BACKGROUND
a number of very serious human disorders are characterized by the appearance of protein aggregates, including neurodegenerative diseases such as huntington’s disease , metabolic disorders, and certain developmental disorders  <cit> .
hereditary forms of these diseases are caused by de novo gain-of-function mutations that result in expression of destabilized, aggregation-prone proteins  <cit> , accompanied by the disruption of protein-folding homeostasis  and cellular dysfunction  <cit> .
although these mutations themselves are causative, many disease characteristics such as age of onset, penetrance, and severity of specific symptoms can vary widely between individuals.
for example in hd, the age of neurological onset is strongly associated with the length of polyglutamine  expansion in huntingtin protein.
yet age of onset can vary by several decades in people carrying the same length polyglutamine expansion, and a large proportion of this residual variation is genetic in nature and may be due to polymorphisms in other genes  <cit> .
this variability is also seen in animal models of disease; for example, different genetic backgrounds of common laboratory mouse strains differentially modify somatic cag repeat expansion and the onset of nuclear accumulation of mutant huntingtin in a knock-in hd mouse model  <cit> .
it is generally well appreciated that genetic variation influences the phenotypic expression of mutations and transgenes, and the effects of environmental perturbations  <cit> .
however, the nature of modifying alleles segregating in populations remains elusive for protein conformational diseases  <cit> .
much of the current knowledge about modifiers of conformational diseases has been gathered from linkage/association studies, candidate approaches, and unbiased genetic screens.
the latter two approaches often use induced mutations, including knockouts/knockdowns and overexpression, in fixed genetic backgrounds.
although genetic variants identified in this way are enormously informative about pathways that affect disease processes and that can, when perturbed, modify disease phenotypes  <cit> , they are likely to be different from the naturally occurring variations.
first, these approaches are designed to identify individual modifier genes with strong phenotypic effects.
by contrast, a naturally occurring genetic variation, in addition to the singleton genes with strong effect, is likely to be represented by complex networks of interactions of multiple common variants with small effects, and/or of rare or even private alleles with larger contributions  <cit> , as well as cryptic genetic variation  <cit> , thus making association of phenotypic differences with their underlying genotypes very challenging.
protein aggregation diseases present an additional unique challenge, owing to the sheer number of different processes and pathways that control proteostasis  <cit> .
modulation of proteostasis has been shown to buffer the expression of certain mutations and coding polymorphisms into phenotypes.
for example, expression of aggregation-prone polyq and sod <dig> proteins  <cit> , or down-modulation in plants and animals of activity of molecular chaperones, such as heat shock protein  <dig> , unmasks buffered phenotypes caused by mildly destabilizing mutations segregating in populations  <cit> , a phenomenon similar to the heat-induced phenocopies  <cit> .
at the same time, the presence of such mild destabilizing variants in the genetic background modulates the aggregation and toxicity of aggregation-prone proteins, including polyq  <cit> .
an additional consideration in protein conformation diseases is the possibility of certain induced mutations, especially knockouts/knockdowns, to imbalance the proteostasis directly, for example by targeting a member of a multimeric complex and inducing a folding stress in the cell.
because imbalance in proteostasis can indirectly affect the aggregation phenotype of the disease-causing mutation, such genetic intervention may appear as a true modifier.
secondly, because natural variants have been acted upon by selection, they are largely compatible with normal organismal development and function  <cit> , in contrast to many of the modifiers of the induced-mutation type, which are not phenotypically innocuous.
moreover, genetic manipulations that protect cells and organisms from the toxic effects of protein aggregation can themselves be deleterious to the organism; many of the lifespan-extending mutations protect against proteotoxicity, but are incompatible with normal development, or are maladaptive under competitive conditions  <cit> .
similarly, overexpression of molecular chaperones or heat shock factor, which consistently protects against proteotoxicity, can be accompanied by slow growth and development, decreased fecundity, and aberrant signaling, and may even support transformed phenotypes  <cit> .
thus, identifying proteostasis modifiers from among natural variants shaped by selection may pinpoint the potential genes and networks that are naturally plastic  <cit>  and thus can be pharmacologically modulated without negative effects on the organism.
in contrast to induced-mutation approaches, association studies do look at natural variants.
however, the complexity of the genetic variation involved in control of proteostasis, as discussed above, makes these studies very difficult.
indeed, only a small number of genes have been firmly identified as modifiers of aggregation diseases by either candidate or association approaches  <cit> .
furthermore, these modifiers are often not replicated in different populations, and replicated modifiers account for only a small fraction of the heritable variation  <cit> .
genetic tractability of model animals, together with the ability to control the contribution of the environment, offers a unique opportunity to examine how natural variation in the genetic background of individuals affects their ability to resist protein aggregation; what is the nature of the genetic variants that modify proteostasis; whether these are distinct from the spectrum of induced mutations; and whether natural variation can predict/modify the susceptibility to protein conformation diseases.
recombinant inbred lines  have been used successfully, from yeast to mice, to answer similar questions about non-transgenic physiological traits, including complex traits  <cit> .
here we established the polyglutamine transgenic caenorhabditis elegans as one such model, taking advantage of the availability of genetically diverse wild isolates, and the ability to detect and measure protein aggregation in a live animal throughout its lifespan.
in addition to the short generation time and lifespan of c. elegans, and the wealth of knowledge and reagents available for the pathways known to modulate aging and proteostasis, the facultative sexual reproduction mode of this organism allows for multifactorial perturbation of genetic variants through outcrossing, combined with the capture of true-breeding genes by self-reproduction, thus providing a rich resource for future mapping of the natural modifier alleles of protein aggregation and toxicity.
