Destabilizing Protein Polymorphisms in the Genetic Background Direct Phenotypic Expression of Mutant SOD1 Toxicity

Genetic background exerts a strong modulatory effect on the toxicity of aggregation-prone proteins in conformational diseases. In addition to influencing the misfolding and aggregation behavior of the mutant proteins, polymorphisms in putative modifier genes may affect the molecular processes leading to the disease phenotype. Mutations in SOD1 in a subset of familial amyotrophic lateral sclerosis (ALS) cases confer dominant but clinically variable toxicity, thought to be mediated by misfolding and aggregation of mutant SOD1 protein. While the mechanism of toxicity remains unknown, both the nature of the SOD1 mutation and the genetic background in which it is expressed appear important. To address this, we established a Caenorhabditis elegans model to systematically examine the aggregation behavior and genetic interactions of mutant forms of SOD1. Expression of three structurally distinct SOD1 mutants in C. elegans muscle cells resulted in the appearance of heterogeneous populations of aggregates and was associated with only mild cellular dysfunction. However, introduction of destabilizing temperature-sensitive mutations into the genetic background strongly enhanced the toxicity of SOD1 mutants, resulting in exposure of several deleterious phenotypes at permissive conditions in a manner dependent on the specific SOD1 mutation. The nature of the observed phenotype was dependent on the temperature-sensitive mutation present, while its penetrance reflected the specific combination of temperature-sensitive and SOD1 mutations. Thus, the specific toxic phenotypes of conformational disease may not be simply due to misfolding/aggregation toxicity of the causative mutant proteins, but may be defined by their genetic interactions with cellular pathways harboring mildly destabilizing missense alleles

HSP-4/BiP expression in secretory cells is regulated by a developmental program and not by the unfolded protein response.

Differentiation of secretory cells leads to sharp increases in protein synthesis, challenging endoplasmic reticulum (ER) proteostasis. Anticipatory activation of the unfolded protein response (UPR) prepares cells for the onset of secretory function by expanding the ER size and folding capacity. How cells ensure that the repertoire of induced chaperones matches their postdifferentiation folding needs is not well understood. We find that during differentiation of stem-like seam cells, a typical UPR target, the Caenorhabditis elegans immunoglobulin heavy chain-binding protein (BiP) homologue Heat-Shock Protein 4 (HSP-4), is selectively induced in alae-secreting daughter cells but is repressed in hypodermal daughter cells. Surprisingly, this lineage-dependent induction bypasses the requirement for UPR signaling. Instead, its induction in alae-secreting cells is controlled by a specific developmental program, while its repression in the hypodermal-fated cells requires a transcriptional regulator B-Lymphocyte-Induced Maturation Protein 1 (BLMP-1/BLIMP1), involved in differentiation of mammalian secretory cells. The HSP-4 induction is anticipatory and is required for the integrity of secreted alae. Thus, differentiation programs can directly control a broad-specificity chaperone that is normally stress dependent to ensure the integrity of secreted proteins

A Bystander Mechanism Explains the Specific Phenotype of a Broadly Expressed Misfolded Protein

<div><p>Misfolded proteins in transgenic models of conformational diseases interfere with proteostasis machinery and compromise the function of many structurally and functionally unrelated metastable proteins. This collateral damage to cellular proteins has been termed 'bystander' mechanism. How a single misfolded protein overwhelms the proteostasis, and how broadly-expressed mutant proteins cause cell type-selective phenotypes in disease are open questions. We tested the gain-of-function mechanism of a R37C folding mutation in an endogenous IGF-like <i>C</i>.<i>elegans</i> protein DAF-28. DAF-28(R37C) is broadly expressed, but only causes dysfunction in one specific neuron, ASI, leading to a distinct developmental phenotype. We find that this phenotype is caused by selective disruption of normal biogenesis of an unrelated endogenous protein, DAF-7/TGF-β. The combined deficiency of DAF-28 and DAF-7 biogenesis, but not of DAF-28 alone, explains the gain-of-function phenotype—deficient pro-growth signaling by the ASI neuron. Using functional, fluorescently-tagged protein, we find that, in animals with mutant DAF-28/IGF, the wild-type DAF-7/TGF-β is mislocalized to and accumulates in the proximal axon of the ASI neuron. Activation of two different branches of the unfolded protein response can modulate both the developmental phenotype and DAF-7 mislocalization in DAF-28(R37C) animals, but appear to act through divergent mechanisms. Our finding that bystander targeting of TGF-β explains the phenotype caused by a folding mutation in an IGF-like protein suggests that, in conformational diseases, bystander misfolding may specify the distinct phenotypes caused by different folding mutations.</p></div

mCherry::DAF-7 is expressed in multiple neurons and secreted early in embryonic development.

<p><b>A,B</b>. L2 animal expressing UPR reporter p<i>hsp-4</i>::GFP together with mCherry::DAF-7 protein. The UPR reporter is induced in the intestine and seam cells, but not in the ASI neuron. <b>C</b>. Comma-stage embryo expressing the SMAD::GFP reporter. The area of developing pharynx is indicated. <b>D-G</b>. Comma-stage (D,E) and 3-fold (F,G) embryos expressing mCherry::DAF-7 protein and p<i>daf-7</i>::GFP,<i>rol-6</i> transgene (ASI::GFP). GFP fluorescence can be seen in multiple cells in the head region of developing comma-stage embryo, and secreted mCherry::DAF-7 protein has already accumulated in the extraembryonic fluid at this stage. D is a close-up of the boxed area in E, green channel only. Bracket in F indicates position of amphid sensory neurons, including ASI. Note a strong mCherry fluorescence in coelomocytes in three-fold embryo (F,G). E and G are single plane images. <b>H,I</b>. 2.5-fold embryo expressing DAF-28::mCherry protein together with ASI::GFP transgene. DAF-28::mCherry protein is visible in coelomocytes. I is a single plane image.</p

<i>daf-28(sa191)</i> gain-of-function mutation phenocopies <i>daf-7</i> loss-of-function under growth-promoting conditions.

