Anti-aging Treatments Slow Propagation Of Synucleinopathy By Restoring Lysosomal Function

Introduction:

Accumulation of specific protein aggregates is characteristic of neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). Proteins that are aggregated in these diseases include Ab and MAPT/ tau in AD, SNCA/a-synuclein in PD, and TARDBP/TDP-43 in ALS. Protein aggregation is a consequence of impaired proteostasis, which is maintained by coordinated regulation of protein synthesis, folding, and degradation. Mutations in protein synthesis and degradation machinery cause neurodegeneration in animal models and are often linked to human neurodegenerative diseases. Protein aggregates in neurodegenerative diseases spread progressively from limited brain regions to larger areas as the diseases progress. In the case of PD, SNCA aggregates initially occur in the lower brain stem nuclei and olfactory bulb and sequentially spread through the ascending pathways through the upper brain stem regions to the cortical areas. Cell-to-cell transmission of protein aggregates has been suggested as the underlying mechanism for this pathological dissemination in the brains of patients with neurodegenerative diseases.1-3 The details of the mechanism of cell-to-cell aggregate transmission remain elusive, however, evidence suggests that the unconventional exocytosis and subsequent endocytosis in the neighboring cells constitute the main frame of the mechanism. Under the premise that aggregate propagation leads to the progression of disease phenotypes, the disruption of intercellular aggregate transmission is emerging as a promising strategy for stopping disease progression.4,5 Cell-to-cell transmission of protein aggregates is a 2-step process: transfer of aggregates from donor cells to recipient cells, and co-aggregation of transferred proteins and endogenous proteins.6 Here, we developed Caenorhabditis elegans (C. elegans) models that utilize bimolecular fluorescence complementation (BiFC)7 to exhibit fluorescence in pharyngeal muscles and their associated neurons only when SNCA proteins interact with each other transcellularly. Using these animal models, we investigated the effects of aging on cell-to-cell aggregate transmission and the mechanism underlying the aging effects.

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Results Generation and characterization of the C. elegans model for transmission of synucleinopathy

In order to develop an animal model for a convenient assay of cell-to-cell protein transmission, we produced C. elegans transgenic lines expressing SNCA fused to either the N-terminal or C-terminal fragment of Venus, a variant of yellow fluorescence protein (Fig. 1A). The N-terminal part of Venus (V1) was attached to the SNCA N terminus (V1S), and the C-terminal part of Venus (V2) to the C terminus of SNCA (SV2). In the C. elegans model, V1S was expressed in the pharynx muscle using the myo-2 promoter (Pmyo-2),8 and SV2 and DsRed were coexpressed in neurons connected to the pharynx using the flip- 21 promoters (Pflflp-21) 9 (Fig. 1B). 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 Pflflp- 21 has been described9 and our own marker for flip-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 was labeled with DsRed (Fig. 1C and D; Fig. S1E and G). The data indicated that protein transmission occurred in both directions. The co-expression of Pflflp-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 Pflflp-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 the 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 FL 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 FL 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 a 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 a pharyngeal pumping analysis. The pumping rates of the wild-type N2 did not change signifificantly with aging until d 16. A 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 signifificant 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. A comparison of the timelines indicates that the 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.

Effects of aging-related genetic factors on cell-to-cell SNCA transmission

Next, we examined the effects of aging-related genetic variations on aggregate transmission and degenerative phenotypes. The BiFC SNCA constructs were injected into daf-2(e1370) and daf-16(mu86) mutants (Fig. S2A and B), which model aging effects, with daf-2 (e1370) mutants showing a slower aging rate and extended life span while daf-16(mu86) mutants age faster than the wild type and have a shortened life span.11 The daf-2(e1370); V1SCSV2 animals showed a reduced BiFC signal (Fig. 2A and B; Fig. S2D), a smaller number of inclusion bodies (Fig. 2C and D; Fig. S2E), less nerve degeneration (Fig. 2E and F; Fig. S2F and G) improved pumping behavior (Fig. 2G; Fig. S2H), and extended life span than the V1SCSV2 line (Fig. 2H; Fig. S2I). Conversely, in the daf-16(mu86); V1SCSV2 animals, BiFC-positive inclusion bodies appeared much earlier than in the V1SCSV2 animals; as early as 2-d post the L4- stage (Fig. 2C and D; Fig. S2E). The BiFC signal itself was lower in the daf-16(mu86); V1SCSV2 than in the V1SCSV2 (Fig. 2B; Fig. S2D), probably due to early and robust formation of inclusion bodies. The daf-16(mu86); V1SCSV2 animals showed more severe nerve degeneration (Fig. 2E and F; Fig. S2F and G), more decreased pumping behavior (Fig. 2G; Fig. S2H), and shorter life span than the V1SCSV2 animals (Fig. 2H; Fig. S2I). 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. 