RESULTS
genetic background modulates age of onset and cell-specific susceptibility to polyq aggregation
as described previously, expression of the near-threshold yellow fluorescent protein -tagged polyglutamine expansion polyq40-yfp  in muscle cells of c. elegans shows a striking age-dependent onset of aggregation, regulated by heat shock factor  <dig>  and the insulin signaling pathway  <cit> .
however, unlike the variability of polyq-associated disease phenotypes in the human population, the behavioral, biochemical, and aggregation phenotypes in the c. elegans laboratory bristol  strain are homogeneous.
to investigate whether natural genetic variation in c. elegans modifies polyq aggregation and associated phenotypes, we introgressed the polyq <dig> transgene from the bristol background into three wild strains: a california-derived isolate dr <dig>  the madeira isolate ju <dig>  and a genetically distant hawaii isolate cb <dig>  <cit> .
the introgression approach ensures that the transgene is present in the same copy number and genomic location in each of the resulting strains , while long-term backcrossing  ensures a high degree of genetic homogeneity within each strain.
polyq aggregation and toxicity was strongly affected by introgression into wild strains.
we observed an early onset of aggregation in dr <dig> background, with on average eight-fold higher number of aggregates in the second larval stage  of q40dr <dig> animals than in q40bristol animals , and a twofold to threefold increase at the late larval stage  <dig>  .
these early onset and increased aggregation were not due to any changes in polyq <dig> expression .
by contrast, the hawaii and madeira backgrounds had only mild effects on polyq aggregation .
the relative susceptibility of subsets of muscle cells to polyq aggregation was markedly different between the bristol and dr <dig> backgrounds.
head muscle cells were more resistant to aggregation than body wall muscles in q40bristol animals, whereas the inverse was true in the q40dr <dig> animals, with the head muscles exhibiting the earliest onset of polyq aggregation.
the head muscle cells of l2/l <dig> q40dr <dig> animals contained  <dig> ± 4% of all aggregates, compared with  <dig> ± 12% for q40bristol animals .
the q40dr <dig> strain retained this cell type-preferential aggregation phenotype throughout development .
the allele or alleles that confer increased susceptibility of this subset of muscle cells to aggregation appear to be unique to these two strains, as neither the q40hawaii nor q40madeira strains exhibited this phenotype.
genetic background differentially modulate polyq aggregation and toxicity
consistent with the earlier onset of aggregation, the q40dr <dig> strain exhibited an earlier onset of toxicity, with a twofold to threefold reduction in mean motility relative to q40bristol animals at days  <dig> to  <dig> of adulthood .
this was in contrast to the hawaiian background where this relationship was inverted, and reduced and delayed toxicity was apparent despite slightly increased aggregation.
the q40hawaii strain had on average twofold improved motility relative to q40bristol on day  <dig> of adulthood, and this protection against polyq toxicity was further enhanced to fivefold on day  <dig> of adulthood .
these results reveal that the hawaiian background is highly protective against the toxic effects of polyq <dig>  without having any obvious effect on the visual appearance of aggregates .
this demonstrates that genetic polymorphisms that are present in the wild strains, and, therefore, acted upon by selection, are able to separately regulate the aggregation and resulting toxicity of polyglutamine protein.
thus, the genetic variants present in outbred populations provide natural protection against, or susceptibility to, protein aggregation and toxicity.
multiple alleles control polyq aggregation in c. elegans
because introgression of the polyq <dig> transgene into different genetic backgrounds revealed the presence of natural variation in both aggregation and associated toxicity, we tested whether these phenotypes are controlled by the same or distinct genetic networks.
to address this, we established a panel of rils, each carrying the same polyq <dig> transgene in the same genomic location, using the two backgrounds, that were most divergent for these phenotypes: bristol and dr <dig> .
although each ril carried a randomly generated assortment of parental alleles, the long-term inbreeding of each ril ensured within-line homogeneity.
this approach allowed for separation in the progeny of alleles that act epistatically or in concert in parental backgrounds, and for new configurations of genetic networks.
the  <dig> rils exhibited a wide range of phenotypes from delayed onset and suppressed extent of aggregation in rils  <dig> and  <dig>  to strongly enhanced aggregation in rils  <dig> and  <dig> .
rils  <dig> and  <dig> showed transgressive behavior, with the number of aggregates in the majority of animals  being more than 2sd away from the mean of either of the parental lines, indicating contributions from multiple modifier alleles.
the onset and extent of protein aggregation can be dependent on the expression levels, and we have previously reported delay in onset of aggregation in animals heterozygous for polyq <dig>  <cit> .
