Cysteine cathepsins are essential in lysosomal degradation of α-synuclein

Significance

Identifying factors that regulate the degradation of α-synuclein, the protein at the center of Parkinson’s disease etiology, is vital in designing therapeutic strategies. This study provides new mechanistic insights into α-synuclein clearance in the lysosome, a cellular site for proteolysis by using purified mouse lysosomes and purified lysosomal proteases. Cathepsins B and L are identified to be vital through peptide mapping by mass spectrometry. Importantly, cathepsin L degrades α-synuclein amyloid fibrils, materials associated with neurodegeneration, and, thus, changes in its activity or levels could provide a promising avenue for intervention. Additional results on the stimulatory effect of anionic phospholipids on cathepsin activity suggest that changes in lipid metabolism may also contribute to lysosomal dysfunction leading to pathogenesis.

Abstract

A cellular feature of Parkinson’s disease is cytosolic accumulation and amyloid formation of α-synuclein (α-syn), implicating a misregulation or impairment of protein degradation pathways involving the proteasome and lysosome. Within lysosomes, cathepsin D (CtsD), an aspartyl protease, is suggested to be the main protease for α-syn clearance; however, the protease alone only generates amyloidogenic C terminal-truncated species (e.g., 1–94, 5–94), implying that other proteases and/or environmental factors are needed to facilitate degradation and to avoid α-syn aggregation in vivo. Using liquid chromatography–mass spectrometry, to our knowledge, we report the first peptide cleavage map of the lysosomal degradation process of α-syn. Studies of purified mouse brain and liver lysosomal extracts and individual human cathepsins demonstrate a direct involvement of cysteine cathepsin B (CtsB) and L (CtsL). Both CtsB and CtsL cleave α-syn within its amyloid region and circumvent fibril formation. For CtsD, only in the presence of anionic phospholipids can this protease cleave throughout the α-syn sequence, suggesting that phospholipids are crucial for its activity. Taken together, an interplay exists between α-syn conformation and cathepsin activity with CtsL as the most efficient under the conditions examined. Notably, we discovered that CtsL efficiently degrades α-syn amyloid fibrils, which by definition are resistant to broad spectrum proteases. This work implicates CtsB and CtsL as essential in α-syn lysosomal degradation, establishing groundwork to explore mechanisms to enhance their cellular activity and levels as a potential strategy for clearance of α-syn.

A presynaptic neuronal protein, α-synuclein (α-syn), is linked genetically and neuropathologically to Parkinson’s disease (PD) (1). The protein exists in a multitude of conformations that likely dictates the physiological function of α-syn (2–6). Upon membrane association, α-syn adopts an α-helical structure (7), whereas in solution, the protein is disordered (3, 6, 8). Elucidation of molecular mechanisms underlying the transformation of α-syn to a disease-associated species is still the subject of intense research. However, the deposition of insoluble α-syn fibrils rich in β-sheet structure, generally referred to as amyloid is a hallmark of the disease (9, 10).

Understanding the cellular pathways that control α-syn aggregation is critical in establishing ways to circumvent the progression of PD. A primary cause leading to PD is associated with impaired α-syn turnover (11–13). Hence, boosting the degradation of α-syn could be an effective way to alleviate this burden. Both in vitro and in vivo data support the involvement of the proteasome and lysosome in α-syn degradation (14, 15). The proteasome pathway is generally considered to be responsible for removal of soluble α-syn, whereas the lysosome eliminates aggregation-prone species or excess levels of α-syn. Dysfunction of either system has been shown to increase α-syn levels (16).

α-syn enters via two main autophagic pathways (17), either through macroautophagy (14) or chaperone-mediated autophagy (18) into the lysosome, where the protein is degraded. The main class of lysosomal proteases is the cathepsins (Cts), which are subdivided based on the active-site amino acids that confer catalytic activity. These are cysteine (CtsB, CtsC, CtsF, CtsH, CtsK, CtsL, CtsS, CtsV, and CtsX), serine (CtsA and CtsG), and aspartyl (CtsD and CtsE) proteases (19). Activity of cathepsins is optimal in acidic pH, and most are inactive at neutral pH. CtsD (20) is the only protease implicated to date in the lysosomal degradation of α-syn (21–24).