Figure 1. Generation and characterization of the C. elegans model for transmission of synucleinopathy. (A) Postulated events leading to the generation of BiFC fluorescence via cell-to-cell transmission of SNCA. (B) Transgenes used in C. elegans. DsRed is expressed as an independent translational unit, which is used to label neurons. (C) BiFC fluorescence in pharyngeal muscles and neurons. All pictures contain DIC and fluorescence images. The arrows indicate BiFC signals from neurons (see also Fig. S1E to H). Scale bars: 200 mm. (D) Quantification of BiFC fluorescence in (C) and the V1S C SV2 double-transgenic integrated line. Twenty-five worms from each line were used. (E) Increase in BiFC fluorescence with aging. White arrows indicate the BiFC signals in neurons, and red arrowheads indicate inclusions in the pharynx. Scale bars: 200 mm. (F) Quantification of BiFC fluorescence in (E) and the V1SCSV2 double transgenic integrated line. Twenty-five worms from each line were used;


, P < 0.001. (G) Nerve processes expressing DsRed were analyzed for neurodegeneration. Scale bars: 200 mm. (H) A 3-dimensional reconstruction of axonal processes from URA motor neuron containing DsRed fluorescence. N, normal axonal process; F, fragmented axonal process. “Fragmented” represents the complete degeneration of nerves. Scale bars: 40mm. (I and J) Blebbing phenotype. Percentage of worms that have blebs at d 8 (I) and blebbing phenotype with aging (J). Thirty worms from each line were used n D 3;
, P < 0.05;

, P < 0.01. (K) Fragmentation of axonal process at d 8. Thirty worms from each line were used, n D 3;
, P < 0.05. (L) Pharyngeal pumping rate with aging; Twenty-five worms from each line were used, P values are listed in Table S1. (M) Life-span analyses. One hundred fififty worms for each line were used, P values are listed in Table S2.

Figure 2. Effects of daf-2, daf-16 mutants on cell-to-cell SNCA transmission. (A and B) BiFC fluorescence in the aging-related mutants, daf-2(e1370) and daf-16(mu86) (see also Fig. S2D). Scale bars: 200 mm (A). Twenty worms for each line were used n D 3;
, P < 0.05;

, P < 0.01;


, P < 0.001. (C and D) Percentage of worms that have BiFC-positive inclusions with aging (see also Fig. S2E). Twenty worms for each line were used n D 3;
, P < 0.05;

, P < 0.01. (E and F) Quantification of axonal bleb number (E) and nerve fragmentation (F) at d 8 (see also Fig. S2F and G). Twenty worms for each line were used n D 3;
, P < 0.05;

, P < 0.01. (G) Pharyngeal pumping rates at d 13 (see also Fig. S2H). Twenty worms for each line were used n D 3;

, P < 0.01. (H) Life-span analyses (see also Fig. S2I). Three hundred worms for each line were used, P values are listed in Table S2.

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 signifificantly 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.

Figure 3. Effects of GlcNAc in transcellular SNCA transmission. (A) Changes in BiFC fluorescence with GlcNAc treatment at d 13. The white arrows indicate BiFC signals from neurons, and the red arrowheads indicate inclusions in the pharynx. Scale bars: 200 mm. (B) Worms with BiFC-positive inclusions were quantified. Thirty worms for each line were used. (C and D) Axonal bleb numbers in the V1SCSV2 (C) and daf-16(mu86); V1SCSV2 animals (D) with GlcNAc treatment at d 8. Twenty worms for each line were used n D 5;
, P < 0.05. (E and F) Nerve fragmentation in the V1SCSV2 (E) and daf-16(mu86); V1SCSV2 (F) transgenic lines at d 8. Twenty worms for each line were used n D 5;
, P < 0.05. (G) Pharyngeal pumping rates at d 11. Forty worms for each line were used;
, P < 0.05;

, P < 0.01. (H) Life-span analyses with GlcNAc treatment. Five hundred worms for each line were used, P values are listed in Table S2.

