Autophagic stress activates distinct compensatory secretory pathways in neurons

Pathogenic mutations in LRRK2 induce kinase hyperactivity, leading to inappropriate phosphorylation of kinase substrates, predominantly organelle-associated Rab GTPases (15), and a resulting disruption in intracellular trafficking pathways (12), including autophagosome maturation in neurons (7, 16). To test our hypothesis that this disruption might lead to a compensatory increase in secretion, we began by focusing on the most common PD-linked mutation in LRRK2, a G2019S missense mutation. We isolated embryonic cortical neurons from wild type and LRRK2^G2019S mice. Conditioned media were collected from cultured neurons over 10 d and EVs were enriched from the harvested media. We tracked the size and concentration of particles secreted using Nanoparticle Tracking Analysis (NTA) and observed a 15% increase in particles in EV samples isolated from LRRK2^G2019S neurons as compared to EV samples isolated from control neurons (Fig. 1 B and C). No significant changes in size distribution were detected, with the most commonly detected particle size being 135 nm in wild type and 145 nm in LRRK2^G2019S samples (Fig. 1F). Several shoulder peaks were also detected in our analysis of both wild type and LRRK2 particles, suggesting the release of multiple vesicle populations over a range of sizes (Fig. 1C).

To extend this observation, we examined EV secretion from human iPSC-derived neurons [KOLF2.1J (33)] harboring a distinct PD-causing mutation, LRRK2^R1441H. This mutation results in a more profound hyperactivation of the LRRK2 kinase as compared to G2019S, leading to more profound deficits in autophagosome trafficking (15, 16). Again, we observed significantly more particles released from LRRK2^R1441H neurons as compared to isogenic control neurons (a 42% increase) (Fig. 1D), with no change in size distribution detected between the genotypes (Fig. 1F). While this is a cross-species comparison, the effect size on secretion appears to correlate with the extent of kinase hyperactivation.

To confirm these findings, we used CalceinAM to assay intact vesicles collected from WT and LRRK2^G2019S neurons following EV isolation via ultracentrifugation (34). CalceinAM fluoresces after passively entering an EV that is unpermeabilized and thus the fluorescent signal corresponds to intact, membrane-enclosed structures. Consistent with our NTA results, we observed significantly more CalceinAM puncta in samples collected from LRRK2^G2019S mutant neurons (Fig. 1 G and H), indicating increased secretion of extracellular vesicles. Thus, in both mouse and human neurons, hyperactive LRRK2 induces increased secretion of extracellular vesicles.

Extracellular vesicles serve a variety of purposes (35, 36), including waste disposal (23, 37) and transcellular signaling (38), and therefore may contain a wide range of internalized cargos and transmembrane proteins. To characterize the released vesicles, we first performed quantitative proteomic analysis of isolated vesicles from five independent cultures of control and of LRRK2^G2019S primary cortical neurons. Extracellular vesicles were enriched via ultracentrifugation to broadly capture released EVs. The protein concentration of isolated EVs was normalized across samples prior to quantitative mass spectrometry analysis (SI Appendix, Fig. S1 A and B↗). Detected proteins were assigned an expression value based on the number of detected reads within a given sample (median expression value = 0). Vesicle enrichment for both genotypes was confirmed by comparison to previous EV proteomic analysis that characterized proteins found in either small or large vesicle fractions (39) (Fig. 2A and SI Appendix, Fig. S1C↗). We noted the presence of 89/100 of the most common EV-associated proteins, which were readily detected above the median detection threshold in both control and LRRK2 samples (expression value > 0) (40) (Fig. 2E and SI Appendix, Fig. S1D↗). Next, we compared the expression of detected proteins associated with either small EVs (Fig. 2B) or large EVs (Fig. 2C) across genotypes. While we detected no change in the average expression value of small-EV associated proteins, we did detect significantly higher expression values for large-EV associated proteins in LRRK2^G2019S samples, suggesting that large EVs were likely enriched in these samples.

Ontology analysis of the secretomes of LRRK2 ^G2019S and wild type confirmed that proteins associated with secretion, including those associated with exosome formation, e.g., multivesicular body (MVB) proteins, were readily released (Fig. 2D and SI Appendix, Fig. S1E↗). Interestingly, we noted that proteins associated with synaptic function (e.g., Synapsin-1), mitochondria (e.g., TOMM40), and autophagy (e.g., GABARAP) were also enriched in the secretomes from both LRRK2 ^G2019S and wild type neurons (Fig. 2D and SI Appendix, Fig. S1E↗). Recently, we used proteomic analysis to define the basal cargos of neuronal autophagosomes and detected both synaptic proteins and mitochondria as enriched autophagosome cargo (41). Given this commonality, we reasoned that these neurons could be releasing a population of autophagic vesicles via secretory autophagy. To test this possibility, we compared all detected proteins in EVs isolated from LRRK2^G2019S or control neurons to the previously characterized proteome of autophagosomal cargos from the brain (41). In both cases, over 70% of known autophagic cargos from the brain were detected within our EV proteomic datasets (Fig. 2F).

If wild type and LRRK2^G2019S neurons are releasing the inner autophagosome vesicle, members of the ATG8 family, well-established markers of autophagosomes, should be detected among the secreted proteins. Consistent with this, nearly all secretory autophagy pathways have reported the release of the autophagic protein LC3B (17, 21, 23, 25, 26), a member of the ATG8 family. In line with previous work, we detected 5 of 6 ATG8s in both the LRRK2^G2019S and control samples, with the highest detected being LC3B (Fig. 2E and SI Appendix, Fig. S1D↗). Thus, unbiased proteomic profiling of the secretomes from either control or LRRK2^G2019S primary cortical neurons detected markers associated with multiple classes of extracellular vesicles, including exosomes and secreted autophagic vesicles.