to address this, we measured polyq-yfp fluorescence levels in extracts of l <dig> larva, prior to onset of aggregation, to determine whether it correlated with aggregation, and found no correlation between expression levels and aggregation of polyq protein in rils  .
we further selected several rils with similar expression levels, which were either most suppressed or most enhanced in terms of aggregation, and two rils whose aggregation was similar to each of the parental lines , resulting in five rils and two parental lines binned into four aggregation groups.
similar polyq expression levels in these strains were confirmed by measuring total polyq-yfp protein levels in l <dig> animals by western blotting analysis, showing that neither enhancement  and 15), nor suppression  of aggregation in these selected lines was due to a change in expression levels of polyq protein .
furthermore, increased  or decreased  aggregation as detected by visual appearance of fluorescent foci in day  <dig> adult animals correlated with increased or decreased levels of polyq protein in the sds-resistant high molecular weight fraction .
thus, polyq aggregation appears to be a transgressive trait, with some ril progeny occupying phenotypic extremes relative to the parental strains , indicating that multiple alleles with opposing or epistatic effects control protein aggregation.
natural variation independently controls resistance to aggregation and the ability to restrict its associated cellular dysfunction
in both parental lines, we found expression of the aggregation-prone polyq <dig> to be associated with muscle toxicity .
thus, we investigated whether natural variants segregating in the ril progeny population were coordinately regulating polyq aggregation and toxicity.
we reasoned that rils with enhanced aggregation would show earlier or more severe organismal correlates of polyq toxicity, and vice versa.
to monitor toxicity phenotypes, we scored motility and egg-laying, traits that directly report on the dysfunction of two different muscle subtypes, namely, body wall and reproductive muscles.
we also scored shortening of the lifespan, which is a more indirect measure of organismal dysfunction caused by expression of polyq <dig> in the muscle cells.
the ability to simultaneously score these different toxicity measures in the same animals allows comparison of cell type-selective and global consequences of polyq toxicity.
motility was measured by adapting a population-level muscle dysfunction index , that reports on the gradual deterioration of movement over the lifespan  <cit>  .
we found that the mdi overall tracked with enhanced or suppressed aggregation for the five rils scored .
for example, ril  <dig> exhibited the lowest level of aggregation  and was the most protected of the polyq40-containing lines , whereas ril  <dig> had early onset of both aggregation and muscle dysfunction.
however, there was not an absolute correspondence in relative ranking: ril  <dig>  representing the most enhanced aggregation bin , had a similar time to onset of muscle dysfunction as the much less aggregating ril  <dig> , indicating that variants present in ril  <dig> protected muscle cells from the toxic effects of polyq aggregation, but not from the protein aggregation itself.
two of the rils examined, rils  <dig> and  <dig>  exhibited a phenotype intermediate between the non-transgenic bristol and dr <dig> strains and the parental q40bristol and q40dr <dig> strains .
thus, polyglutamine aggregation and the associated muscle dysfunction are both controlled by complex but non-overlapping allelic variation present in c. elegans strains.
shortening of lifespan also showed incomplete correspondence with enhanced or suppressed aggregation.
the reduced aggregation rils  <dig> and  <dig> had the longest lifespan  of the seven strains scored .
however, the highest aggregating ril  <dig> had only intermediate shortening of the lifespan.
furthermore, although q40dr <dig> and ril  <dig> belonged to the same aggregation bin , they had very disparate shortening of the lifespan  , with one having a shorter and one a longer lifespan than that predicted by the aggregation rank .
ril, recombinant inbred line.
because progressive muscle dysfunction in c. elegans has been linked to aging  <cit> , we compared the lifespan and mdi ranking of the same animals, and found that polyq toxicity measured by muscle dysfunction could be uncoupled from the shortening of the lifespan.
for example, relative to its aggregation rank, q40dr <dig> had a high degree of muscle dysfunction , but only mild shortening of lifespan .
overall, these data showed that in some genetic backgrounds there is correspondence between aggregation, cellular dysfunction, and lifespan, whereas other backgrounds appear to contain non-overlapping variation that uncouples aggregation from one or both measures of toxicity.
finally, we scored an internal hatching phenotype  that reports on the function of reproductive muscle cells .
unexpectedly, we found that the expression of polyq <dig> caused significant toxicity in the reproductive tract, irrespective of the overall aggregation status .
the bag phenotype was undetectable or was only at background levels in the first  <dig> days of the reproduction period, suggesting that it was degenerative in nature and not due to a defect in muscle development.
thus, it appears that not only it is possible to uncouple aggregation and toxicity through the influence of natural genetic variation in c. elegans, but there are also intrinsic differences in the relative sensitivity of muscle cell types to the expression of polyglutamine expansion.
discussion
our studies establish c. elegans as a potent model to probe the natural variation that shapes the susceptibility of individual animals to the toxic effects of protein aggregation.
we found that three of the wild backgrounds used in this study had modifying effects on multiple aspects of polyglutamine aggregation and toxicity, including the onset and extent of aggregation, the differential susceptibility of specific subsets of muscle cells to aggregation, and the direct cellular and indirect organismal correlates of dysfunction.
