In 1999, Hart and colleagues described a C. elegans Huntington's disease (HD) model based on expression of a huntingtin fragment (exon 1) containing a 150 repeat polyglutamine (Ht-Q150) expansion 3. Expression of this huntingtin fragment was driven by the osm-10 promoter, resulting in expression specifically in (non-essential) sensory neurons. Ht-Q150 expression was found to induce both neuronal dysfunction (demonstrated by the inability of chemosensory neurons to take up fluorescent dyes) and eventual death of chemosensory neurons (under conditions of osm-10::GFP co-expression). The apparent toxicity of Ht-Q150 was age-dependent but relatively mild, such that 13% of transgenic worms had dye uptake abnormalities at day 8 of development (mid-adulthood). This transgenic phenotype was subsequently used to identify mutations that enhanced Ht-Q150 toxicity, leading to the identification of pqe-1 4. Loss-of-function mutations in pqe-1 were found to dramatically enhance Ht-Q150-dependent chemosensory neuron death. pqe-1 encodes a nuclear protein rich in glutamines and prolines that also contains a conserved exonuclease domain (although PQE-1-homologous protein exist in mammals and flies, these proteins do not appear to contain glutamine/proline-rich domains). Given the cellular distributions of PQE-1 and Ht-Q150, these authors argued that wild type PQE-1 protein may protect from Ht-Q150 toxicity by competing for proteins sequestered by Ht-Q150.

The osm-10/Ht-Q150 model has also been used to investigate the roles of specific histone deacetylases in regulating huntingtin polyglutamine (Ht-polyQ) toxicity 5. Studies in multiple models have demonstrated that expanded polyglutamine huntingtin can sequester CREB-binding protein (CBP) and its associated histone acetyltransferase activity 67. Inhibition of histone deacetylase activity can counter this effect and subsequently reduce Ht-polyQ toxicity 89. The ability to knock down specific gene expression in C. elegans by a simple feeding RNA interference procedure 10 allowed Bates et al to quickly assess the roles of 8 specific histone deacetylases in Ht-polyQ. Interestingly, knock down of one deacetylase (hda-3) reduced polyQ toxicity, while knockdown of the others increased toxicity as expected. Introduction of loss of function mutations for hda-1, hda-4, and sir-2.1 similarly enhanced Ht-polyQ toxicity.

A conceptually similar model for HD was developed by Parker et al 11 using the mec-3 promoter to express an N-terminal 57 residue fragment of huntingtin (with or without expanded polyglutamine repeats) fused to Green Fluorescent Protein (Ht-polyQ::GFP). The mec-3 promoter is active in 10 (non-essential) neurons, including the six touch receptor cells. Ht-polyQ::GFP toxicity was assayed by measuring touch responsivity in individual transgenic worms. As observed in the osm-10 model, increasing the number of polyglutamine repeats in the transgene led to increased deficits in touch sensitivity. Transgene-induced touch insensitivity was not associated with death of the touch cells, but could be associated with visible morphological abnormalities in touch cell axons. This model was subsequently used to demonstrate that over-expression of the sir-2.1 deacetylase could protect against Ht-polyQ::GFP toxicity 12. This result was extended by demonstrating that resveratrol, a demonstrated activator of sirtuin acetylases, could protect against Ht-polyQ toxicity in both worm and neuronal culture models. Interestingly, sir-2.1 or resveratrol protection in the C. elegans mec-3/Ht-polyQ model was dependent on the FOXO transcription factor daf-16, suggesting that this protection was not due to reversal of Ht-polyQ effects on histone acetylation per se. The mec-3/Ht-polyQ:: model has also been used to demonstrate a protective role for hipr-1, a homolog of the HIP1 huntingtin-interacting protein, against Ht-polyQ toxicity 13.

