 hits were categorized into 10 groups (Table 1). The largest group of well-annotated hits comprised molecular chaperones and quality control components, amounting to about a quarter of the hits. These are discussed below. The other large group, of about the same size, comprised a collective of uncharacterized gene products.

21To independently confirm RNAi hits in the parental genetic background, loss-of-function alleles were obtained for 12 hits of interest and were crossed with the parental G85R-YFP strain. 11 out of 12 of these enhanced the inclusion phenotype of G85R-YFP. For example, heat shock factor 1 (HSF1), which transcriptionally regulates a number of stress components, registered very strongly in the RNAi screen in increasing aggregate formation. Consistently, when the sy441 allele of hsf-1 was crossed into G85R-YFP, a strong increase in aggregate formation was observed (Figure S10A), and locomotion of these animals was substantially decreased as compared with either parental strain (Figure S10B).

22Multiple interference hits of strong magnitude were observed in a number of pathways (Table 2), indicating that these pathways are likely playing a role in preventing aggregation of the misfolded G85R-YFP protein or facilitating its turnover. For example, hits of a collective of chaperone components registered strong increases of aggregation, including an Hsp110 (C30C11.4), a DnaJ (A2) (dnj-19), an Hsp70 (stc-1), and a neuron specific Hsp16 (F08H9.4). Three of these were validated as strongly increasing aggregation when mutant alleles were crossed with G85R-YFP. Two hits were identified in the ubiquitin-mediated turnover pathway at the level of E3 ligase SCF complexes, SEL-10, an F-box protein, and RBX-1, a RING finger protein, the latter confirmed with the ok782 knockout allele. Three hits were also observed in the sumoylation pathway by the RNAi screen, uba-2, encoding the E1 enzyme that activates SUMO, ubc-9, which encodes one subunit of the E2 SUMO-conjugating heterodimer, and an E3 SUMO-ligase component gei-17 (homologous to PIAS1). In validation of the interference effects, an allele of SUMO, smo-1 (OK359), increased aggregation when crossed into the parental G85R-YFP strain. The latter hits raised the question of whether G85R-YFP is itself sumoylated, and this is under study. In addition, three components involved with redox regulation were identified, PDI-2, an orthologue of a human thioredoxin domain-containing protein (C30H7.2), and BLI-3, a dual oxidase with both a peroxidase domain and a superoxide-generating oxidase domain.

23Other pathways were also identified through strong effects of interference. For example, a hit in the TGFβ component DBL-1 was confirmed by the allele nk3. DBL-1 is expressed in neuronal cells, e.g. in the ventral cord, has recently been implicated in GABAergic synaptic transmission, and has been shown to affect both body size and male tail development. Notably, in mammalian contexts, TGF-β family members have exhibited effects on axon outgrowth and protection from excitotoxicity. A hit of the dopamine transporter DAT-1 was confirmed with allele tm903. A link between neuronal activity and aggregation in the cases of GABAergic and dopaminergic transmission thus seems a consideration but requires further study. In the DNA replication/repair pathways, interference with top-1, the gene for topoisomerase 1, had a very strong effect, equivalent to the strong effect of hsf-1 interference. How such action could figure into aggregation behavior remains unclear. Similarly, interference of div-1, a subunit of the DNA polymerase α/primase complex, increased aggregation. Also a strong effect was observed with interference of pha-4, a FoxA transcription factor, implicated in calorie-restriction-mediated longevity, at least in part by regulating endogenous C. elegans SOD enzymes.

