All worms were maintained, grown, on nematode growth medium (NGM) plates fed with Escherischia coli (E. coli) strain OP50 at 20℃ and were handled by using standard procedures [19]. Wild-type Bristol N2 was obtained from the Caenorhabditis Genetics Center (CGC, University of Minnesota, St. Paul, MN).

Plasmids, including P_myo-2::V1S, P_flp-21::SV2-ICR-DsRed and P_myo-2::V1Q25 vector, created as previously described [20] were used.

To make vectors expressing the human huntingtin exon 1 with a polyglutamine stretch, a sense primer containing a SalI site, 5′-TAAGCAGTCGACCGCCATGGCGACCCTGGA-3′ and an anti-sense primer containing an XhoI site, 5′-TGCTTACTCGAGAGTGCTTACTCGAGAGGTCGGTGCAGAGGCTCCTC-3′ were used to amplify polyQ products obtained from pcDNA3.1 Myc-His polyQ25 or Q97 vector, which were generous gifts from Dr. George Lawless (UCLA HD group, CA, USA). The human SNCA fragment of P_flp-21::SV2-ICR-DsRed was replaced by the PCR-amplified polyQ fragment to construct P_flp-21::Q25V2-ICR-DsRed, P_flp-21::Q97V2-ICR-DsRed. In addition, to construct P_myo-2::V1Q25 and P_myo-2::V1Q97, a sense primer containing a ClaI site, 5′-TAAGCAATCGATATGGCGACCCTGGAAAAGCTG-3′ and an anti-sense primer containing a ClaI site, 5′-TGCTTAATCGATAGGTCGGTGCAGAGGCTCCTC-3′ were used. The plasmid P_myo-2::V1Q25, P_myo-2::V1Q97 were generated by replacing SNCA with the HTT-exon1 polyQ fragment in P_myo-2::V1S.

The procedure for BiFC transgenic lines was performed as previously described [20]. To analyze the effects of expanded polyglutamine on aggregate transmission, P_myo-2::V1Q97 and P_flp-21::Q97V2-ICR-DsRed plasmids were co-injected into the gonads of late L4-stage N2 worms with a selection marker, pRF4 which expresses a mutant collagen gene, rol-6(su1006) [21], to make a double transgenic line expressing the BiFC pair and huntingtin exon 1 with a polyglutamine stretch. Additionally, to analyze the effects of polyQ length on aggregates transmission, P_myo-2::V1Q25 and P_flp-21::Q25V2-ICR-DsRed plasmids were co-injected into N2 worms with pRF4. All of these transgenic worms showed a roller phenotype and expression of DsRed fluorescence in the pharyngeal neurons.

Single-worm PCR analyses were performed as previously described [20]. The genomic DNA released from a gravid single worm in each line was mixed with Ex Taq™ polymerase (RR001A; Takara Shuzo Co. Ltd, Shiga, Japan) and then single-worm PCR for target genes was performed in Bio-Rad MyCycler PCR Thermal Cycler system (Bio-Rad Laboratories Inc., Hercules, CA, USA).

The procedure for western blotting was performed as previously described [20]. Protein samples obtained from the worm pellets were loaded onto 12% SDS-PAGE gels. Monoclonal anti-polyglutamine primary antibody was used for western blotting (MAB1574; Millipore Corporation, Temecula, CA, USA). Chemiluminescence detection was performed with ECL™ prime solution (RPN2232; GE Healthcare Life Sciences, Marlborough, MA, USA), and images were obtained using the Amersham imager 600 (GE Healthcare Life Sciences, Marlborough, MA, USA) and quantified with Multi Gauge (v3.0) software (Fujifilm, Tokyo, Japan).

Worms were collected, washed with M9 buffer (22 mM KH₂PO₄, 22 mM Na₂HPO₄, 85 mM NaCl, 1 mM MgSO₄), and then immobilized with 10 mM sodium azide (S2002, Sigma-Aldrich) in M9 buffer. After removing the buffer, drop worms were placed on microscope cover glasses (HSU-0101242; Marienfeld Laboratory Glassware, Lauda-Königshofen, Germany), and covered with a coverslip. All images were obtained using Olympus FV1000 confocal laser scanning microscopy (Olympus, Tokyo, Japan).

Pharyngeal pumping rate of each line was monitored for 1 min at room temperature using Axio observer a1 inverted microscope (Carl Zeiss MicroImaging Inc., Göttingen, Germany). The pumping count was symbolized as PPM (Pumps Per Minute).

Life span analyses were performed as previously described [20]. Eggs produced from gravid worms were synchronously grown up to the larval 4 (L4)-stage on NGM plates seeded with E. coli OP50 at 20℃. The L4-stage worms were transferred to NGM plates containing 100 mM 5-fluoro-2′-deoxyuridine (FudR; F0503, Sigma-Aldrich). The number of worms that were alive or dead was recorded every 1~2 days. Worms that ruptured, burrowed, or crawled off the plates were censored but included in the life span analysis as censored animals. The survival data and the mean lifespan assays were analyzed by using online application for the survival analysis (OASIS; http://sbi.postech.ac.kr/oasis/surv/) [22].

