Aging is the major risk factor for neurodegenerative diseases that are also associated with impaired proteostasis, resulting in abnormal accumulation of protein aggregates. However, the role of aging in development and progression of disease remains elusive. Here, we used Caenorhabditis elegans models to show that aging-promoting genetic variations accelerated the rate of cell-to-cell transmission of SNCA/ a-synuclein aggregates, hallmarks of Parkinson disease, and the progression of disease phenotypes, such as nerve degeneration, behavioral deficits, and reduced life span. Genetic and pharmacological anti-aging manipulations slowed the spread of aggregates and the associated phenotypes. Lysosomal degradation was significantly impaired in aging models, while anti-aging treatments reduced the impairment. Transgenic expression of hlh-30p::hlh-30, the master controller of lysosomal biogenesis, alleviated intercellular transmission of aggregates in the aging model. Our results demonstrate that the rate of aging closely correlates with the rate of aggregate propagation and that general anti-aging treatments can slow aggregate propagation and associated disease progression by restoring lysosomal function. 

 The presence and expression of these transgenes were verified using single-worm PCR and immunofluorescence with the anti-SNCA antibody Ab27410 (Fig. S1A to D), verifying specific expressions of proteins exclusively in the intended cell types. The expression pattern of Pflp21 has been described9 and our own marker for flp-21 promoter activity (DsRed) also exhibited the same expression pattern, which includes expression in the ADL, ASE and ASH sensory neurons, the URA motor neurons, the MC, M2 and M4 pharyngeal neurons, and the intestine (Fig. S1E). Expression of either V1S or SV2 alone did not produce BiFC fluorescence. However, coinjection of both constructs produced strong BiFC fluorescence in both the pharyngeal muscle and adjacent neurons, and the latter were labeled with DsRed (Fig. 1C and D; Fig. S1E and G). The data indicated that protein transmission occurred in both directions. The coexpression of Pflp-21::SV2-DsRed and Pmyo-2::V1 (without the SNCA gene) did not generate a BiFC signal (Fig. 1C and D; Fig S1E), indicating that the signal was not due to nonspecific interactions between the Venus fragments. To test the specificity of the BiFC system, we generated Pmyo-2::V1Q25 C Pflp-21::SV2-DsRed line, the transgenic worm expressing huntingtin exon 1 with a 25 glutamine stretch under the control of Pmyo-2 and SV2 in neurons. These worms did not exhibit BiFC signal in either pharyngeal muscle or neurons (Fig. 1D; Fig. S1E). This result validates specificity of the BiFC transgenic worms for SNCA transmission.

 We also established integrated transgenic lines expressing V1S and SV2-DsRed respectively, and crossed them to create an integrated double-transgenic line. As expected, neither V1S nor SV2-DsRed integrated line produced BiFC fluorescence, whereas the integrated double-transgenic line showed strong BiFC fluorescence in both the pharyngeal muscle and adjacent neurons (Fig. S1F and H). Thus, this C. elegans BiFC system can be utilized as an in vivo model in which both protein transfer and coaggregation between SNCA proteins derived from adjacent cells can be accurately and quantitatively analyzed in real time. BiFC fluorescence increased as the worm aged (Fig. 1E and F), and older animals showed clumps of BiFC signal while younger ones showed mostly diffuse patterns (Fig. 1E). These data indicate that SNCA transmission is a continuous process, and that the accumulated aggregates make large inclusions later in life. We then examined the degeneration of axonal processes from the URA motor neuron.9 These nerves were intact in the wild-type N2 at d 8. Expression of SV2 in neurons caused neuritic bleb formation and nerve fragmentation in a small number of worms (Fig. 1G to K), indicating autonomous cellular toxicity of SNCA in neurons. These degenerative phenotypes were further exacerbated when V1S was expressed in the pharyngeal muscle. In the latter case, approximately 15% of nerves were completely lost (Fig. 1K). To verify nerve fragmentation, we performed 3-D reconstruction of the stacked images of nerve processes. This experiment clearly exhibited nerve fragmentation and bleb formation (Fig. 1H).