Although prior literature supports CtsD involvement, the question of whether other lysosomal protease(s) are involved is also raised. For example, a strong correlation exists in vivo between overexpression of CtsD and α-syn levels as well as the neurotoxic potential of overexpressed α-syn (21, 22). However, in vitro CtsD activity yields incomplete proteolysis of α-syn and generates truncated C-terminal (α-synΔC) species (24). More concerning is that both α-synΔC peptides, such as residues 1–87 (10), 1–103 (25), and 1–119 (26) of α-syn (residues 1–140), and the acidic environment of the lysosomal lumen (27) are known to enhance amyloid formation. Interestingly, human tissue samples contain an abundance of α-synΔC species under normal physiological conditions (28). Some of these α-syn fragments (1–115, 1–119, 1–122, 1–133, and 1–135) have also been isolated from Lewy bodies (29), classic PD hallmarks. The significance of these truncations remain ill-defined, but one could speculate the involvement of CtsD and partial lysosomal degradation of α-syn (24).

Here, we sought to identify other lysosomal cathepsins and environmental factors needed to facilitate α-syn degradation and to avoid aggregation. Direct interaction between α-syn and lysosomal proteases was shown by purified mouse brain and liver lysosomal extracts as well as purified human cathepsins and was characterized by peptide mapping using liquid chromatography–mass spectrometry (LC-MS). We found that mouse brain and liver lysosomal extracts harbor mostly cysteine cathepsin activity (namely CtsB and CtsL) that efficiently degrades recombinant α-syn. Lysosomal and individual cathepsin activities on soluble, membrane-bound, and fibrillar α-syn as well as their impact on α-syn amyloid formation have been evaluated.

Results

Mimicking α-syn Degradation by Lysosomes.

Purified lysosomes were isolated from mouse brain and liver and monitored for α-syn degradation (Fig. 1 and SI Appendix, Fig. S1). Reactions contained recombinant monomeric α-syn (α-syn_m, 3.8−60 µM) and lysosomal extracts (18 µg total protein) in pH 5 buffer and were incubated at 37 °C (0–60 h). The addition of liver lysosomal extract completely inhibited α-syn_m (60 µM) aggregation as determined by no changes in turbidity (Fig. 1A) and emission intensity of an amyloid sensitive dye, thioflavin T (ThT) (Fig. 1B). Analysis by SDS/PAGE at 60 h revealed loss of full-length α-syn_m with generation of many smaller peptides (<2 kDa; Fig. 1B, Inset). In buffer alone, only full length protein is present and no degradation occurred during incubation (Fig. 1B, Inset). Transmission electron microscopy (TEM) confirmed that α-syn fibrils (α-syn_f) are not formed in the presence of lysosomal extract compared with the protein alone control (Fig. 1C).

Fig. 1.

Lysosomal degradation of α-syn. Aggregation kinetics of α-syn_m (60 µM) incubated (37 °C with shaking) in the absence and presence of liver lysosomal extract (18 µg total protein) at pH 5 monitored simultaneously by turbidity (λ_obs = 440 nm) (A) and ThT fluorescence (λ_obs = 480 nm) (B). (Inset) Corresponding SDS/PAGE (4–12%) visualized by Coomassie staining after 60 h of incubation of α-syn_m in the absence (Left) and presence (Right) of liver lysosomal extract. (C) Corresponding TEM images taken after 60 h of incubation of α-syn_m in the absence (Left) and presence (Right) of lysosomal extract. Scale bars are as indicated. (D) Schematic representation of the primary amino acid sequence of α-syn with identifiable cleavage sites derived from both brain and liver lysosomal activity (SI Appendix, Table S1). Cleavage sites generated from brain only (green), liver only (magenta), or both brain and liver (black) lysosomal extract are shown. Endogenous CtsD activity is denoted by a blue asterisk. A PK-resistant core (residues 19–89) determined from α-syn_f incubated with PK overnight at pH 5 is colored light gray.

To identify peptide fragments generated, LC-MS was performed at various incubation times (SI Appendix, Fig. S2 and Table S1). Cleavage sites (denoted by X/X, where / indicates the cut) common to both sources occurred mostly in the N-terminal amyloid region of α-syn at M5/K6, G14/V15, K34/E35, G41/S42, H50/G51, K60/E61, Q62/V63, G73/V74, T75/A76, A90/A91, and F94/V95 (Fig. 1D), which is consistent with the observed inhibition of α-syn aggregation.