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 signifificantly 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 signifificantly 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 age-dependent aggregate propagation. Consistent with this interpretation, GlcNAc treatment increased the expression of lysosomal genes such as lamp-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 sqst- 1/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 high-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 the lysosomal gene, asp-1 as well as the gene involved in autophagy, sqft-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 high-30p::hlh- 30 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).

Figure 4. Changes in steady-state levels of polyubiquitinated proteins by anti-aging treatment. (A and B) Levels of SNCA aggregates (Syn-O2) in aging-related lines and with GlcNAc treatment. The data were normalized to total SNCA expression (274). Agg, SNCA aggregates; Total, total SNCA expression, n D 3 (A), n D 5 (B);
, P < 0.05. (C and D) The levels of polyubiquitinated proteins in aging-related models. (E and F) Quantification of the levels in Tx-sol (E) and Tx-insole (F) fractions. The levels were highest in the daf-16(mu86); V1SCSV2 lines. Tx-sol, Triton-soluble; Tx-insole, Triton-insoluble, n D 3;
, P < 0.05. (G and H) The levels of polyubiquitinated proteins with GlcNAc treatment in aging-related models. (I and J) Quantification of the levels in Tx-sol (I) and Tx-insole (J) fractions. n D 5;
, P < 0.05. All the line on the right side of western images indicates the quantified size range in the blots. All the data were normalized to ACTB expression. (K and L) Expression levels of autophagy-related and lysosomal genes in the V1SCSV2 (K) and daf-16(mu86); V1SCSV2 animals (L) with GlcNAc treatment measured by qPCR. n D 5;
, P < 0.05;

, P < 0.01.

Figure 5. Involvement of the endolysosomal pathways in SNCA transmission. (A and B) BiFC fluorescence in wild-type and dyn-1(ky51) transgenic lines. Scale bars: 200 mm (A). Twenty-four worms for each line were used;

, P < 0.01;


, P < 0.001. (C to E) BiFC fluorescence and inclusion body formation in wild-type, asp-4(ok2693), and asp-1(tm666) mutant worms (see also Fig. S3D and E). Scale bars: 200 mm (C). Twenty worms for each line were used n D 3;
, P < 0.05;

, P < 0.01. (F to J) Epistasis analysis of asp-4 and asp-1 transgenic worms with GlcNAc treatment. (F and G) The levels of polyubiquitinated proteins with GlcNAc treatment. (H and I) Quantification of the levels in Tx-sol (H) and Tx-insole (I) fractions. (J) Levels of SNCA aggregates with GlcNAc treatment. The data were normalized to ACTB (F and G) and total SNCA expression (J), n D 3, ns: not signifificant.

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:: huh-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 backgrounds. 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). 

Figure 6. Restoration of lysosomal degradation function by transgenic expression of hlh-30phlh-30. (A) Expression levels of autophagy-related and lysosomal genes measured by qPCR. n D 3;
, P < 0.05. (B and C) The levels of polyubiquitinated proteins in daf-16(mu86); V1SCSV2 in the presence or absence of the high-30phlh- 30 transgenes. (D and E) Quantification of the levels in Triton-soluble (D) and Triton-insoluble (E) fractions. The size ranges quantified are indicated by the line to the right in the blots. All the data were normalized with ACTB, n D 3;
, P < 0.05. (F) Levels of SNCA aggregates in the high-30p::hlh-30 transgenic lines. The data were normalized to total SNCA expression, n D 3;
, P < 0.05.

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 signifificant 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 the neuronal expression of fly-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 similar results, confirming that the double transgenic produced much more aggregates than the sum of every 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 pro-lysosomal treatments used in the current study exert their effects on intercellular aggregate transmission.

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