All experiments were performed blind-coded and repeated at least three times. The graphs were drawn using Prism 5 software (Graphpad Software Inc., San Diego, CA, USA) and the values in the figures are represented as mean±s.e.m. All data were analyzed, compared for statistical significance by one-way ANOVA with Tukey's post-hoc test using Graphpad InStat version 3.05 software (Graphpad Software Inc., San Diego, CA, USA).

BiFC has been applied to visualization of dimerization and oligomerization of proteins in cells [23]. This technique has also been successfully used to investigate the cell-to-cell transmission of α-synuclein in mammalian cells [24] and C. elegans [20]. To investigate cell-to-cell transmission of HTT aggregates in vivo, we generated transgenic C. elegans lines in which the transmission can be detected real-time in a quantitative manner (Fig. 1A). For the generation of transgenic lines, we constructed a several vectors expressing polyQ proteins conjugated to either N-terminal or C-terminal fragment of Venus. The N-terminal part of Venus (V1) fused to the huntingtin exon 1 with a polyQ tract was expressed in pharyngeal muscle under the myo-2 promoter (P_myo-2) [25]. And the C-terminal part of Venus (V2) linked to the huntingtin exon 1 with a polyQ stretch and DsRed, which used as a neuronal marker, were co-expressed using the flp-21 promoter (P_flp-21) in neurons that are connected with the pharyngeal muscle (Fig. 1B) [26]. The wild type (V1Q25 and Q25V2) and the mutant pair (V1Q97 and Q97V2) were injected into the L4-stage N2 worms, and several extrachromosomal lines were obtained for each model. The presence of transgenes in these lines was confirmed by single-worm PCR (Fig. 1C). Expression levels of polyQ proteins in pharyngeal muscles were measured by western blotting. We selected three lines that have the similar expression levels in each model for experimental analysis (Fig. 1D).

To determine the effects of the length of polyQ on aggregate transmission, we analyzed the BiFC signals in the pharynx using confocal microscopy. The BiFC fluorescence in our system solely depends on the extent of transmission rather than the aggregation propensity, because the cell-autonomous protein aggregation would not generate BiFC fluorescence. All lines we generated produced BiFC fluorescence in both the pharyngeal muscle and neighboring neurons (Fig. 2A). This suggests that the transmission does occur between these two cell types and in a bidirectional manner. However, the lines with long polyQ tracts (Q97) exhibited stronger BiFC fluorescence than the lines with short polyQ length (Fig. 2A and B).

The transmission of both wild type and mutant polyQ proteins was increased with age. At all ages, the Q97 lines showed higher BiFC signal than the Q25 lines. While transgenic animals with Q25 exhibited largely diffuse patterns in the pharynx until day 8 (Fig. 2A), some inclusions did appear at day 13 in these animals (Fig. 2B). On the other hand, the Q97 animals developed the inclusion bodies as early as two day after the L4-stage (Fig. 2C~E), and the numbers of BiFC-positive inclusion bodies were considerably higher in the Q97 lines compared to the Q25 lines at all ages (Fig. 2C and D). These data indicate that both transgenic animals showed an age-dependent increase in aggregate transmission with polyQ expansion accelerating the rate of aggregate transmission between muscles and adjacent neurons.

In order to investigate the effects of different polyQ lengths on the degenerative phenotypes in C. elegans, we first examined nerve degeneration of axonal processes from the URA motor neuron [26] that were labeled with DsRed. While the wild-type N2 (Non-tg) worms showed undamaged axonal processes at day 8 and 13, transgenic animals expressing Q25 protein exhibited a small degree of nerve degeneration, determined by neuritic bleb formation and fragmentation of axonal process, on both day 8 and day13 (Fig. 3A and B). The nerve degeneration was further worsened at all ages when pathogenic huntingtin mutant, Q97, was expressed in muscle and neurons (Fig. 3A and B), showing correlation between the extents of polyQ transmission and those of nerve degeneration.

To analyze behavioral changes in these animals, we measured the pharyngeal pumping rates. The pumping rates of transgenic worms containing Q25, compared to the wild-type N2, did not change significantly at day 8 (Fig. 3C). However, at day 13, the reduction of pumping rates in Q25 animals had become significant (Fig. 3C). The pumping rates of the Q97 animals were significantly decreased already at day 8 and exhibited more deficits at all ages than the Q25 animals (Fig. 3C).

In life span analyses, transgenic worms with Q25 exhibited a slightly, but not significantly, decreased life span compared to the N2 worms at day 8 (Fig. 3D), whereas the life span of Q97 animals were significantly decreased than the other lines (Fig. 3D). There is no significant difference of the survival rates among three independent lines in each transgenic model, validating consistency of the data. When average life span was concerned, the transgenic lines with Q25 exhibited shortened life span compare with N2 worms, while the Q97 worms showed further reduced mean lifespan (Fig. 3E).