Cathepsin D Incompletely Degrades α-syn.

Unexpectedly, none of the major cleavage sites corresponded to previously reported CtsD activity (24, 30); therefore, we revisited α-syn degradation by CtsD. Upon incubation of α-syn_m with human CtsD at pH 5, two main species with molecular weights of ∼12 and ∼8 kDa were detected by SDS/PAGE and Coomassie staining (SI Appendix, Fig. S3A). LC-MS analysis of α-syn_m (2−60 µM) incubated with CtsD (0.1 µM) revealed peptide masses corresponding to fragments 1−94, 5–94, and 95–140, indicating the primary cleavage sites were F4/M5 and F94/V95 (SI Appendix, Fig. S3 B–D and Table S2). There are also minor cuts at M116/P117, A124/Y125, and G132/Y133. No further cleavage occurred within the amyloidogenic N-terminal fragment 5−94. Although the cleavage sites at F4/V5 and F94/V95 were not previously identified for CtsD (30), these cuts were found in low levels from lysosomal degradation of α-syn_m (Fig. 1D). Because in vivo experiments (21–24) rely heavily on immunoblotting with α-syn antibodies that have C-terminal epitopes such as the widely used Syn-1 (31), the fate of the N-terminal α-syn peptides (e.g., 5–94) as a result of CtsD activity would not have been previously observed (SI Appendix, Fig. S4). Hence, interpretations based on Western blot analysis that CtsD fully degrades α-syn in vivo are incorrect.

Aggregation of α-syn_m (3.8–60 µM) still occurs in the presence of CtsD and the presence of fibrils were confirmed by TEM (SI Appendix, Fig. S5). Interestingly, N- and C-terminal truncations by CtsD did not accelerate aggregation as would be anticipated from previously reported data at neutral pH (10, 25, 26). Instead, the lag phases were unchanged with the exception of α-syn concentrations <15 µM, where the lag times actually lengthened. This may be explained by the presence of in situ-generated C-terminal peptides, which could modulate aggregation propensity, whereas previous studies used recombinant α-synΔC constructs. Nevertheless, CtsD alone cannot degrade α-syn_m and prevent fibril formation. In accordance, limited proteinase K (PK) digestion of fibrils at pH 5 showed the smallest PK-resistant core contains residues 19–89 (SI Appendix, Table S3), a region that CtsD does not cleave within. Clearly, these results strongly suggest the involvement of other proteases in lysosomal clearance of α-syn_m.

Cysteine Cathepsins Are Responsible for α-syn Degradation.

To identify the class of protease(s) responsible for α-syn_m degradation, lysosomal extracts were treated with protease inhibitors. Of the four known protease types (serine, cysteine, aspartyl, and metalloproteases), we elected to focus on cysteine and aspartyl proteases because they represent the majority of lysosomal activity (19). α-syn_m in the presence of liver lysosomal extracts was treated with leupeptin (LeuP) and pepstatin A (PePA), cysteine and aspartyl protease inhibitors, respectively. In the presence of LeuP, the inhibition of α-syn_m aggregation by lysosomal extract was reverted, exhibiting both apparent lag and elongation phases (Fig. 2A). Corresponding LC-MS analysis (SI Appendix, Fig. S6 and Table S4) showed that the majority of cleavage sites were eliminated by the addition of LeuP to both liver and brain lysosomal extracts, supporting the observation that α-syn_m can now aggregate. The only activity from these samples resulted in cuts at F4/M5, F94/V95, N103/E104, and N122/E123 for brain and K6/G7, L8/S9, N103/E104, and E139/A140 for liver lysosomal extracts. F4/M5 and F94/V95 sites are consistent with CtsD activity, whereas cuts at N103/E104 and N122/E123 could be attributable to asparagine endopeptidase (AEP) activity. The presence of these cuts is revealed because AEP hydrolysis of substrates C-terminal of Asn residues is insensitive to LeuP (32). The origin of K6/G7, L8/S9, and E139/A140 cleavage sites could not be identified.

Fig. 2.