These data clearly demonstrate nonautonomous cellular effects on neuronal viability. Nerve degeneration worsened as the transgenic worms aged (Fig. 1J). In order to assess behavioral changes due to the transmission of SNCA aggregates, we performed pharyngeal pumping analysis. The pumping rates of the wild-type N2 did not change significantly with aging until d 16. Single expression of V1S or SV2-DsRed in the pharyngeal muscle and adjacent neurons, respectively, resulted in a slight decline in pumping rates in old age (Fig. 1L and Table S1).  The reduction in pumping rates of all the single expressers became significant on d 13 (Fig. 1L and Table S1). Coinjection and double integrated lines showed more severe phenotypes for pumping rates, with the decline becoming apparent as early as d 2 and progressively deteriorating as the worms aged (Fig. 1L and Table S1). In longevity assays, the single-transgenic animals showed a slightly decreased life span compared to the N2 worms, whereas the life span of the double-transgenic animals was shorter than the single-transgenic lines (Fig. 1M and Table S2). Thus, aggregate transmission and inclusion body formation, as well as the associated degenerative phenotypes, progress with aging. Comparison of the timelines indicates that death of the organism is preceded by the accumulation of BiFC signal, nerve degeneration, and a decline in pumping behavior. These results were replicated with the worms expressing untagged SNCA in the same cell types as the BiFC model (Fig. S1I to L), suggesting that the phenotypes observed in the BiFC model is attributed to SNCA. 

Similar results were obtained in 3 independent lines for each genotype. These results indicate that genetic factors affecting aging processes might change the rate of cell-to-cell transmission of SNCA aggregates and the associated degenerative phenotypes in vivo. Effects of pharmacological anti-aging treatment on transcellular SNCA transmission We then sought to determine the effects of the anti-aging agent, N-acetylglucosamine (GlcNAc),12 on aggregate transmission. When GlcNAc was administered to the V1SCSV2 and daf-16 (mu86); V1SCSV2 animals, both animals showed reduced formation of BiFC-positive inclusion bodies (Fig. 3A and B) and significantly alleviated phenotypes for nerve degeneration (Fig. 3C to F), pumping behavior (Fig. 3G), and life span (Fig. 3H). These results suggest that pharmacological anti-aging treatments can slow the progress of synucleinopathy. Changes in steady-state levels of polyubiquitinated proteins by anti-aging treatment To confirm the microscopy data for changes in the levels of aggregates, we performed a dot blot analysis with an antibody specific to b-sheet-rich SNCA multimers (Syn-O2).13 Consistent with the BiFC inclusion analysis, the dot blot analysis showed that b-sheet-rich SNCA aggregates were reduced by the daf-2 mutation and by GlcNAc, whereas the daf-16 mutation increased the aggregates (Fig. 4A and B). Aging causes a progressive decline in protein homeostasis.12,14,15 This led us to examine the steady-state levels of polyubiquitinated proteins, which represent the activities of major protein degradation systems, such as the ubiquitin-proteasome system and autophagy. 

