Polyglutamine proteins at the pathogenic threshold display neuron-specific aggregation in a pan-neuronal Caenorhabditis elegans model

Abstract

The basis of neuron-specific pathogenesis, resulting from the expression of misfolded proteins, is poorly understood and of central importance to an understanding of the cell-type specificity of neurodegenerative disease. In this study, we developed a new model for neuron-specific polyQ pathogenesis in Caenorhabditis elegans by pan-neuronal expression that exhibits polyQ length-dependent aggregation, neurotoxicity, and a pathogenic threshold at a length of 35-40 glutamines. Analysis of specific neurons in C. elegans revealed that only at the threshold length, but not at shorter or longer lengths, polyQ proteins can exist in a soluble state in certain lateral neurons or in an aggregated state in motor neurons of the same animal. These results provide direct experimental evidence that the expression of a single species of a toxic misfolded protein can exhibit a range of neuronal consequences.

Figure 1.

Pan-neuronal Q19::CFP has a soluble distribution pattern (A, B), whereas Q86::CFP is distributed into discrete foci (C, D). Thin arrows indicate commissural neurons. Thick arrow indicates DNC; triangle indicates VNC. Scale bar, 100 μm. E, Flattened z-stacks of C. elegans head. Arrow indicates circumpharyngeal nerve ring; triangle indicates processes of chemosensory neurons. Scale bar, 50 μm. Expression of a range of polyQ lengths reveals that proteins with tracts that are equal to or less than that of Q40 maintain a soluble distribution pattern, whereas those equal to or more than Q67 form foci. All animals depicted are young adults (4 d post-hatch).

Figure 2.

FRAP in C. elegans neurons expressing Q19::YFP, a photobleached area (box), recovers rapidly (B, D), similar to Q0 (A, D), indicating soluble proteins, whereas bleached Q86::YFP foci do not recover (C, D). Therefore, Q86::YFP foci are insoluble, consistent with aggregation. Quantification (D) is five or more experiments with SEM. Signal intensity is measured in the color scale (A), where blue is least intense and red is most intense. Scale bar, 5 μm. RFI, Relative fluorescence intensity.

Figure 3.

Q86 protein in neuronal aggregates exhibits FRET, indicating close and roughly ordered interactions at the molecular level. A, C, E, G, YFP channel, before and after photobleaching. B, D, F, H, CFP intensity in the same cell before and after YPP photobleaching. Animals expressing CFP::YFP FRET (A, B), E_i = 0.248 (± 0.089). CFP and YFP coexpression (C, D) do not produce FRET, E_i = 0.001 (± 0.025). Neurons coexpressing Q19::CFP and Q19::YFP (E, F) do not produce FRET, E_i = −0.090 (± −0.057), whereas coexpression of Q86::CFP and Q86::YFP (G, H) does produce FRET, E_i = 0.224 (± 0.076). Cells shown are representative of FRET experiments. Intensity is by a color scale (G): blue is least intense and red is most intense. Arrows indicate the cell being analyzed for FRET. Scale bar, 2 μm.

Figure 4.

Increases in polyQ length correlate with neuronal dysfunction. Each point represents the average thrashing rate of a single young adult animal (4 d) over a period of 30 s. A bar graph and statistical analysis of these data can be found in supplemental Figure 3 (available at www.jneurosci.org as supplemental material).

Figure 5.

FRAP experiments reveal that Q40 solubility in the VNC varies widely (A–D). Data shown are representative of three independent Q40 lines tested. Quantification of data is from FRAP experiments in a single Q40 animal (D). Each line, including those with open symbols, is an individual experiment rather than an average of several cells, as shown Figure 3D. Q40 protein can recover rapidly from photobleaching [A, D; Q40(s)], similar to Q19, or not at all [C, D; Q40(a)] similar to Q86. Additionally, Q40 animals displayed areas with intermediate recovery rates [B, D; Q40(i)]. Scale bar, 2 μm. Filled triangles indicate the vulva. Q19 distribution is diffuse, with rounded cell bodies (E), whereas Q86 protein is contained in well defined foci (G). Q40 is similar to Q19 in appearance except in the VNC, where sharply tapered, intensely fluorescent areas can be detected (F). Filled arrows indicate areas soluble by FRAP (E, F), whereas the open arrow indicates the area with decreased solubility (F). Scale bar, 20 μm. RFI, Relative fluorescence intensity; (a), aggregated; (i), intermediate; (s), soluble.

Figure 6.

FRET analysis of Q40 proteins in C. elegans neurons reveals two distinct populations of intermolecular interactions in young adult animals. A, B, FRAP was performed to determine protein solubility before FRET analysis. Photobleaching was performed inside the boxes. Recovery from FRAP (A) was rapid, indicating soluble protein and labeled Q40(s). A second area in the same animal [Q40(a)] did not recover from photobleaching (B), indicating insoluble protein, consistent with aggregation. FRET was performed on the areas of interest, indicated by arrows. C, D, YFP channel; E, F, CFP channel. Photobleaching of YFP (C) has little effect on CFP intensity; therefore, no strong FRET was occurring (E). In contrast, YFP photobleaching (D) of insoluble Q40 proteins causes an increase in CFP intensity (F), indicating that intermolecular interactions between Q40 proteins are sufficient to generate FRET. Scale bars, 2 μm. G, Simple scatter plot of E_i from FRET experiments shows two populations of FRET-positive areas in a single Q40 animal. Average ± SD for Q19: E_i, −0.090 (± 0.05), n = 15; Q40: E_i, 0.180 (± 0.13), n = 18; Q86: E_i, 0.224 (± 0.07), n = 17. (a), Aggregated; (s) soluble.

Figure 7.

A, Specific neurons are identified in the flattened z-stack of a representative Q40 animal (8 d old); B, FRAP results are shown for each neuron or area identified in A. C, FRAP data shown are the average for each specific neuron, with SD, analyzed in animals from 4 to 10 d of age. In these neurons, Q40 maintains a soluble state even in aged animals. D, In the DNC and VNC, solubility is polymorphic: soluble and immobile states can be observed in young adult animals or those aged to 8 d. Data shown are the average, with SD of data grouped by recovery of >50% for soluble and <50% for immobile.