Perhaps a more general approach towards understanding polyglutamine repeat toxicity was initiated by the Morimoto lab, which examined the effects of short (Q19) and long (Q82) polyglutamine repeat lengths fused directly to GFP and expressed in C. elegans body wall muscle cells 14. Expression of GFP::Q82 resulted in aggregate formation and induction of heat shock proteins. Subsequent studies with a series of YFP-polyQ fusions demonstrated a narrow threshold of polyQ repeat size (35-40) for induction of aggregation and toxicity 15. Examination of transgenic worms expressing threshold-level glutamine repeats (Q40) demonstrated a strong age dependence for aggregation as well as significant individual variability. This model system was employed in an elegant series of studies to demonstrate that formation of polyQ aggregates generally disrupted protein homeostasis 16. Introduction of an aggregation-prone YFP::polyQ transgene into a collection of temperature-sensitive missense mutations was found to dramatically enhance the phenotypes of these mutants, while conversely the presence of these missense mutations was found to enhance the aggregation of YFP::polyQ. These studies are consistent with a general "chaperone depletion" model that posits that aggregating protein toxicity results from competition for limited components of the protein homeostasis machinery. Indeed, a large scale RNAi screen for modifiers of YFP::polyQ aggregation identified a large set of genes involved in protein folding or degradation that increased polyQ aggregation when their expression was knocked down 17.

Additional C. elegans polyQ models have been generated with muscle-expressed polyglutamine repeats associated with either 17 amino acid residues derived from the dentatorubural pallidoluysian atrophy protein (DRPLAP) 18 or huntingtin exon 1 19. The former model has been used to demonstrate protective effects of C. elegans p97 homologs CDC-48.1 and CDC-48.2, while the latter model was used to show ubiquilin protection against polyQ toxicity. The muscle Ht-polyQ model has recently been used to implicate mitochondrial fission/fusion in huntingtin toxicity 20.

The studies described above illustrate the advantages (and disadvantages) of C. elegans neurodegeneration model systems, and the type of findings that can be made. The ability to undertake unbiased forward genetic screens enabled the identification of a novel gene involved in Ht-polyQ toxicity (pqe-1). However, vertebrate homologs of pqe-1 do not contain the relevant glutamine/proline-rich portion of this protein, so the direct relevance to HD is unclear. The optical transparency and short lifespan of C. elegans readily allowed the demonstration of an age-dependence for polyQ aggregation, something that is difficult to do directly in other model systems. Similarly, the existence of a large collection of characterized C. elegans mutations enabled the important demonstration of polyQ aggregation-dependent perturbation of protein homeostasis. The technically simple feeding RNAi protocol has enabled a number of candidate genes (e.g., hda-1, hda-4, sir-2.1, hipr-1, cdc-48.1,2 and dpr-1) to be implicated in in vivo polyQ toxicity. As is the case in other model systems, however, the use of different transgenic constructs by different research groups does confound making direct parallel between some studies. For example, it is unclear if toxicity in the neuronal expression osm-10/Ht-Q150 model is due to the disruption of protein homeostasis, or if the huntingtin sequences themselves play an important role.

α-synuclein is a major component of Lewy bodies found in dystrophic dopaminergic neurons in Parkinson's disease (PD), and α-synuclein mutations have been found to be casual in a relatively small number of familial PD cases. C. elegans models of synucleinopathy have been established with human α-synuclein expression in either dopaminergic neurons 212223 or pan-neuronally 2123. Lasko et al observed movement deficits in worms with pan-neuronal expression of wild type or A53T α-synuclein, while Kuwahara did not observe any phenotypic effects of pan-neuronal wild type, A30P, or A53T α-synuclein expression. (These differences may be a result of the specific promoters used in the transgene constructions; Lasko et al used the aex-3 promoter, while Kuwahara et al used the unc-51 promoter). However, all three groups have reported loss of either dopaminergic neuron cell bodies or dendrites when α-synuclein expression is driven by the dopaminergic-specific dat-1 promoter. Kuwahara et al also observed preferential neuronal dysfunction in worms expressing mutant α-synuclein (A53T or A30P) relative to wild type, measuring either dendritic loss or deficits in behaviors (slowing upon food sensation) known to be dopamine-dependent. The dat-1/wt α-synuclein model of Cao et al was used to demonstrate the protective effects of the chaperone protein torsin A 22 and Rab1, a protein believed to function in Golgi vesicular trafficking 24. The pan-neuronal α-synuclein model of Kuwahara et al has been used in a large-scale feeding RNAi screen to identify enhancers of α-synuclein toxicity, leading to the identification of a number of genes (e.g., apa-2, aps-2, eps-8 and rab-7) involved in the endocytic pathway 25. Studies of C. elegans α-synuclein models have thus strongly supported the link between α-synuclein toxicity and intracellular (synaptic?) vesicle trafficking.