The levels of polyubiquitinated proteins were increased in the daf-16 transgenic animals, while they were decreased in the daf-2 transgenic animals (Fig. 4C to F). Similarly, the treatment of animals with GlcNAc decreased the levels of polyubiquitinated proteins (Fig. 4G to J). These results suggest that the effects of aging and anti-aging treatments on the propagation of synucleinopathy are mediated by the changes in the capacity of protein degradation systems. The endolysosomal pathway in SNCA transmission Previous studies in cell models have shown that intercellular SNCA transmission is mediated by endocytosis, and the transferred proteins are delivered to lysosomes for degradation.16-19 When V1SCSV2 was introduced into dynamin mutants, dyn-1 (ky51), 20 BiFC fluorescence was significantly reduced compared to that in the wild type (Fig. 5A and B). Reduction of the BiFC signal in the dyn-1(ky51) transgenic animals was greater at d 5 than at d 2, suggesting that the effect was cumulative. Our previous studies have shown that lysosomal function is important for clearing the “seeds” in the process of intercellular SNCA transmission, and lysosomal dysfunction resulted in an enhancement of aggregate transmission.17,19,21 Consistent with these studies, when V1SCSV2 was introduced into the asp-4 (ok2693) and asp-1(tm666) mutants, carrying mutations in cathepsin genes,22 BiFC fluorescence was significantly increased in both mutants, often in the form of inclusion bodies (Fig. 5C to E; Fig. S3D and E).

 These results suggest that lysosomal responses are crucial for protecting the animals from agedependent aggregate propagation. Consistent with this interpretation, GlcNAc treatment increased the expression of lysosomal genes such as lmp-1/LAMP1, sul-3/ARSB and asp-1/ CTSD (Fig. 4K and L). This was further validated by epistasis analysis, where asp-4(ok2693); V1SCSV2 and asp-1(tm666); V1SCSV2 worms were treated with GlcNAc. In contrast to the V1SCSV2 worms, aging-related phenotypes were not rescued by GlcNAc treatment in the asp-1 and asp-4 mutant transgenic animals (Fig. 5F to J). We also analyzed the expression of sqst1/p62, a key component of the autophagic process, and its expression seemed to be upregulated, although the change failed to exhibit statistical significance (Fig. 4K and L). The effects of anti-aging treatments on aggregate transmission are associated with enhanced lysosomal function To verify the role of lysosome in protection against aggregate propagation, we generated the hlh-30 transgenic lines overexpressing the vector hlh-30p::hlh-30, 14 an ortholog of TFEB, the master control transcription factor for lysosome biogenesis,23 into the daf-16(mu86); V1SCSV2 transgenic animals (Fig. S4A).

 In addition to lysosomal and autophagic genes, downstream target genes for HLH-30/TFEB include genes involved in metabolism, apoptosis, and signaling.24 Expression of hlh-30p::hlh-30 in the daf-16(mu86); V1SCSV2 animals increased the expression of lysosomal gene, asp-1 as well as the gene involved in autophagy, sqst-1 (Fig. 6A), and reduced and the steady-state levels of polyubiquitinated proteins (Fig. 6B to E), which indicate the restoration of protein degradation, and the formation of SNCA aggregates (Fig. 6F). The hlh-30p::hlh30 transgenic lines showed reduced BiFC signal (hence, reduced aggregate propagation), decreased nerve degeneration, increased pumping rates, and increased life span (Fig. 7A to H; Fig. S4B to D).Cell-autonomous aggregation vs. intercellular transmission The results presented above do not differentiate between intercellular aggregate transmission and cell-autonomous aggregation. To address this issue, we have generated 4 transgenic lines expressing V1S or SV2 alone in N2 and daf-16(mu86) mutant worms. Also 2 transgenic worms overexpressing the hlh-30p:: hlh-30 transgene with V1S or SV2 were generated. Expression levels were normalized with single-worm PCR and western analysis (in case of V1S lines) or DsRed fluorescence (in case of SV2 lines). Nerve degeneration, pumping behavior, and life span of the transgenic worms in mutant backgrounds were compared with the ones in normal genetic background. We did not find significant differences in the phenotypes (Fig. S5C to G), suggesting the genetic modification we investigated does not have a large impact on SNCA aggregation in the respective tissues (Fig. S5H). 