24We have described here that pan-neuronal expression of a human ALS-associated mutant SOD in C. elegans produces substantial locomotor defects associated with macroscopic aggregation in neuronal cell bodies. By contrast, a wild-type human SOD produced neither locomotor defects nor aggregation. Further testing indicated that the human SOD mutant, G85R, unable to fold properly and producing both soluble oligomers and aggregates, appears to produce neuronal dysfunction in C. elegans at the presynaptic level. This was indicated by three types of observation. First, there was a reduced number and brightness of puncta of fluorescently-labeled synaptobrevin, RAB-3, and synapsin, as well as of the presynaptic active zone protein RIM1 in the dorsal nerve cord in transgenic G85R animals as compared with wild-type SOD transgenic animals. Second, mutant transgenic animals but not wild-type exhibited resistance to aldicarb paralysis, consistent with deficient acetylcholine release at cholinergic synapses in the mutant. Third, EM studies on a small number of animals revealed paucity of vesicles in the presynaptic region of nerve ring synapses of the mutant animals. In addition, EM showed reduced numbers of vesicles and mitochondria in ventral nerve cord processes (axons) of transgenic mutant animals. In contrast with the foregoing findings pointing to presynaptic dysfunction, there was a lack of postsynaptic effects. The cholinergic agonist levamisole produced the same efficient paralysis of both mutant G85R and wild-type SOD transgenic animals. In addition, EM inspection of muscle revealed normal morphology.

25The putative presynaptic defect could result, in the first instance, from defective biogenesis, axonal transport, or recycling of synaptic vesicles. The diminution of vesicles in dorsal and ventral nerve cord processes and their sparsity in the presynaptic regions as demonstrated from the EM and fluorescence studies suggest that biogenesis or transport is affected. Because accumulations of vesicles were not detected in cell bodies, it seems less likely that transport is affected. Yet the failure of recovery of the GFP-synaptobrevin fluorescence following photobleaching could be consistent with an ongoing transport or recycling defect. Further studies will be required to resolve the steps affected.

26Excitingly, effects of expression of human SOD1 on synaptic transmission in another invertebrate system have recently been reported. Watson et al observed that expression of human SOD1 (wild-type or mutant) in motor neurons of Drosophila produced an age-progressive climbing defect that was associated in electrophysiological studies, stimulating muscle through the giant fiber circuit, with progressive loss of muscle response during high frequency stimulation.

27Presynaptic effects have also been observed in several contexts in C. elegans expressing a number of other neurodegeneration-associated proteins. Animals transgenic for both wild-type and FTDP-associated mutant forms of tau presented with locomotor defects, associated with aldicarb resistance (and levamisole sensitivity), followed by appearance of macroscopic aggregates and then apparent neuronal loss. Animals transgenic for α-synuclein exhibited locomotor defects when any of a number of synaptic proteins were knocked down, associated with aldicarb resistance and levamisole sensitivity. In contrast with these studies, however, the studies of SOD1 presented here indicate a direct and specific effect of the mutant G85R SOD1 on presynaptic function.

28So far it is not evident how or whether the presence of G85R-SOD aggregates in cell bodies of specific neurons, comprising a variety of neuronal cell types in the transgenic mutant animals, directly relates to the apparent presynaptic defect. We note, however, that the degree of locomotor defect correlates roughly with the degree of aggregation observed. For example, for the G85R-YFP transgene, out of multiple stable integrant lines, those with the highest level of fluorescence and greatest level of aggregation exhibited the strongest locomotor defect. Thus, either the aggregates themselves or perhaps the apparent precursors, soluble oligomers (Figure 3B), may be directly responsible for the presynaptic defect. The defect could come at the level of physical interactions of oligomers or aggregates either directly with synaptic vesicles themselves or with soluble or cytoskeletal components that are involved with vesicle biogenesis and traffic. A recent study producing pan-neuronal expression of dimeric versions of human wild-type and G85R SOD1 in C. elegans also observed locomotor defects associated with aggregation, but, in contrast to the direct correlation above, observed that a heterodimeric G85R-WTSOD-GFP molecule, while producing less aggregation than G85R-G85R-GFP, led to a greater paraquat-induced reduction of animal survival. Thus in the context of a heterodimer, residual SOD enzymatic activity may contribute to toxicity.

29Notably, despite evident protein aggregation and synaptic dysfunction, neurons in the mutant animals studied here were not subject to cell death, even during later adult life. This resembles the observation in the recent report of Watson et al where human SOD1 expressed in Drosophila motor neurons produced both focal SOD protein accumulation and measurable electrical dysfunction but no observable cell death. Similarly, in earlier studies of C. elegans PLM neurons transgenic for polyglutamine expansion, touch sensitivity function was abolished but cell death did not occur. This lack of cell death may either relate to the state of disease progression or be a function of the invertebrate neuronal systems themselves. For example, concerning stage of disease, in the mammalian context, SOD1-affected motor neurons appear likely to be functionally affected for a period of time before being subject to cell death. In C. elegans or Drosophila, by contrast, the trajectory may not extend sufficiently in time to produce cell death, albeit that added insults such as oxidative toxicity may be capable of producing cell death. Alternatively, these systems may differ from that of mammals. C. elegans has, for example, a limited number of glia, and they may not function as in the mammalian context to hasten death of affected neurons –. Alternatively, the absence of cell death could be a function of a different neuronal response to chronic exposure to misfolded protein as compared with mammalian neurons.