C. elegans models have also been constructed to look for genes specifically influencing α-synuclein aggregation, following the approach initially developed for polyQ-induced aggregation 17. The Caldwell and Nollen groups both demonstrated that α-synuclein::GFP (or YFP) fusion protein expressed in C. elegans muscle cells leads to the formation of visible fluorescent aggregates, as previously observed for GFP::polyQ (2627. Hamamichi et al initially tested 868 "hypothesis based" RNAi clones for enhancement of α-syn::GFP aggregation. (This screen was sensitized by co-expression of TOR-2 to reduce initial aggregation levels.) Twenty genes were ultimately identified that increased α-syn::GFP aggregation when their expression was knocked down early in development by feeding RNAi. Seven of these genes were tested for effects on α-synuclein toxicity (not necessarily equivalent to aggregation) by transgenic overexpression in the dat-1/α-synuclein model; five were found to partially suppress dopaminergic neuron loss. Interestingly, these validated aggregation/toxicity enhancer genes included the vacuolar assembly/sorting protein vps-41 and the autophagy-related atgr-7. van Ham et al similarly screened for genes that increased α-syn::YFP aggregation after RNAi knockdown, in this case using the "whole genome" Ahringer feeding RNAi library, which contains ~15,000 clones (~85% of the genome represented). 80 genes were identified, with an over-representation of lipid- and vesicle- associated genes. For three of these genes (sir-2.1, lagr-1, and ymel-1) suppression of α-syn::YFP aggregation was independently confirmed using genetic loss-of-function mutations.

Perhaps the most interesting result from these two large-scale screens is that essentially no overlap was identified between genes that suppressed GFP::polyQ and α-syn::GFP aggregation. This observation is consistent with results from yeast studies and supports the view that aggregation of different disease-associated proteins is not equivalent. However, it should be pointed out that there is also no overlap in the genes identified in the Hamamichi et al and van Ham et al studies. This lack of overlap could be the result of technical differences between the two screens, and in fact non-congruence between similar C. elegans RNAi screens is not uncommon (e.g., 2829). Large scale RNAi screens in C. elegans appear to have an inherent variability, and thus negative results from these screens should be interpreted cautiously.