Identification of cysteine cathepsins in lysosomal degradation of α-syn. (A) Aggregation kinetics of α-syn_m (53 µM) incubated with liver lysosomal extract (18 µg total protein) (black) in the presence of LeuP, a cysteine protease inhibitor (red), or PePA, an aspartyl protease inhibitor (green), respectively. (Inset) Western blot analysis of liver lysosomal extracts probed with antibodies against endogenous CtsB (Upper) and CtsL (Lower). (B) Aggregation kinetics of α-syn_m (10, 20, and 40 µM) incubated (37 °C with shaking) in the presence of 0.15 µM CtsB (green), CtsL (black), and CtsD (gray to pink to blue) at pH 5 and monitored by ThT fluorescence (λ_obs = 480 nm). (C) SDS/PAGE (4–12%) visualized by Coomassie staining showing degradation of α-syn_m (5, 10, and 20 µM) by 0.15 µM CtsB (green), CtsL (black), and CtsD (purple) after 30 min at pH 5 and 37 °C; α-syn_m incubated alone is also shown. (D) Location of the corresponding cleavage sites generated by reactions shown in C as identified by LC-MS analysis (SI Appendix, Tables S7 and S8) are shown as triangles. Primary cleavage sites (denoted by X/X, where / indicates the cut) are highlighted.

In contrast, samples with PePA behaved as the control (no inhibitor added) with no detectable changes even after 60 h (Fig. 2A). In the presence of PePA, most of the lysosomal activity was retained with the exception of cleavages at F4/M5 and F94/V95 (SI Appendix, Fig. S6 and Table S5). These data establish that cysteine cathepsins are involved in lysosomal degradation of α-syn_m, and more importantly, unlike CtsD, they degrade the central portion of α-syn_m and circumvent protein aggregation.

Lysosomal Cysteine Cathepsin Activity Involves CtsB and CtsL.

Because cysteine cathepsins CtsB and CtsL have been associated with other neurodegenerative amyloid diseases (33–35), we tested their specific involvement in α-syn degradation. The presence of both mature forms of CtsB and CtsL in liver lysosomal extracts was confirmed by Western blot analysis (Fig. 2A, Inset). Activities of purified human CtsB and CtsL were evaluated and compared with the lysosomal data. To ensure that the purchased enzymes were active, each cathepsin was validated using a fluorescence-based assay (SI Appendix, Fig. S7 A–C) and Western blots (SI Appendix, Fig. S7D). In addition, aggregation of α-syn (10, 20, and 40 µM) in the presence of each cathepsin (0.15 µM) at pH 5 was monitored by ThT fluorescence (Fig. 2B). As expected from our lysosomal inhibition data, α-syn aggregation is completely suppressed in the presence of cysteine cathepsins, CtsB and CtsL but proceeds in the presence of CtsD.

Limited proteolysis experiments at different α-syn_m concentrations (5, 10, and 20 µM) clearly showed that CtsL is the most proficient followed by CtsB and then CtsD (Fig. 2C). After 30 min of incubation, full-length protein was depleted by CtsL at all α-syn_m concentrations. Lower-molecular-weight peptides were only observed at the highest α-syn concentration of 20 µM. Consistent results were also observed for an overnight reaction for 60 µM α-syn (SI Appendix, Fig. S8). LC-MS data taken at different times for the reactions of CtsB and CtsL with α-syn (SI Appendix, Fig. S9 and Tables S6–S8) were used to identify cleavage sites (Fig. 2D). Initial cuts were G14/V15 and A90/A91 for CtsB, M5/K6 and N103/E104 for CtsL, and F4/M5 and F94/V95 for CtsD. Remarkably, all cuts from lysosomal degradation can be assigned to a combination of CtsB, CtsL, and CtsD activity with the exception of K12/E13 and N122/E123 (previously suggested as AEP activity).

Distinct cleavage sites for CtsB (e.g., G14/V15 and A90/A91) and for CtsL (e.g., M5/K6 and N103/E104) affirmed both their presence in the lysosomal extracts. By comparing multiple LC-MS experiments from both lysosomal sources, brain lysosomal extracts harbor relatively more CtsB activity as noted by the significant presence of peptides 1–90 and 91–140. CtsB and CtsL activities were comparable in liver lysosomal extracts (SI Appendix, Fig. S10).

Effect of Lipids on the Lysosomal Activity.