another important finding in this model is that both polyq aggregation and toxicity behaved as complex traits, suggesting that multiple additive or interacting alleles from wild c. elegans backgrounds are contributing to this phenotypic variation.
thus, this model will provide an invaluable tool to investigate the nature of alleles that contribute to the robustness of proteostasis in genetically diverse but phenotypically normal individuals.
much progress has been made in understanding the mechanisms by which protein misfolding and aggregation causes cellular toxicity, and the pathways and networks that can be protective  <cit> .
however, because the common candidate approaches , or overexpression) are designed to detect genes that individually have large phenotypic effects, they often identify modifier genes that are not easy targets for intervention.
many of the genes identified as important for maintenance of proteostasis, including molecular chaperones, regulators of stress responses, insulin signaling, and autophagy, are themselves either essential, or have additional strong effects on development and/or normal physiology  <cit> .
nevertheless, modifier backgrounds do exist in the outbred human population, as evidenced by the large variability in age of onset or even penetrance of protein misfolding diseases.
this suggests that natural genetic variants that are shaped by selection can both provide protection against protein aggregation and toxicity, and, at the same time, be compatible with normal development and physiology.
thus, natural variation may tag the genetic and molecular pathways whose inherent plasticity allows for development of intervention strategies without the negative or toxic effects.
previous mutational studies have shown that protein aggregation can be uncoupled from toxicity  <cit> .
for example, decreased insulin signaling causes increased aggregation but decreased toxicity of aβ in c. elegans <cit> , whereas overexpression of hsp <dig> rescues polyq toxicity in drosophila by affecting the properties but not the extent of aggregation  <cit> .
these studies suggest that changing the aggregation pathway may translate into changes in toxicity; however, the question remains whether the two are always related causally, or whether they are controlled by molecular pathways that can be modified independently of each other.
studies of plant-pathogen and animal-pathogen interactions show that resistance  and tolerance  can have distinct genetic bases, and that genetic variation exists for both  <cit> .
we suggest that a similar logic could be applied to proteotoxic mutations.
we found that identical genetic lesions  produced vastly different aggregation phenotypes, supporting variation in resistance to aggregation, including at the level of cell type-specific resistance in head versus body wall muscle cells.
at the same time, strains that exhibited similar levels of polyq aggregation differed widely in toxicity correlates, such as muscle dysfunction and shortening of lifespan.
although further research will be needed to characterize these phenomena, these findings indicate a remarkable variation at the organismal level in the ability to tolerate protein aggregation.
this is clearly evident in natural isolates of c. elegans, as the hawaiian strain showed increased tolerance of polyq aggregation as measured by the loss of motility, whereas dr <dig> showed both decreased resistance and decreased tolerance in terms of muscle dysfunction, but increased tolerance when measured by the shortening of lifespan.
furthermore, it appears that natural variants contributing to resistance and tolerance can be found even within the limited ril panel used in this study; relative to the parental strains, ril  <dig> demonstrated remarkable resistance and tolerance, the latter in both of the phenotypic measures for which variation was present.
our data thus reveal that distinct genetic networks control protein aggregation and the various cellular and organismal consequences of such aggregation.
one potential scenario for this distinction is that pathways that affect resistance to aggregation act directly on the mutant aggregation-prone protein, for example, by changing its folding trajectory or degradation rate, whereas those that affect tolerance of protein aggregates also affect specific cellular pathways that are disrupted in disease, or affect parallel or compensatory pathways.
this suggests that, depending on the individual’s genetic background, disease intervention strategies may need to distinguish between the strategy of prevention of misfolding and aggregation from an alternative strategy of strengthening specific cellular pathways that mediate disease phenotypes.
CONCLUSIONS
these results establish c. elegans as a potent model to study how natural variation shapes the susceptibility to proteotoxicity in genetically diverse but phenotypically normal individuals, and support the view that intrinsic aggregation propensity of the mutant disease-related proteins is not, by itself, sufficient to explain disease onset and phenotypes.
we found that both protein aggregation and toxicity behave as complex traits, suggesting that multiple additive or interacting alleles contribute to the variation in these phenotypes.
the ability of standing genetic variation in wild isolates of c. elegans to independently modify the age of onset, the cell type-specific susceptibility to protein aggregation, and several independent toxicity correlates of the polyglutamine expansion, suggests not only multiple paths to the expression of toxicity, but also multiple potential routes of intervention.
because natural variation represents genetic and molecular pathways that are inherently plastic, identifying these pathways will allows for development of intervention strategies that avoid the negative or toxic effects.