The β-amyloid peptide (Aβ) is a primary component of senile plaques found in the brains of Alzheimer's disease (AD) patients, and the existence of mutations in the gene encoding Aβ (Amyloid Precursor Protein, APP) in a subset of familial AD cases argues for a causal role for this peptide in this disease. Aβ has also been found to accumulate in muscles of patients with Inclusion Body Myositis, a debilitating myopathy 30. Initial attempts to use C. elegans to understand Aβ toxicity employed the unc-54 promoter to constitutively express a signal peptide::Aβ minigene in body wall muscle 31. Transgenically-expressed Aβ was found to accumulate intracellularly in muscle cells, and result in an age-dependent paralysis phenotype. This intracellular Aβ was able to form amyloid dye-reactive deposits with a fibrillar ultrastructure (i.e., amyloid deposits) 32. Single amino acid substitutions (e.g., Leu¹⁷Pro) were identified that blocked amyloid formation in this model but did not reduce toxicity, suggesting that amyloid itself is not the toxic species 33. The HSP-16 family of small heat-shock proteins was found to be induced by and co-immunoprecipitate with Aβ in this model 34, and ectopic expression of HSP-16 could suppress Aβ toxicity 35. Cohen et al 36 used the unc-54/signal peptide::Aβ 1-42 model to investigate the roles of two important modulators of C. elegans lifespan, the insulin growth factor 1-like signaling (ILLS) pathway and heat shock factor (HSF), in Aβ toxicity. RNAi knockdown of either daf-16 (the FOXO transcription factor that controls gene expression downstream of ILS) or hsf-1 enhanced Aβ toxicity, while activation of DAF-16 (via RNAi knockdown of daf-2, a negative regulator of daf-16) suppressed toxicity. Bacterial deprivation, a form of dietary restriction that extends lifespan in C. elegans, has also been found to reduce Aβ (and polyQ) toxicity in a HSF-dependent manner 37.

Overall, the results of studies with worms with constitutive muscle expression of either Aβ or YFP::polyQ are similar, and suggest that in both cases the observed toxicity primarily results from a general perturbation of protein homeostasis (proteostasis). Interestingly, neither DAF-16 activation nor bacterial deprivation was observed to reduce the overall accumulation of Aβ, despite being protective. These results suggest that upregulation of the chaperone/protein folding machinery is protective either because this blocks the formation of some specific toxic form of Aβ (supported by the results of Cohen et al, who found changes in the aggregation state of Aβ correlating with toxicity) and/or because this compensates for a general depletion of chaperone capacity by deposition of Aβ aggregates. It is an open question whether the unc-54/signal peptide::Aβ 1-42 model is directly relevant to Alzheimer's disease, as the levels of intraneuronal Aβ accumulation in the brain are unlikely to reach the intracellular Aβ levels generated in this model. However, results from this model may be generally instructive in that they suggest that the age-dependence of AD on other neurodegenerative conditions may stem from age-dependent loss of the ability to maintain cellular protein homeostasis. It should be noted that this model may be more directly relevant to inclusion Body Myositis, given the parallels of intramuscular Aβ accumulation.

Transgenic worms have also been engineered to inducibly express Aβ upon temperature upshift, either in muscle 38 or pan-neuronally 39. This temperature inducibility was engineered by using transgene constructs with abnormally long 3' untranslated regions, resulting in transgenic transcripts that are subject to degradation by the mRNA surveillance system. Introduction of these transgenes into worms containing a temperature-sensitive mutation in a gene essential for mRNA surveillance (smg-1) resulted in strains that had a ~5-fold increase in transgenic transcripts when shifted from the permissive (16°C) to non-permissive (23°C) conditions 37. An advantage of this system is that, at least for engineered muscle expression, transgenic strains have been constructed that have wild type movement at the permissive temperature but become rapidly (~24 hr) and uniformly paralyzed upon temperature upshift. The reproducible phenotype of the myo-3/signal peptide::Aβ 1-42/long 3' UTR model has allowed straightforward quantification of the effects of treatments on Aβ toxicity, and has been used to demonstrate the protective effects of specific gingkolides 39. This inducible model has also been used to demonstrate a role for autophagy in countering Aβ toxicity 40. Microarray studies with this model identified AIP-1, a conserved protein thought to be a positive regulator of proteasome function 4142, as an Aβ-induced protein that protects against toxicity by reducing Aβ accumulation 43. Interestingly, a human homolog of AIP-1, AIRAPL, also reduces toxicity when co-expressed with Aβ.