Because membranes modulate α-syn conformation and its propensity to form amyloid (36), the effect of phospholipid vesicles on α-syn degradation by lysosomal extracts were investigated. Two lipids abundant in intraluminal vesicles in lysosomes (37, 38) were used, zwitterionic phosphatidylcholine (PC) and anionic bis(monoacylglycero)phosphate (BMP). Anionic lipids are of particular interest because α-syn prefers to associate with negatively charged membranes in forming α-helical structures (39). Secondary structural changes of α-syn_m were probed by circular dichroism spectroscopy. In solution, α-syn_m is highly disordered with a negative maximum at 198 nm (Fig. 3A, red). As expected, no apparent changes are detected upon the addition of 100% PC vesicles (Fig. 3A, gray lines), whereas spectroscopic signature of double-negative maxima (208 and 222 nm) affirms the formation of α-helical structure in the presence of BMP-containing (30 mol %) PC (PC/BMP; Fig. 3A, blue) vesicles (average diameter, ∼100 nm).

Fig. 3.

Effect of phospholipids on cathepsin degradation of α-syn. (A) Far-UV CD spectra of α-syn_m (5 µM) in the presence of PC (gray) and PC/BMP (yellow to green-to-blue) vesicles at varying lipid concentrations [lipid-to-protein molar ratio (L/P) = 1, 60, 100, 400, and 600] at pH 5 and 37 °C. (B) SDS/PAGE (4–12%) visualized by Coomassie staining showing degradation of α-syn_m (20 µM) by liver lysosomal extract (18 µg) in the presence (Left) and absence (Right) of PC/BMP at an L/P of 60 (where P is α-syn_m) after 20 h at pH 5 and 37 °C. The addition of protease inhibitors PePA and LeuP to lysosomal extracts is also shown. (C) SDS/PAGE (4–12%) of α-syn_m (5, 10, and 20 µM) incubated with PC/BMP at an L/P of 60 in the presence of 0.15 µM CtsB, CtsL, and CtsD for 30 min at pH 5 and 37 °C; α-syn_m (5, 10, and 20 µM) incubated alone is also shown. (D) Schematic representation of the primary amino acid sequence of α-syn_m with identifiable cleavage sites generated from CtsD activity in the presence of PC (magenta and black) or PC/BMP (black) vesicles (SI Appendix, Tables S10 and S11); α-syn membrane binding region is depicted as spanning residues 1–100, whereas C-terminal residues 101–140 are denoted as the acidic region.

The addition of PC/BMP vesicles to α-syn_m [20 μM and lipid-to-protein (L/P) molar ratio of 60] and liver lysosomal extracts yielded slightly less full-length and <12-kDa peptides (Fig. 3B and SI Appendix, Fig. S11A) compared with that in solution alone, suggesting a modest enhancement of degradation. This stimulatory effect is more evident in the data obtained from brain lysosomal extracts (SI Appendix, Fig. S11B). In contrast, the addition of PC vesicles to liver lysosomal extracts and α-syn_m inhibited activity slightly because <12-kDa peptides are more abundant than that of buffer alone (SI Appendix, Fig. S11A). These results show the impact of phospholipids on α-syn conformation and its degradation process. Activity from all three cathepsins was observed by LC-MS analysis (SI Appendix, Table S9).

Effect of Lipids on Individual Cathepsin Activity.

To delineate whether aspartyl and/or cysteine cathepsins are modulated by the presence of PC/BMP vesicles, inhibition experiments were conducted (Fig. 3B). When LeuP is added to inhibit cysteine cathepsins, the degradation of the full length protein is suppressed and most of the lower-molecular-weight bands disappear with the exception of an 8-kDa fragment, which we attribute to CtsD activity. Consistently, lysosomal degradation of α-syn is unchanged upon PePA inhibition of aspartyl protease activity, namely CtsD. These results affirm that cysteine cathepsins are essential in lysosomal degradation of α-syn and indicate that cathepsin activity, especially CtsD, may be enhanced by anionic lipids.

To test the assertion that CtsD is stimulated by lipids, α-syn_m (5, 10, and 20 µM) was incubated with PC/BMP and PC vesicles at an L/P of 60 in the presence of purified CtsB, CtsL, and CtsD (0.15 µM) (Fig. 3C and SI Appendix, Figs. S12–S15). Changes in α-syn_m degradation were noticeable in the presence of PC/BMP vesicles (Fig. 3C). Consistent with the solution data, CtsL remains the most efficient protease in the presence of PC/BMP vesicles; however, CtsD proteolysis of α-syn_m is dramatically stimulated by PC/BMP and is now comparable to CtsB. We note that there is also a small enhancement in CtsB and CtsL degradation because the abundance of <12 kDa peptides was diminished in the presence of PC/BMP.