 a number of very serious human disorders are characterized by the appearance of protein aggregates, including neurodegenerative diseases such as huntington’s disease, metabolic disorders, and certain developmental disorders. hereditary forms of these diseases are caused by de novo gain-of-function mutations that result in expression of destabilized, aggregation-prone proteins, accompanied by the disruption of protein-folding homeostasis and cellular dysfunction. although these mutations themselves are causative, many disease characteristics such as age of onset, penetrance, and severity of specific symptoms can vary widely between individuals. for example in hd, the age of neurological onset is strongly associated with the length of polyglutamine expansion in huntingtin protein. yet age of onset can vary by several decades in people carrying the same length polyglutamine expansion, and a large proportion of this residual variation is genetic in nature and may be due to polymorphisms in other genes. this variability is also seen in animal models of disease; for example, different genetic backgrounds of common laboratory mouse strains differentially modify somatic cag repeat expansion and the onset of nuclear accumulation of mutant huntingtin in a knock-in hd mouse model. it is generally well appreciated that genetic variation influences the phenotypic expression of mutations and transgenes, and the effects of environmental perturbations. however, the nature of modifying alleles segregating in populations remains elusive for protein conformational diseases. much of the current knowledge about modifiers of conformational diseases has been gathered from linkage/association studies, candidate approaches, and unbiased genetic screens. the latter two approaches often use induced mutations, including knockouts/knockdowns and overexpression, in fixed genetic backgrounds. although genetic variants identified in this way are enormously informative about pathways that affect disease processes and that can, when perturbed, modify disease phenotypes, they are likely to be different from the naturally occurring variations. first, these approaches are designed to identify individual modifier genes with strong phenotypic effects. by contrast, a naturally occurring genetic variation, in addition to the singleton genes with strong effect, is likely to be represented by complex networks of interactions of multiple common variants with small effects, and/or of rare or even private alleles with larger contributions, as well as cryptic genetic variation, thus making association of phenotypic differences with their underlying genotypes very challenging. protein aggregation diseases present an additional unique challenge, owing to the sheer number of different processes and pathways that control proteostasis. modulation of proteostasis has been shown to buffer the expression of certain mutations and coding polymorphisms into phenotypes. for example, expression of aggregation-prone polyq and sod proteins, or down-modulation in plants and animals of activity of molecular chaperones, such as heat shock protein, unmasks buffered phenotypes caused by mildly destabilizing mutations segregating in populations, a phenomenon similar to the heat-induced phenocopies. at the same time, the presence of such mild destabilizing variants in the genetic background modulates the aggregation and toxicity of aggregation-prone proteins, including polyq. an additional consideration in protein conformation diseases is the possibility of certain induced mutations, especially knockouts/knockdowns, to imbalance the proteostasis directly, for example by targeting a member of a multimeric complex and inducing a folding stress in the cell. because imbalance in proteostasis can indirectly affect the aggregation phenotype of the disease-causing mutation, such genetic intervention may appear as a true modifier. secondly, because natural variants have been acted upon by selection, they are largely compatible with normal organismal development and function, in contrast to many of the modifiers of the induced-mutation type, which are not phenotypically innocuous. moreover, genetic manipulations that protect cells and organisms from the toxic effects of protein aggregation can themselves be deleterious to the organism; many of the lifespan-extending mutations protect against proteotoxicity, but are incompatible with normal development, or are maladaptive under competitive conditions. similarly, overexpression of molecular chaperones or heat shock factor, which consistently protects against proteotoxicity, can be accompanied by slow growth and development, decreased fecundity, and aberrant signaling, and may even support transformed phenotypes. thus, identifying proteostasis modifiers from among natural variants shaped by selection may pinpoint the potential genes and networks that are naturally plastic and thus can be pharmacologically modulated without negative effects on the organism. in contrast to induced-mutation approaches, association studies do look at natural variants. however, the complexity of the genetic variation involved in control of proteostasis, as discussed above, makes these studies very difficult. indeed, only a small number of genes have been firmly identified as modifiers of aggregation diseases by either candidate or association approaches. furthermore, these modifiers are often not replicated in different populations, and replicated modifiers account for only a small fraction of the heritable variation. genetic tractability of model animals, together with the ability to control the contribution of the environment, offers a unique opportunity to examine how natural variation in the genetic background of individuals affects their ability to resist protein aggregation; what is the nature of the genetic variants that modify proteostasis; whether these are distinct from the spectrum of induced mutations; and whether natural variation can predict/modify the susceptibility to protein conformation diseases. recombinant inbred lines have been used successfully, from yeast to mice, to answer similar questions about non-transgenic physiological traits, including complex traits. here we established the polyglutamine transgenic caenorhabditis elegans as one such model, taking advantage of the availability of genetically diverse wild isolates, and the ability to detect and measure protein aggregation in a live animal throughout its lifespan. in addition to the short generation time and lifespan of c. elegans, and the wealth of knowledge and reagents available for the pathways known to modulate aging and proteostasis, the facultative sexual reproduction mode of this organism allows for multifactorial perturbation of genetic variants through outcrossing, combined with the capture of true-breeding genes by self-reproduction, thus providing a rich resource for future mapping of the natural modifier alleles of protein aggregation and toxicity.  