We also treated the single-tissue expression lines carrying V1S or SV2 alone in N2 and daf-16(mu86) mutant worms with GlcNAc and compared the same battery of phenotypic assays with untreated animals. Unlike the transmission models, the single-tissue expression lines did not exhibit significant changes in pathogenic phenotypes upon treatment with GlcNAc (Fig. S6D to M). To examine the effects of GlcNAc on the expression levels of SNCA, we measured the levels of SNCA by dot blot (Fig. S6N and O). The data showed that the expression levels were not changed by GlcNAc treatment. We also examined neuronal expression of flp-21 promoter upon GlcNAc treatment by monitoring DsRed. Expression of DsRed was strictly confined in neuronal cells with or without GlcNAc treatment (Fig. S6P), indicating that the treatment does not change the cell-type specificity of the promoter. Finally, we examined the detergent solubility of SNCA in the single (V1S or SV2) and double (V1SCSV2) transgenic worms with or without GlcNAc treatment. Triton X-100-insoluble to -soluble ratio was drastically increased in the double transgenics compared with the single transgenic, even though the expression level of each protein is supposedly the same (Fig. 8A to E; Fig. S8). This result implicates transcellular aggregate amplification between the pharyngeal muscle and the associated neurons. Dot blot with the aggregate-specific Syn-O2 antibody showed the similar results, confirming that the double transgenic produced much more aggregates than the sum of each single transgenic (Fig. 8F). Another important point here is that the single transgenic indeed produced SNCA aggregates.

 This confirms that cell-autonomous aggregation does occur, so transmission of the aggregates can be initiated. In the detergent solubility experiment, the Triton-insoluble to -soluble ratio was not changed upon GlcNAc treatment in single transgenics, whereas the same treatment greatly reduced the ratio (Fig. 8C to E; Fig. S8), suggesting that the anti-aging treatment affected primarily the transmission process, rather than the de novo aggregation of each protein. Dot blot analysis with the Syn-O2 antibody showed largely the same results (Fig. 4B; Fig. S6N). These results suggest that the anti-aging and prolysosomal treatments used in the current study exert their effects on intercellular aggregate transmission. Discussion We have developed a quantitative, real-time technique for the study of cell-to-cell transmission of SNCA in C. elegans. The model exhibits many key features of synucleinopathy, including the progressive accumulation of SNCA aggregates, nerve degeneration, behavioral deficits, and reduced life span. Both the genetic and pharmacological manipulations of animals have shown strong correlations among aging rates, transcellular SNCA transmission, and neurodegenerative phenotypes, and likewise, various manipulations associated with anti-aging effects can slow the progression of these events. Aging-dependent progression of synucleinopathy is accompanied by a decline in protein degradation. Anti-aging treatments can restore the systems mediating protein degradation.

 Furthermore, restoration of lysosomal function alleviated the propagation of aggregates and the accompanying neurodegeneration in these aging models. BiFC is a widely used fluorescence technique that has been successfully applied to assess protein-protein interactions and protein dimerization and/or oligomerization in living cells.7 The V1S-SV2 BiFC pair has been shown to fluoresce upon dimerization/oligomerization of SNCA when these proteins are coexpressed in mammalian cells.7 In a previous study,17 by generating neuroblastoma cell lines expressing either one of V1S and SV2, we have shown that cell-to-cell transfer of SNCA proteins and coaggregation of the transferred proteins with the endogenous SNCA can be visualized with BiFC fluorescence during coculture of these cells.One of the important technical issues of the BiFC system is to verify the cell type-specific expression of each protein. Although quantitative analysis of SNCA transgenic expression in each cell type is not technically feasible, we have a series of data supporting the argument that SNCA is exclusively expressed only in the intended cell types. First, DsRed fluorescence is detected only in flp-21 neurons, not in the pharyngeal muscle cells at all. Second, immunofluorescence staining of SNCA with a highly specific and sensitive antibody exhibited the expression of this protein specifically in the intended cell types. Third, expression of the same BiFC pair in the worms with the dyn-1 mutation significantly reduced the BiFC fluorescence signal, indicating that generation of the BiFC fluorescence requires endocytosis.