The observed stimulatory effect of anionic phospholipids on CtsD degradation of α-syn could imply that in vivo CtsD acts on lipid-bound α-syn. Recall that this activity enhancement was revealed when cysteine cathepsin activity was inhibited in lysosomal extracts. Although anionic lipids have been shown to enhance CtsD activity (40), the mechanism of stimulation is not known. In the presence of anionic phospholipids, many more N-terminal cleavage sites were identified in addition to those previously identified in solution for CtsD (Fig. 3D). To evaluate the role of protein conformation, the concentration of PC/BMP was varied (SI Appendix, Fig. S14). Even at a low L/P of 1, where no helicity is detected (Fig. 3A, yellow), increased proteolysis of α-syn_m is still observed (SI Appendix, Fig. S14). Interestingly, whereas increasing the amount of PC/BMP vesicles had little effect on the rate of proteolysis, an enhancement was observed with PC vesicles at higher L/P conditions with prolonged incubation (SI Appendix, Fig. S14). Although most CtsD cleavage sites of α-syn_m were common in the presence of both PC/BMP (SI Appendix, Table S10) and PC (SI Appendix, Table S11), the abundance of these generated peptide fragments were significantly different (SI Appendix, Fig. S15). Interestingly, CtsD prefers to cleave within the C terminus in the presence of PC vesicles and unique sites were observed (Fig. 3D).

We infer from these data that α-syn conformation may be less important than BMP stimulation of CtsD activity. It is plausible that there is a direct modulation of the active site by lipids or perhaps enhancement arises because of an increase in the effective concentration on the membrane surface. More work is clearly needed to discern what factors are involved in the stimulation mechanism of CtsD by anionic lipids.

Cathepsin L Degrades α-syn Fibrils.

Because both CtsB and CtsL efficiently degraded monomeric α-syn, we investigated whether they would also have capabilities in digesting preformed fibrils. For comparison, CtsD was also tested. Remarkably, CtsL degraded α-syn_f (20 µM), whereas both CtsB and CtsD had moderate effects (Fig. 4). Significant losses were observed in both turbidity and ThT fluorescence (Fig. 4 A and B) after 5 h in the presence of CtsL. The amount of proteins that persist is miniscule (Fig. 4C and SI Appendix, Table S12), and no intact fibrils were found by TEM (Fig. 4D). For comparison, TEM image of α-syn_f alone is also shown in Fig. 4D. Although α-syn_f remains in the presence of CtsB, it is noteworthy that TEM images indicate thinner fibrils, suggesting that the enzyme is trimming solvent accessible regions. Supporting this assertion, remaining peptide fragments identified by LC-MS (e.g., 15–114 and 20–114; SI Appendix, Table S13) still retained the PK-resistant core. Similarly, some degradation by CtsD also did occur (Fig. 4B and SI Appendix, Table S14); fragments were identical to those observed with α-syn_m (SI Appendix, Table S2). In contrast to α-syn_m, the inclusion of anionic lipids did not have a dramatic effect on CtsD activity on α-syn_f (SI Appendix, Fig. S16).

Fig. 4.

Effect of cathepsins on α-syn fibril stability. (A) Turbidity measurements (λ_obs = 440 nm) of preformed α-syn fibrils (α-syn_f, 20 µM) incubated with 0.15 µM CtsD (purple), CtsB (green), and CtsL (black) as a function of time at pH 5 and 37 °C. (B–D) Representative ThT fluorescence (error bars represent SD; n = 3) (B), SDS/PAGE (4–12%) (C), and TEM images (D) taken after 20 h of incubation. Scale bars are as indicated. Results for α-syn_f alone are also shown in red.

Our data suggest that only CtsL is capable of disassembling α-syn_f. To test whether there is synergy between the enzymes, we performed experiments using liver lysosomal extracts. Although there was an initial drop in turbidity, no differences were observed with further incubation (SI Appendix, Fig. S17). ThT fluorescence and SDS/PAGE showed similar results relative to the α-syn_f control, indicating little change in the amounts of α-syn_f (SI Appendix, Fig. S17). The change in turbidity could be attributable to some fibril proteolysis but insufficient to change ThT intensity. In accordance, LC-MS analysis shows only minor amounts of α-syn peptides were generated (SI Appendix, Table S15). The addition of lipids did not have a significant effect (SI Appendix, Fig. S17). A concentration dependence of purified CtsL on α-syn_f degradation (SI Appendix, Fig. S18) indicate that results obtained with lysosomal extracts are comparable to those obtained for nanomolar CtsL (<9.4 nM), which are not sufficient to fully digest the fibrils under these experimental conditions.