Transgenic worms with pan-neuronal expression (signal peptide::Aβ driven by the snb-1 promoter) were found to have intraneuronal accumulation of Aβ 44 but relatively mild phenotypes, including altered chemotaxis to benzaldehyde and hypersensitivity to exogenous serotonin 39. It is unclear if the apparent differences in the severity of phenotypes resulting from muscle or neuronal expression reflect differences in the manner by which C. elegans muscle and neuronal cells respond to Aβ, or simply quantitative differences in the effective expression levels resulting from the use of different promoters. McColl et al 45 have recently demonstrated by mass spectrometry that in the unc-54/signal peptide::Aβ 1-42 constitutive model the species of Aβ that accumulates is actually Aβ 3-42, likely due to signal peptidase cleavage after Ala² of the Aβ sequence. As the inducible models utilize the same signal peptide::Aβ minigene, it is likely that all of these models produce the truncated Aβ 3-42. The 3-42 form of Aβ is readily found in senile plaques, where the N-terminal glutamate residue is often converted to pyroglutamate 46. However, McColl and colleagues did not detect pyroglutamate-modified Aβ in the transgenic worm model.

Alzheimer's disease, as well as Pick's disease and some forms of frontotemporal lobar dementia (FTLD), are associated with intraneuronal accumulation of neurofibrillary tangles (NFTs) composed of the microtubule binding protein tau. Mutations in tau have been demonstrated to underlie familial FTLD linked to chromosome 17 (FTLD-17) 474849, causally linking tau to some forms of neurodegeneration. Kraemer et al established a C. elegans tauopathy model by expressing wild type or FTLD-17-mutant tau pan-neuronally using the aex-3 promoter 50. This model system replicated key observations of the human disease: accumulation of insoluble, phosphorylated tau, evidence of age-dependent neuronal degeneration and loss, a clear organismal phenotype (uncoordinated movement), and greater toxicity in worms expressing FTLD-17-mutant tau (V³³⁷M and P³⁰¹L) than in worms expressing wild type tau. The uncoordinated phenotype of the aex-3/tau worms has been used for both classic forward genetic and RNAi-based reverse genetic screens, resulting in interesting (and perhaps unexpected) findings. Genetic screens based on chemical mutagenesis and positional cloning have identified two genes, sut-1 51 and sut-2 52, whose loss of function suppresses the tau-induced uncoordinated phenotype. sut-1 encodes a nematode-specific protein that binds Sm proteins and SmY, a small nematode-specific RNA of unknown function 53. However, Kraemer and Schellenberg were able to use yeast two-hybrid and genetic studies to demonstrate that sut-1 interacts with UNC-34, a member of the conserved Ena/VASP protein family. They speculated that this interaction may modulate actin dynamics and thereby ultimately suppress tau pathology. sut-2 is a zinc finger-containing protein homologous to yeast Nab2 and human ZC3H14. Nab2 has demonstrated roles in the nuclear export of mRNA 54, and both Nab2 and ZC3H14 have been shown to bind polyadenosine sequences 55. Guthrie et al also found by two yeast hybrid studies that C. elegans protein ZYG-12, and its human homolog HOOK2, can interact with SUT-2. Given that HOOK2 is a component of aggresomes, these authors suggested that sut-2 suppression of tau pathology could involve either aggresome effects on tau accumulation or, conversely, tau effects on proper aggresome formation.

The aex-3/tau V³³⁷M model was used as the basis for a full genome feeding RNAi screen (16,757 RNAi clones assayed), looking for modifiers of the tau-induced uncoordinated phenotype 56. Although no suppressor RNAi clones were found, 60 genes were eventually identified that specifically enhanced the tau-induced phenotype when their expression was knocked down by RNAi. These genes encoded proteins with a surprising range of functions, including phosphorylation, chaperone activity, neurotransmission and signaling, RNA processing, and various enzymatic functions. Seven of these genes (sir-2.3, vap-1, lin-44, aex-1, acr-14, pxn-1, and mut-14) were independently tested by introducing genetic loss-of-function mutations into the aex-3/tau V³³⁷M background; in all cases the enhancement of the uncoordinated phenotype was recapitulated. The range of identified enhancer genes may reflect a multitude of steps by which neurons normally counter either toxic protein accumulation (e.g., altering tau modification, aggregation, or degradation) or its downstream consequences. Alternatively, the apparent interaction of these genes with tau pathology could result from a rather indirect synergism in which a reduction of gene function that normally results in a phenotypically undetectable compromise of neuronal function (e.g., reduction of acr-14, one of the many acetylcholine receptors in C. elegans) nevertheless significantly exacerbates tau pathology in a perhaps additive fashion. In any case, the interacting genes identified by both forward and reverse genetic approaches with this model suggest potentially novel mechanisms of tau pathology that warrant investigation.