<p><b>A</b>. IGF-like protein DAF-28 and TGF-β protein DAF-7 are secreted from the ASI sensory neuron under growth-promoting conditions and bind to their respective receptors on target cells. This triggers transcriptional programs that promote reproductive development and prevent dauer. INS-4 and INS-6 proteins also contribute to the pro-growth insulin/IGF signaling. <b>B</b>. Possible developmental trajectories and their outcomes. Green arrow indicates normal development. Activation of dauer signaling and a brief transient entry into L2d/pre-dauer stage results in ~ 1 day developmental delay (yellow). A longer L2d duration (dashed red) or commitment to dauer development produce more severe delay (red). <b>C</b>. Percent animals in each developmental outcome for indicated genotypes. <i>sa191</i> animals activate dauer signaling even under growth-promoting conditions, as do <i>daf-7</i> loss-of-function mutants. At least three repetitions of 100–200 animals with per genotype; the raw data available in the Supplemental Table.</p

DAF-28::mCherry is expressed in punctate pattern in axons of head neurons.

<p><b>A,B</b>. Single plane images of same animal expressing p<i>daf-28</i>::GFP transcriptional reporter (<i>wwEx85)</i> and DAF-28::mCherry protein (low expression transgene). p<i>daf-28</i>::GFP expression is seen in multiple neuronal cell bodies (c.b.), in axons (ax) and dendrites (d), in hypodermis (hyp), and pharynx (ph). <b>C,D</b>. DAF-28::mCherry protein is bright in coelomocytes (cc) of mid-L1 larva relative to its intensity in neuronal cell bodies (c.b.), indicating its efficient secretion. DAF-28::GFP accumulates in cell bodies. Both can be seen in a regular punctate pattern in axonal region. p.i. indicates posterior intestine. D, D' and D'' are enlarged areas boxed in C. D' and D'' are green and red channels of the same image. <b>E</b>. DAF-28::mCherry protein rescues persistent dauer entry of the 4xDel strain (missing four pro-growth IGF-like proteins, including DAF-28, INS-4 and INS-6). <b>F</b>. DAF-28::mCherry protein (red) localizes in a regular pattern in large puncta adjacent to the ASI axon, as well as in pharyngeal and hypodermal tissues. Green, p<i>daf-7</i>::GFP;<i>rol-6</i> array used to visualize ASI neuron (ASI::GFP). Orientation of the ASI neurons is shown in panel H. <b>G</b>. Inactivation of the <i>C</i>. <i>elegans</i> homologue of axonal kinesin KIF1A, <i>unc-104</i>, eliminated axon-adjacent puncta of DAF-28::mCherry. Accumulation of mCherry-positive vesicles, presumably representing the DAF-28::mCherry secreted from the cell body, can be seen instead. <b>H</b>. Schematic representation of orientation of the ASI neuron relative to the pharynx. Anterior is to the left, dorsal up. The ASI cell body, dendrite, and the proximal and synapse-rich distal axonal regions are indicated. All confocal images of ASI neurons follow this orientation.</p

DAF-7/TGF-β protein function is disrupted in <i>daf-28</i> mutant animals.

<p><b>A</b>. Fluorescent and transmitted light micrographs of late L1 larvae of the indicated genotypes, carrying SMAD::GFP (<i>cuIs5</i>) transgene. Bright fluorescence in the pharynx indicates DAF-7 activity. Animals were picked under transmitted light to provide an unbiased sample. Left panels correspond to boxed areas showing fluorescent pharynxes. <b>B</b>. Box-and-whisker plot of GFP intensity in animals of indicated genotypes (<i>n</i> = 1351, 770, and 118 animals, respectively; L1/L2 animals) carrying the SMAD::GFP transgene. Boxes show interquartile range, whiskers are 1-99^th percentile. The outliers are shown as individual data points outside the whisker range. <b>C</b>. Development of <i>sa191</i> animals with bright <i>vs</i>. dim SMAD::GFP fluorescence. Decreased reporter activity correlates with stronger activation of dauer signaling (χ² = 38.81, df = 2). Animals were separated into dim and bright populations by eye using fluorescent stereo microscope. <b>D</b>. Over-expression of DAF-7 cDNA in the ASI neuron (ASI::DAF-7) (χ² = 81.71, df = 2) or expression of mCherry::DAF-7 fusion protein (χ² = 427.3, df = 2) rescues <i>sa191</i> dauer phenotype at 20°C. Since this is a non-integrated transgene, non-transgenic siblings (N-Tg sib.) were used as internal controls. 4xDel strain lacks four pro-growth IGF-like proteins, including DAF-28, INS-4 and INS-6. <b>E</b>. mCherry::DAF-7 fusion protein expressed from its cognate promoter rescues <i>daf-7(e1372)</i> loss-of-function mutation at 20°C. Non-transgenic siblings (N-Tg sib.) in the first generation only are also rescued. Data in B were analyzed by ANOVA followed by Bonferroni’s multiple comparison test, α = 0.05, ****<i>P</i><0.0001, ***<i>P</i><0.001. Data in C, D were analyzed by Chi-square test, α = 0.05, ****<i>P</i><0.0001.</p