Discussion

Accumulation of aggregated α-syn is a feature in PD pathogenesis, and therefore enhancement in lysosomal degradation could prove beneficial. Our work points to a direct role for cysteine cathepsins in lysosomal clearance of α-syn. Cysteine cathepsins CtsB and CtsL alone are capable of proteolyzing the central and amyloidogenic region of α-syn. In contrast, the current cathepsin thought to be responsible for lysosomal degradation of α-syn, CtsD, requires the presence of anionic lipids to degrade that same region. Interestingly, CtsL effectively degrades α-syn amyloid fibrils, materials that are closely associated with neurodegeneration. Collectively, this work offers a new perspective on how lysosomes deal with α-syn, where the protein conformational state dictates the role of individual cathepsins (Fig. 5). Specifically, soluble and membrane-bound α-syn is readily digested by both CtsB and CtsL, whereas CtsD is only effective in processing the membrane-bound form. Finally, CtsL would be capable of proteolyzing aggregated α-syn. Whereas CtsD activity is stimulated by anionic lipids in vitro, CtsL is still the most efficient, with CtsD comparable to CtsB activity under these conditions. Even in the presence of anionic lipids, CtsD activity in lysosomal extracts are insignificant compared with that of cysteine cathepsins (Fig. 3B). Thus, we conclude cysteine cathepsins are essential in the lysosomal degradation of α-syn.

Fig. 5.

Schematic representation of lysosomal degradation of α-syn involving cathepsins. In functioning lysosomes, CtsB (green) and CtsL (black) efficiently degrade monomeric α-syn, whereas CtsD can only generate N- and C-terminal truncations (not shown). For CtsD (purple) to have maximum activity, anionic lipids are needed (not shown) and α-syn is membrane-bound. Depicted are intraluminal vesicles with α-syn adopting a highly helical structure (49) upon membrane association. CtsD activity results in degradation of α-syn mainly in the N-terminal region. CtsB and CtsL can also proteolyze membrane-bound α-syn (not shown). When α-syn is in a fibrillar state, CtsL (black) is the only cathepsin capable of fully dissecting these aggregates; α-syn fibrils are shown in a parallel-in-register conformation, with each monomer comprising five β-strands (50).

Cysteine cathepsins have been associated with other neurodegenerative ailments (34), including amyloid disorders such as Alzheimer’s (33) and prion diseases (35). Evidence directly linking these proteases to α-syn has not been demonstrated until now. Interestingly, one study showed an abnormal distribution of CtsL in postmortem human brain tissues of PD patients (41), and in another, treatment with a potent cysteine cathepsin inhibitor, cystatin C, induced insoluble α-syn accumulation in primary cultured neurons from mice (42). Because lysosomal malfunction is associated with PD, we propose that CtsB and CtsL could act as disease modifiers.

The finding that cysteine cathepsins are essential in lysosomal degradation of α-syn is at variance with previous literature reporting CtsD as the main protease involved (21–24). We propose different interpretations of prior literature that might reconcile these differences. First, the reliance on C-terminal α-syn antibodies to monitor degradation of α-syn gives no information regarding the fate of the N-terminal amyloidogenic region because this epitope is lost during CtsD activity. Hence, the conclusion that CtsD is the protease responsible for complete degradation of α-syn needs readdressing. Second, a more indirect role for CtsD is plausible such as CtsD activation of CtsB (43, 44), which then is responsible for α-syn proteolysis. Another indirect mechanism involves CtsD degradation of cystatins (45), inhibitors of cysteine cathepsins, which would enhance CtsB and CtsL activity upon CtsD up-regulation.

This work also reveals an important role for phospholipids in α-syn degradation. As lipid metabolism changes with age and/or with disease (46–48), it is thus conceivable that subtle alterations in phospholipid membrane compositions could impact not only membrane binding of α-syn but also its proteolysis and could thereby enhance α-syn accumulation and aggregation in the lysosome. In a broader context, membranes may also play a role in the turnover of other aggregation-prone proteins. Protein–lipid interactions could have both positive and negative effects in modulating proteolysis efficiency, depending on what peptide regions are exposed to proteases and whether the resulting fragments have reduced or increased aggregation propensity. Finally, membranes themselves could either promote or inhibit protein aggregation, which would further impact proteostasis.