Tau pathology has also been engineered in C. elegans by expressing wild type and FTLD-17-mutant tau specifically in touch neurons, using the mec-7 promoter 57. Transgenic expression of tau in the touch neurons resulted in an age-dependent loss of touch sensitivity, again with FTLD-17-mutant tau (P³⁰¹L and R⁴⁰⁶W) showing enhanced toxicity. This model was used to test the effects of HSP70 overexpression (mild suppression of mutant tau toxicity), GSK-3 overexpression (mild enhancement of tau toxicity), or genetic blockage of apoptosis (no effect on tau toxicity). Brandt et al 58 examined the role of phosphorylation in tau toxicity by using the rgef-1 promoter to engineer pan-neuronal expression of wild type, pseudophosphorylated (10 specific kinase target serine residues changed to glutamate), or phosphorylation-resistant (10 specific kinase target serine residues changed to alanine) tau. Both wild type and glutamate-substituted tau were observed to have similar age-dependent uncoordinated phenotypes, although worms expressing the glutamate-substituted tau had higher levels of abnormal motorneurons. (The alanine-substituted tau also induced an uncoordinated phenotype, although this was hard to interpret because transgenic lines expressing this modified tau all had significantly higher levels of tau expression.) Neuronal death was not observed in either the mec-7/tau or rgef-1/tau models. However, in both models neuronal outgrowth abnormalities were observed. While this could be indicative of developmental effects of tau expression on axon pathfinding, similar abnormal outgrowth has been observed as a result of regeneration of broken axons 59, suggesting the alternative possibility that tau expression results in fragile axonal processes more susceptible to movement-induced breakage.

Mutations in superoxide dismutase (SOD1) are the most common known cause of familial Amyotrophic Lateral Sclerosis (fALS). Oeda et al 60 generated the first C. elegans model of SOD1 toxicity by expressing wild type or fALS mutant SOD1 (A⁴V, G³⁷R, or G⁹³A), using either the muscle-specific myo-3 promoter or the heat-shock inducible hsp-16.2 promoter. While these transgenic worms were not reported to have discernable phenotypes under standard conditions, worms expressing the fALS mutant SOD1 were found to be preferentially sensitive to paraquat. Wang et al 61 engineered pan-neuronal SOD1 expression using the snb-1 (synaptobrevin) promoter, expressing both wild type and G⁸⁵R fALS mutant SOD1. Expression of SOD1 G⁸⁵R (fused to YFP or unfused), but not wild type SOD1, resulted in a clear age-dependent inhibition of locomotion. Transgenic worms expressing G⁸⁵R SOD1::YFP also had visible fluorescent aggregates in neurons, allowing a full genome feeding RNAi screen in a sensitized eri-1; lin-15b genetic background. As observed in the RNAi screens of the aex-3/tau model, both expected (e.g., chaperone-related genes such as hsf-1) and unexpected (topoisomerase gene top-1, TGF β component dbl-1) were recovered as aggregation/toxicity enhancers. (One aggregation/toxicity suppressor was also identified but not described in this study.) Transgenic worms expressing SOD1::YFP fusions have also been constructed by Gidalevitz et al 62, who used the unc-54 muscle-specific promoter to express wild type and G⁸⁵R, G⁹³A, and truncated (¹²⁷X) fALS mutant SOD1. As reported by Wang et al, fALS SOD1::YFP, but not wild type, formed visible aggregates. While transgenic worms expressing fALS mutant SOD1::YFP had relatively mild phenotypes, introduction of temperature-sensitive missense mutations (the same mutant alleles that are enhanced by YFP::polyQ aggregation) strongly exacerbated SOD1::YFP toxicity in an fALS mutant-specific manner. This result nicely complements the previous YFP::polyQ study by the Morimoto group, and supports the idea that misfolded proteins (i.e., destabilized missense mutant proteins and fALS mutant SOD1) compete for limiting protein homeostasis machinery.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common known cause of familial Parkinson's disease (fPD). LRRK2 is highly conserved, and C. elegans contains a clear ortholog, lrk-1. Saha et al 63 have recently shown that knockdown of lrk-1 sensitizes worms to the mitochondrial toxic rotenone, while pan-neuronal expression of wild type human or G²⁰¹⁹S fPD LRRK2 (driven by the snb-1 promoter) protects against rotenone toxicity. However, transgenic LRRK2 expression also led to a preferential loss of dopaminergic neurons, with G²⁰¹⁹S fPD LRRK2 being measurably more toxic than wild type LRRK2. This model should be amenable to the approaches previously used in the transgenic α-synuclein models.