30The RNA interference screen conducted here, examining relative levels of G85R-YFP protein aggregation in relation to knockdown of various gene products, validated in many cases by crosses with corresponding mutant alleles (Table 2), provided evidence that a proteostatic network similar to that present in body wall muscles of C. elegans, and elsewhere is operative in C. elegans neurons. Whether it is induced in response to mutant human SOD1 remains to be determined, and whether a higher level of expression of some or all members of the network could be protective remains to be tested. Two transcriptional regulators that lie at the top of such a network were identified, HSF-1 and PHA-4/FoxA, which have a broad range of targets in, for example, chaperone pathways and redox regulation, respectively. Components within the network were also identified, including chaperones, an E3 ligase, and redox components. Additional components had effects on aggregation but their mechanism of action remains unclear, including SUMO, the TGF-β homologue, DBL-1, expressed mainly in neurons, and two components of DNA maintenance, topoisomerase I and a subunit of polα/primase complex, implicating DNA integrity in the response. Notably absent from both the interference screen and from EM studies was evidence for involvement of the autophagy system. Consistent with this, administration of rapamycin was without effect on the extent of aggregation (data not shown).

31Do the phenotypic properties observed here bear any relation to mammalian disease induced by expression of mutant SOD1? Could the transgenic C. elegans inform usefully about mammalian disease? As mentioned, the G85RSOD-YFP fusion protein expressed in mice indeed produces an ALS-like disease whereas WTSOD-YFP fusion produces no ill effect. Interestingly, the presynaptic effects observed here with the G85R transgenic C. elegans may have a parallel in mutant G85R SOD transgenic mice, as reported recently by Caroni and colleagues, who examined motor neuron axons and NMJs in hind limb muscle of mutant transgenic animals of varying age. In vulnerable motor neuron axons (FF and FR), they observed at early time (7 months of age; 2 months before end-stage) localized synaptic vesicle accumulation associated with diminished overall density of vesicles, reflecting apparent stalling of vesicle traffic, followed at a later stage by severe loss of synaptic vesicles associated with loss of presynaptic active zone markers. The findings at later time generally agree with the observations here in dorsal cord of our G85R C. elegans, where both fluorescent synaptic vesicle protein markers and an active zone marker (RIM1) appear to be reduced (Figure 6). The nature of the vesicle trafficking defect in either setting remains to be elucidated. Does it reflect failure of vesicles to be produced in the first instance, as suggested by the lack of organelles in ventral cord processes here (Figure 5), and/or is it a block of recycling, as might be suggested by the FRAP analysis of GFP-synaptobrevin (Figure 6C)? Is it a direct effect of mutant SOD, forming physical association with synaptic vesicles? Or is it a secondary effect, mediated e.g. via the motor/cytoskeletal trafficking system? Questions concerning both the basis to vesicular defects and the overall pathway of toxicity of mutant SOD protein remain to be resolved.

32The C. elegans snb-1 promoter, a PCR-amplified genomic DNA segment extending from minus 3021 bp to just upstream of the SNB start codon, was inserted in place of the unc-54 enhancer/promoter in the plasmid pPD30_38 (Fire Lab Vector Kit, Addgene Inc., Cambridge, MA), and the various human SOD cDNA-containing segments were then adjoined. SOD mutations were generated by PCR, and the derived coding sequences confirmed by sequencing them in entirety. Fusion constructs joining human SOD with YFP via a linker segment (LQLQASAV) were kindly provided by Dr. R. Morimoto, Northwestern University.