genetic background modulates age of onset and cell-specific susceptibility to polyq aggregation as described previously, expression of the near-threshold yellow fluorescent protein -tagged polyglutamine expansion polyq40-yfp in muscle cells of c. elegans shows a striking age-dependent onset of aggregation, regulated by heat shock factor and the insulin signaling pathway. however, unlike the variability of polyq-associated disease phenotypes in the human population, the behavioral, biochemical, and aggregation phenotypes in the c. elegans laboratory bristol strain are homogeneous. to investigate whether natural genetic variation in c. elegans modifies polyq aggregation and associated phenotypes, we introgressed the polyq transgene from the bristol background into three wild strains: a california-derived isolate dr the madeira isolate ju and a genetically distant hawaii isolate cb. the introgression approach ensures that the transgene is present in the same copy number and genomic location in each of the resulting strains, while long-term backcrossing ensures a high degree of genetic homogeneity within each strain. polyq aggregation and toxicity was strongly affected by introgression into wild strains. we observed an early onset of aggregation in dr background, with on average eight-fold higher number of aggregates in the second larval stage of q40dr animals than in q40bristol animals, and a twofold to threefold increase at the late larval stage. these early onset and increased aggregation were not due to any changes in polyq expression. by contrast, the hawaii and madeira backgrounds had only mild effects on polyq aggregation. the relative susceptibility of subsets of muscle cells to polyq aggregation was markedly different between the bristol and dr backgrounds. head muscle cells were more resistant to aggregation than body wall muscles in q40bristol animals, whereas the inverse was true in the q40dr animals, with the head muscles exhibiting the earliest onset of polyq aggregation. the head muscle cells of l2/l q40dr animals contained ± 4% of all aggregates, compared with ± 12% for q40bristol animals. the q40dr strain retained this cell type-preferential aggregation phenotype throughout development. the allele or alleles that confer increased susceptibility of this subset of muscle cells to aggregation appear to be unique to these two strains, as neither the q40hawaii nor q40madeira strains exhibited this phenotype. genetic background differentially modulate polyq aggregation and toxicity consistent with the earlier onset of aggregation, the q40dr strain exhibited an earlier onset of toxicity, with a twofold to threefold reduction in mean motility relative to q40bristol animals at days to of adulthood. this was in contrast to the hawaiian background where this relationship was inverted, and reduced and delayed toxicity was apparent despite slightly increased aggregation. the q40hawaii strain had on average twofold improved motility relative to q40bristol on day of adulthood, and this protection against polyq toxicity was further enhanced to fivefold on day of adulthood. these results reveal that the hawaiian background is highly protective against the toxic effects of polyq without having any obvious effect on the visual appearance of aggregates. this demonstrates that genetic polymorphisms that are present in the wild strains, and, therefore, acted upon by selection, are able to separately regulate the aggregation and resulting toxicity of polyglutamine protein. thus, the genetic variants present in outbred populations provide natural protection against, or susceptibility to, protein aggregation and toxicity. multiple alleles control polyq aggregation in c. elegans because introgression of the polyq transgene into different genetic backgrounds revealed the presence of natural variation in both aggregation and associated toxicity, we tested whether these phenotypes are controlled by the same or distinct genetic networks. to address this, we established a panel of rils, each carrying the same polyq transgene in the same genomic location, using the two backgrounds, that were most divergent for these phenotypes: bristol and dr. although each ril carried a randomly generated assortment of parental alleles, the long-term inbreeding of each ril ensured within-line homogeneity. this approach allowed for separation in the progeny of alleles that act epistatically or in concert in parental backgrounds, and for new configurations of genetic networks. the rils exhibited a wide range of phenotypes from delayed onset and suppressed extent of aggregation in rils and to strongly enhanced aggregation in rils and. rils and showed transgressive behavior, with the number of aggregates in the majority of animals being more than 2sd away from the mean of either of the parental lines, indicating contributions from multiple modifier alleles. the onset and extent of protein aggregation can be dependent on the expression levels, and we have previously reported delay in onset of aggregation in animals heterozygous for polyq. to address this, we measured polyq-yfp fluorescence levels in extracts of l larva, prior to onset of aggregation, to determine whether it correlated with aggregation, and found no correlation between expression levels and aggregation of polyq protein in rils. we further selected several rils with similar expression levels, which were either most suppressed or most enhanced in terms of aggregation, and two rils whose aggregation was similar to each of the parental lines, resulting in five rils and two parental lines binned into four aggregation groups. similar polyq expression levels in these strains were confirmed by measuring total polyq-yfp protein levels in l animals by western blotting analysis, showing that neither enhancement and 15), nor suppression of aggregation in these selected lines was due to a change in expression levels of polyq protein. furthermore, increased or decreased aggregation as detected by visual appearance of fluorescent foci in day adult animals correlated with increased or decreased levels of polyq protein in the sds-resistant high molecular weight fraction. thus, polyq aggregation appears to be a transgressive trait, with some ril progeny occupying phenotypic extremes relative to the parental strains, indicating that multiple alleles with opposing or epistatic effects control protein aggregation. natural variation independently controls resistance to aggregation and the ability to restrict its associated cellular dysfunction in both parental lines, we found expression of the aggregation-prone polyq to be associated with muscle toxicity. thus, we investigated whether natural variants segregating in the ril progeny population were coordinately regulating