Pathological forms of the prion protein (PrP) are believed to be the cause of fatal spongiform encephalies, including Creutzfeldt-Jakob disease (CJD), kuru, and Bovine Spongiform Encephaly (BSE). There is a single report describing the expression of mouse prion protein (residues 23-231) in C. elegans 64. Muscle expression of a MoPrP(23-231)::CFP fusion protein (but not the CFP-only control) was found to induce visible fluorescent aggregates and a variable "Dumpy" phenotype, associated with poor locomotion and sarcomere disruption. Parallel expression of PrP expressing the P¹⁰²L mutation associated with Gerstmann-Straussler-Scheinker disease resulted in a more severe movement deficit, while co-expression of PrP containing a dominant negative mutation (MoPrP Q167R::YFP) with MoPrP::CFP reduced apparent toxicity. However, proteinase K-resistant MoPrP could not be detected in transgenic worms, suggesting that the infectious scrapie form of PrP (PrP^sc) was not being formed. Given the lack of PrP^sc formation and the fact that the transgenic constructs lack the N-terminal lipidation sequences of PrP likely important in pathology, it is unclear if this model is directly relevant to infectious prion diseases or is instead more akin to the general protein aggregation toxicity models.

New C. elegans neurodegeneration models are likely to be developed in the near future, taking advantage of both technical advantages (directed single copy transgene insertions, tetracycline-induced transgene expression), and the identification of new neurodegeneration-associated genes (e.g., TDP-43). Existing approaches have certainly not been exhausted, as genetic modifier screens have not been done to saturation, and some published large scale RNAi screens could still be expanded to full-genome screens. Clearly, some of the C. elegans findings still await validation in mammalian models. This is particularly important with respect to unexpected modifier genes identified in RNAi screens, given the possibility that these genes could be an artifactual result of worm-specific biology or the highly engineered nature of the model system. An important question that has not been rigorously addressed is to what degree underlying pathological processes overlap between the various neurodegeneration models. While some studies point to potential overlap of pathological processes (e.g., the common demonstration that loss of hsf-1 function enhances aggregation/toxicity), it remains to be determined if any suppressors in specific models also have effects in other neurodegeneration models (e.g., do sut-1 or sut-2 suppress α-synuclein or polyQ toxicity?). Technical improvements in C. elegans handling (e.g., the COPAS worm sorter and microfluidic worm manipulation) will also likely enable more efficient use of transgenic worm models in compound screening.