Further investigation is clearly needed to determine whether and to what extent CtsB and CtsL and/or the interplay among the cathepsins is involved in PD. Of particular interest would be the quantification of the physiological concentrations of individual cathepsins as well as their activities through careful proteomics characterization of brain samples of PD patients and healthy individuals. Nevertheless, with the identification of CtsB and CtsL, increasing their activities and/or levels should have positive results toward the clearance of α-syn. This would offer a targeted therapeutic strategy which could have advantages over a general modulation of autophagy. Future in vivo studies modulating CtsB and CtsL will further demonstrate whether cysteine cathepsins would be able to ameliorate α-syn pathology and should provide insights into relevant therapeutic strategies.

Materials and Methods

Details regarding proteins and reagents, experimental methods for lysosomal extract and lipid vesicle preparations, cathepsin activity assays, TEM, gel electrophoresis and immunoblotting, and CD spectroscopy are provided in the SI Appendix. All animal studies were approved by the National Heart, Lung, and Blood Institute (NHLBI) Animal Care and Use Committee (protocols #H-0050 and H-0018).

Fibril Formation.

α-syn was exchanged into pH 5 buffer [20 mM sodium acetate (NaOAc); 50 mM NaCl] using a PD-10 column (GE Healthcare) and filtered through Amicon Ultra 0.5 mL Ultracel 30k membrane centrifugal filters (Millipore) to remove any preformed aggregates before aggregation. Aggregation was performed in sealed clear, polystyrene, 384-well flat-bottom microplates (781185; Greiner Bio-One) containing 40 µL of solution ([α-syn], 50–60 µM) with continuous orbital shaking (1.0 mm, ∼87.6 rpm) at 37 °C using a microplate reader (Tecan Infinite M200 Pro). Turbidity (440 nm) and ThT (10 µM) fluorescence (excitation and emission wavelengths at 415 and 480 nm, respectively) was recorded as a function of time. For experiments with lysosomal extracts and purified cathepsins, buffer also contains 5 mM EDTA and 5 mM DTT.

LC-MS.

Samples (18 µL) were separated using a Vydac 218TP C₁₈ reverse-phase column (2.1 × 50 mm; 5 µm) and an Agilent 1100 series HPLC (Agilent Technologies) coupled to an Agilent G1946D mass selective detector (MSD) equipped with an electrospray ionization interface (Agilent Technologies). Mass spectra were obtained using positive ion mode. The HPLC system and MSD were controlled and data were analyzed using LC/MSD ChemStation software (Rev. A.10.02; Agilent Technologies).

Degradation Reactions of α-syn.

In microcentrifuge tubes (Safe-Lock tubes; 1.5 mL; Eppendorf), α-syn_m (2–60 µM) was incubated with cathepsins (0.10–0.15 µM) in reaction buffer (20 mM NaOAc, 50 mM NaCl, 5 mM DTT, 5 mM EDTA; pH 5) in a total volume of 40 µL and agitated at 300 rpm at 37 °C in a Mini-Micro 980140 shaker (VWR). Reactions were terminated with either PePA (1 µL; 73 µM) or LeuP (1 µL; 100 µM) at various time points. For lysosomal extracts, 18 µg of total protein was used. The same conditions were performed for α-syn_f (20 and 60 µM) reactions. To prepare fibrils, α-syn_m (144 µM) was agitated at 300 rpm in microcentrifuge tubes for 3 d at pH 5 and 37 °C in a Mini-Micro 980140 shaker (VWR). Aggregated samples were centrifuged at 16,100 × g for 10 min, and the supernatant was discarded. Fibril pellets were resuspended in reaction buffer at desired concentration. Fibril disassembly was performed in sealed clear, polystyrene, 384-well flat-bottom microplates (781185; Greiner Bio-one) containing 40 µL of solution ([α-syn_f], 20 and 60 µM) incubated with continuous orbital shaking (1.0 mm; ∼87.6 rpm) at 37 °C using a microplate reader (Tecan Infinite M200 Pro). Turbidity (440 nm) was recorded as a function of time.