33The N2 Bristol strain of C. elegans was used as the wild-type strain. Standard culturing and genetic methods were used. Animals were maintained at 20°C unless otherwise indicated. Mutant strains obtained from the Caenorhabditis Genetics Center (CGC), the National Bioresource Project in Japan, and the lab of Joshua Kaplan are listed in Suppl. Table 2. Germline transformation was performed by injecting DNA solution containing 20 ng/µl of an SOD construct and 5 ng/µl of myo2::GFP into hermaphrodite gonads. Multiple extrachromosomal lines were established based on the fluorescent markers. They were further treated with trimethylpsoralen/UV to generate integrated lines that stably expressed the transgenes. At least three independent stable lines were produced for each variant, and each line was backcrossed with the N2 strain four times. The transgenic SOD and SOD-YFP lines used in these studies are designated in the legend to Figure 1. nuIs152 has the transgene Punc-129::GFP-snb-1. nuIs168 has the transgene Punc-129::YFP-Rab-3. nuIs163 and nuIs165 strains containing Punc-129::snn-1-YFP and Punc-129::unc-10-GFP were the kind gift of Joshua Kaplan.

34For high resolution imaging, animals were immobilized with levamisole and examined by either differential interference contrast (DIC) or fluorescence with an Olympus IX81 microscope equipped with spinning disk confocal illumination. For fluorescence recovery after photobleaching (FRAP), a laser confocal microscope was used (Zeiss LSM510 META).

35For transmission electron microscopy, animals were prepared by conventional two-step chemical immersion fixation or high-pressure-freezing,. Serial thin sections were prepared and post-stained with heavy metals. At least four animals of each genotype were analyzed using a Tecnai 12 Biotwin at 80 kV.

36A video-based assay was used to assess the locomotion speed of C. elegans. Animals were transferred to a plate with a fresh bacterial lawn on which movement tracks could be traced. Immediately upon release, worms exhibit a maximum movement response for a short duration. A 30 second movie was shot for each worm, and the ratio of the movement distance to the body length, measured by the NIH ImageJ software, was used as a movement index (see Video S1).

37Mid L4 animals were transferred to freshly made NGM agar plates without bacterial food containing 1 mM aldicarb, and at different time points the animals were prodded on the nose to determine whether they had reached complete paralysis. An identical experiment with 1 mM levamisole was also performed.

38To assess solubility of SOD protein, animals were disrupted by sonication on ice in 0.5 ml extraction buffer (PBS, 1 mM EDTA, 1 mM EGTA, 1 mM TCEP, with half a tablet of Complete Mini protease inhibitor cocktail (Roche)) and left on ice for 10 min to allow large debris, including cuticle, to sediment. The supernatant fraction was then centrifuged in a Beckman TLA-100 rotor at 53,000 rpm (>120,000×g) for 15 min at 4°C. Pellets were washed once by resuspension in the extraction buffer and sedimentation. Supernatant and SDS-solubilized pellet fractions were analyzed in SDS-PAGE under reducing conditions. Supernatant fractions (1 mg total protein) were also subjected to gel filtration chromatography on a Superose 6 gel filtration column (GE Healthcare) and eluted with PBS supplemented to 0.1 mM TCEP at 0.5 ml/min. Individual fractions (0.5 ml) were examined by SDS-PAGE and Western blotting. For Western analysis, antibodies to human SOD1 (SOD-100, Stressgen, Canada), antibodies raised in rabbits against purified YFP (Cocalico), or antibodies to actin (C4, MP Biomedicals, Inc., Aurora, Ohio) were used.

39An RNAi feeding library of 16,757 bacterial clones was employed for screening (GeneService, Cambridge, UK). Animals at mixed ages were screened in 96-well plates at 15°C as described. “Hits” were identified by an increased number and intensity of fluorescent neuronal inclusions using a Leica fluorescence stereoscope with a 2.0× PLANAPO lens. All positives were subjected to secondary screening at both 15°C and 20°C in 6-well plates. The identities of all positive RNAi clones were confirmed by DNA sequencing of the plasmid insert. For selected hits, where loss-of-function alleles were available, the corresponding strains were crossed to the G85R-YFP parental strain (line 8). The genotypes of the product strains were verified by PCR or PCR/DNA sequencing and the phenotypes studied.