C. elegans neurodegeneration models have been used to test the effects of individual drugs (e.g., resveratrol in a polyQ model, 12; acetaminophen in multiple PD models, 72) or relatively small collections of compounds (e.g., subcomponents of Gingko biloba extracts in Aβ toxicity models, 39, candidate Huntington's drugs in a polyQ model, 73). Although large numbers of replicate C. elegans populations can be readily grown in small volumes (e.g., microtiter wells), a previous hindrance in moving from low- to high-throughput screenings (HTS) was the requirement for manual manipulation and phenotypic scoring of worms. Recent technical advances have now overcome many of the practical limitations in using C. elegans models for HTS, most prominently the development of the Complex Object Parametric Analyzer and Sorter (a.k.a. the COPAS "worm sorter", 74). This instrument can rapidly sort individual worms on the basis of length, optical density, and fluorescence, and can be used to automate both manipulation and scoring. Burns et al 75 demonstrated how this technology could be incorporated with a high-throughput digital imager and data management software for precisely scoring phenotype. This protocol was used by Kwok et al 76 to effectively screen four libraries comprised of 14,100 small molecules, which resulted in their discovery of a new L-type calcium channel antagonist. The COPAS instrument has recently been used for high-throughput neurotoxin screening 77, suggesting that this instrument should be equally applicable for screening for neuroprotective compounds in various neurodegeneration models.

For some relevant phenotypes, such as axonal regeneration after microsurgery 78 or maintenance of motorneuron synapses, subcellular observations are required. Techniques are currently being developed that may allow these challenging measurements to be automated, thereby further expanding HTS possibilities. In 2007 the Yanik group at MIT introduced a series of microfluidic devices, otherwise known as lab-on-chip technology, applicable for manipulating C. elegans 79. Microfluidic devices maneuver fluids and specimens on a sub-millimeter scale via the use of layered control channels and valves. Given the size of a C. elegans adult (~1 mM in length and 0.1 mM in diameter), and that they are able to live out their 2-3 week lifespan suspended in liquid media, worms are an ideal animal model for this technology. These microfluidic devices can be configured with 3-dimensional imaging that allows for subcellular resolution. Additional key components of these devices are the manner with which immobilization of worms is achieved as well as the capability of continuous recirculation. A worm is captured by way of a suction channel, which restricts its movement and positions the worm in a linear fashion providing for optimal imaging. Based upon its phenotype, the animal is then either discarded or collected in a separate chamber through a series to flow valves controls. In 2008, the Hang Lu group demonstrated that integrating this lab-on-chip technology and automated multiparametric analysis could effectively identify and sort worms according to cellular and subcellular phenotypes with 95% accuracy 80. This technology has also facilitated procedures such as those used in the in vivo ultrafast laser nanosurgery study conducted by Ben-Yakar and Bourgeois 81. By integrating lab-on-chip technology and femtosecond laser nanosurgery they were able to increase the level of precision and speed with which cellular and subcellular laser ablations can be performed, suggesting the feasibility of large-scale in vivo neural degeneration and regeneration studies.

Despite the technical advances relative to HTS in C. elegans, biological considerations may still be the most important factor in the potential usefulness of C. elegans neurodegeneration models for drug identification. The C. elegans cuticle is impermeant to many compounds that readily diffuse into tissue culture cells, and thus compound entry may require ingestion along with the standard E. coli food source. For a given compound, there is currently no way to predict what final tissue concentration will result from a given external compound dose, so dose/responses need to be tested empirically. It can be argued that the major advantage of compound screening in C. elegans is also the major disadvantage: protective compounds can be identified without any assumptions about neurotoxic mechanisms, however, when protective compounds are identified, the molecular mechanism of protection may be unknown. This ignorance can be a major hurdle in further drug development, as it limits both compound optimization and predictions about possible side effects. Nevertheless, investigations of protective compounds can be a powerful approach to ultimately understanding the molecular mechanisms of neurodegeneration.