polyq aggregation and toxicity. we reasoned that rils with enhanced aggregation would show earlier or more severe organismal correlates of polyq toxicity, and vice versa. to monitor toxicity phenotypes, we scored motility and egg-laying, traits that directly report on the dysfunction of two different muscle subtypes, namely, body wall and reproductive muscles. we also scored shortening of the lifespan, which is a more indirect measure of organismal dysfunction caused by expression of polyq in the muscle cells. the ability to simultaneously score these different toxicity measures in the same animals allows comparison of cell type-selective and global consequences of polyq toxicity. motility was measured by adapting a population-level muscle dysfunction index, that reports on the gradual deterioration of movement over the lifespan. we found that the mdi overall tracked with enhanced or suppressed aggregation for the five rils scored. for example, ril exhibited the lowest level of aggregation and was the most protected of the polyq40-containing lines, whereas ril had early onset of both aggregation and muscle dysfunction. however, there was not an absolute correspondence in relative ranking: ril representing the most enhanced aggregation bin, had a similar time to onset of muscle dysfunction as the much less aggregating ril, indicating that variants present in ril protected muscle cells from the toxic effects of polyq aggregation, but not from the protein aggregation itself. two of the rils examined, rils and exhibited a phenotype intermediate between the non-transgenic bristol and dr strains and the parental q40bristol and q40dr strains. thus, polyglutamine aggregation and the associated muscle dysfunction are both controlled by complex but non-overlapping allelic variation present in c. elegans strains. shortening of lifespan also showed incomplete correspondence with enhanced or suppressed aggregation. the reduced aggregation rils and had the longest lifespan of the seven strains scored. however, the highest aggregating ril had only intermediate shortening of the lifespan. furthermore, although q40dr and ril belonged to the same aggregation bin, they had very disparate shortening of the lifespan, with one having a shorter and one a longer lifespan than that predicted by the aggregation rank. ril, recombinant inbred line. because progressive muscle dysfunction in c. elegans has been linked to aging, we compared the lifespan and mdi ranking of the same animals, and found that polyq toxicity measured by muscle dysfunction could be uncoupled from the shortening of the lifespan. for example, relative to its aggregation rank, q40dr had a high degree of muscle dysfunction, but only mild shortening of lifespan. overall, these data showed that in some genetic backgrounds there is correspondence between aggregation, cellular dysfunction, and lifespan, whereas other backgrounds appear to contain non-overlapping variation that uncouples aggregation from one or both measures of toxicity. finally, we scored an internal hatching phenotype that reports on the function of reproductive muscle cells. unexpectedly, we found that the expression of polyq caused significant toxicity in the reproductive tract, irrespective of the overall aggregation status. the bag phenotype was undetectable or was only at background levels in the first days of the reproduction period, suggesting that it was degenerative in nature and not due to a defect in muscle development. thus, it appears that not only it is possible to uncouple aggregation and toxicity through the influence of natural genetic variation in c. elegans, but there are also intrinsic differences in the relative sensitivity of muscle cell types to the expression of polyglutamine expansion. discussion our studies establish c. elegans as a potent model to probe the natural variation that shapes the susceptibility of individual animals to the toxic effects of protein aggregation. we found that three of the wild backgrounds used in this study had modifying effects on multiple aspects of polyglutamine aggregation and toxicity, including the onset and extent of aggregation, the differential susceptibility of specific subsets of muscle cells to aggregation, and the direct cellular and indirect organismal correlates of dysfunction. another important finding in this model is that both polyq aggregation and toxicity behaved as complex traits, suggesting that multiple additive or interacting alleles from wild c. elegans backgrounds are contributing to this phenotypic variation. thus, this model will provide an invaluable tool to investigate the nature of alleles that contribute to the robustness of proteostasis in genetically diverse but phenotypically normal individuals. much progress has been made in understanding the mechanisms by which protein misfolding and aggregation causes cellular toxicity, and the pathways and networks that can be protective. however, because the common candidate approaches, or overexpression) are designed to detect genes that individually have large phenotypic effects, they often identify modifier genes that are not easy targets for intervention. many of the genes identified as important for maintenance of proteostasis, including molecular chaperones, regulators of stress responses, insulin signaling, and autophagy, are themselves either essential, or have additional strong effects on development and/or normal physiology. nevertheless, modifier backgrounds do exist in the outbred human population, as evidenced by the large variability in age of onset or even penetrance of protein misfolding diseases. this suggests that natural genetic variants that are shaped by selection can both provide protection against protein aggregation and toxicity, and, at the same time, be compatible with normal development and physiology. thus, natural variation may tag the genetic and molecular pathways whose inherent plasticity allows for development of intervention strategies without the negative or toxic effects. previous mutational studies have shown that protein aggregation can be uncoupled from toxicity. for example, decreased insulin signaling causes increased aggregation but decreased toxicity of aβ in c. elegans, whereas overexpression of hsp rescues polyq toxicity in drosophila by affecting the properties but not the extent of aggregation. these studies suggest that changing the aggregation pathway may translate into changes in toxicity; however, the question remains whether the two are always related causally, or whether they are controlled by molecular pathways that can be modified independently of each other. studies of plant-pathogen and animal-pathogen interactions show that resistance and tolerance can have distinct genetic bases, and that genetic variation exists for both. we suggest that a similar logic could be applied to proteotoxic mutations. we found that identical genetic lesions produced vastly different aggregation phenotypes, supporting variation in resistance to aggregation, including at the level of cell type-specific resistance in head versus body wall muscle cells. at the same time, strains that exhibited similar levels of polyq aggregation differed widely in toxicity correlates, such as muscle dysfunction and shortening of lifespan. although further research will be needed to characterize these phenomena, these findings indicate a remarkable variation at the organismal level in the ability to tolerate protein aggregation. this is clearly evident in natural isolates of c. elegans, as the hawaiian strain showed increased tolerance of polyq aggregation as measured by the loss of motility, whereas dr showed both decreased resistance and decreased tolerance in terms of muscle dysfunction, but increased tolerance when measured by the shortening of lifespan. furthermore, it appears that natural variants contributing to resistance and tolerance can be found even within the limited ril panel used in this study; relative to the parental strains, ril demonstrated remarkable resistance and tolerance, the latter in both of the phenotypic measures for which variation was present. our data thus reveal that distinct genetic networks control protein aggregation and the various cellular and organismal consequences of such aggregation. one potential scenario for this distinction is that pathways that affect resistance to aggregation act directly on the mutant aggregation-prone protein, for example, by changing its folding trajectory or degradation rate, whereas those that affect tolerance of protein aggregates also affect specific cellular pathways that are disrupted in disease, or affect parallel or compensatory pathways. this suggests that, depending on the individual’s genetic background, disease intervention strategies may need to distinguish between the strategy of prevention of misfolding and aggregation from an alternative strategy of strengthening specific cellular pathways that mediate disease phenotypes.  these results establish c. elegans as a potent model to study how natural variation shapes the susceptibility to proteotoxicity in genetically diverse but phenotypically normal individuals, and support the view that intrinsic aggregation propensity of the mutant disease-related proteins is not, by itself, sufficient to explain disease onset and phenotypes. we found that both protein aggregation and toxicity behave as complex traits, suggesting that multiple additive or interacting alleles contribute to the variation in these phenotypes. the ability of standing genetic variation in wild isolates of c. elegans to independently modify the age of onset, the cell type-specific susceptibility to protein aggregation, and several independent toxicity correlates of the polyglutamine expansion, suggests not only multiple paths to the expression of toxicity, but also multiple potential routes of intervention. because natural variation represents genetic and molecular pathways that are inherently plastic, identifying these pathways will allows for development of intervention strategies that avoid the negative or toxic effects. 
resistance to protein aggregation and the ability to restrict its associated cellular dysfunction are independently controlled by the natural variation in genetic background, revealing important new considerations in the search for targets for therapeutic intervention in conformational diseases. we found that aggregation is a transgressive trait, and does not always correlate with measures of toxicity, such as early onset of muscle dysfunction, egg-laying deficits, or reduced lifespan. monogenic gain-of-function protein aggregation diseases, including huntington’s disease, exhibit substantial variability in age of onset, penetrance, and clinical symptoms, even between individuals with similar or identical mutations. to address this, we examined the role of natural variation in defining the susceptibility of genetically diverse individuals to protein aggregation and toxicity, using the caenorhabditis elegans polyglutamine model. introgression of polyq into three wild genetic backgrounds uncovered wide variation in onset of aggregation and corresponding toxicity, as well as alteration in the cell-specific susceptibility to aggregation. to further dissect these relationships, we established a panel of recombinant inbred lines that showed a broad range of aggregation phenotypes, independent of differences in expression levels. this difference in phenotypic expression of proteotoxic mutations is proposed to be due, at least in part, to the variability in genetic background. thus, our c. elegans model can serve as a powerful tool to dissect the contribution of natural variation to individual susceptibility to proteotoxicity. moreover, distinct measures of proteotoxicity were independently modified by the genetic background.
 monogenic gain-of-function protein aggregation diseases, including huntington’s disease, exhibit substantial variability in age of onset, penetrance, and clinical symptoms, even between individuals with similar or identical mutations. this difference in phenotypic expression of proteotoxic mutations is proposed to be due, at least in part, to the variability in genetic background. to address this, we examined the role of natural variation in defining the susceptibility of genetically diverse individuals to protein aggregation and toxicity, using the caenorhabditis elegans polyglutamine model.  introgression of polyq into three wild genetic backgrounds uncovered wide variation in onset of aggregation and corresponding toxicity, as well as alteration in the cell-specific susceptibility to aggregation. to further dissect these relationships, we established a panel of recombinant inbred lines that showed a broad range of aggregation phenotypes, independent of differences in expression levels. we found that aggregation is a transgressive trait, and does not always correlate with measures of toxicity, such as early onset of muscle dysfunction, egg-laying deficits, or reduced lifespan. moreover, distinct measures of proteotoxicity were independently modified by the genetic background.  resistance to protein aggregation and the ability to restrict its associated cellular dysfunction are independently controlled by the natural variation in genetic background, revealing important new considerations in the search for targets for therapeutic intervention in conformational diseases. thus, our c. elegans model can serve as a powerful tool to dissect the contribution of natural variation to individual susceptibility to proteotoxicity. please see related commentary by kaeberlein, http://www.biomedcentral.com/1741-7007/11/ protein aggregationpolyglutamineconformational diseasegenetic modifiernatural variation 