To determine how the secretory proteome was altered in LRRK2^G2019S mutants, we performed differential expression analysis and observed 250 upregulated proteins and 80 downregulated proteins in samples from LRRK2^G2019S neurons as compared to control neurons (Fig. 2G). Ontology analysis of the differentially expressed proteins suggests that mitochondrial proteins are the most prominent cargo upregulated in LRRK2^G2019S EVs (SI Appendix, Fig. S1F↗). Indeed, 92/250 upregulated secreted proteins are classified as mitochondrial by MitoCarta3.0 (42). This observation could indicate that secretory autophagy is upregulated by LRRK2^G2019S neurons as 20% of all neuronal autophagosome cargo is mitochondrially derived (41). The contents of LRRK2^G2019S autophagosomes are largely unchanged from wild type, with mitochondria being readily targeted for autophagosome engulfment (43). Further, while autophagosome maturation is impaired in LRRK2^G2019S neurons, overall numbers of autophagosomes are not significantly impacted. Thus, our findings suggest that LRRK2^G2019S neurons, which exhibit autophagosome maturation defects, may be redirecting unacidified autophagosomes toward extracellular release.

In addition to proteins, many extracellular vesicles contain RNAs, including miRNAs, to mediate transcellular communication. We therefore performed unbiased small RNA (smRNA) sequencing of isolated vesicles from both LRRK2^G2019S and wild type neurons. Secreted extracellular vesicles were isolated via ultracentrifugation followed by RNA extraction, smRNA library preparation, and subsequent sequencing of the normalized libraries. In EVs isolated from both wild type and LRRK2^G2019S, miRNAs were detected, with 391 and 499 miRNAs reaching our detection threshold (average of at least two reads across five samples), respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the downstream targets of the top 25 miRNAs detected in EVs isolated from both wild type and LRRK2^G2019S neurons confirmed that the secreted vesicles were likely mediating transcellular communication (Fig. 2H). Many signaling pathways, including those that impact inflammatory responses, e.g., MAPK, were enriched in both the wild type and LRRK2^G2019S secretome (44). In both genotypes, 4 of the top 5 detected miRNAs were of the let-7 miRNA family (Fig. 2I), which play a role in many processes, including autophagy and inflammation (45, 46). Thus, both wild type and LRRK2^G2019S neurons are offloading miRNAs which could be relevant to neuronal homeostasis. We next asked whether any miRNAs were differentially expressed, and detected no miRNAs significantly upregulated in either genotype (SI Appendix, Fig. S2A↗). While no individual miRNA was significantly upregulated in LRRK2 EVs, the average levels of the top 50 detected miRNAs were all elevated in LRRK2 compared to wild type (Fig. 2I and SI Appendix, Fig. S2B↗). Together, our immunoblots and transcriptomics results argue that it is the overall quantity, and not the contents, of released vesicles that are altered in LRRK2^G2019S neurons.

To identify the class(es) of extracellular vesicles being offloaded from LRRK2^G2019S neurons, we performed differential ultracentrifugation of conditioned media to separate EV populations based on density. Conditioned medium was collected and then subjected to a 500× g spin to remove cellular debris and apoptotic bodies (SI Appendix, Fig. S3A↗). Large extracellular vesicles, including microvesicles and secreted autophagic vesicles, were enriched via a 20,000× g spin (large EVs/P20), followed by sequential enrichment of small EVs, including exosomes, via a 100,000× g spin (small EVs/P100). Large (P20) and small (P100) EV pellets were isolated and analyzed by immunoblot using antibodies against known EV-associated transmembrane proteins and cargos (Fig. 3A). Given that our proteomics results closely mirrored previous proteomic profiling of brain autophagosomes, we first probed for LC3/ATG8 which is offloaded in many secretory autophagy pathways. LC3-I is conjugated to phosphatidylethanolamine to form LC3-II, which marks the forming autophagosome and stays associated throughout autophagosome maturation (47, 48). Across both genotypes, the majority of both LC3-I and LC3-II was detected in the large EV/P20 fraction. Of note, we observed significantly more LC3-II in EVs isolated from LRRK2^G2019S neurons as compared to control neurons in the P20 fraction (2.1-fold increase, P = 0.0089) (Fig. 3 A and B).

Most extracellular vesicles are enriched for a family of transmembrane proteins, the tetraspanins, which form microdomains on vesicles important for biogenesis and cargo sorting. We compared the expression of CD81 and CD63, the two most highly enriched tetraspanins on exosomes (49), on EVs isolated from control vs. LRRK2^G2019S neurons and observed no detectable CD81 or CD63 in the large EV/P20 fraction. Thus, the large LC3-II+ vesicles being secreted from LRRK2 mutant neurons lack tetraspanins, an observation consistent with their identification as secreted autophagic vesicles. Strikingly, in the P100 fraction enriched for small EVs, we observed significantly more CD81 (8.4-fold increase) and CD63 (2.6-fold increase) in EVs released from LRRK2 mutant neurons as compared to wild type neurons (Fig. 3 C and D). Changes in the relative enrichment of these proteins were not due to changes in protein expression as no significant changes were observed by immunoblot analysis of neuronal lysates (SI Appendix, Fig. S3 C and D↗).

Together, these observations suggest that two populations of EVs are upregulated in LRRK2 mutants: 1) larger EVs that are positive for LC3-II but lack canonical tetraspanins and 2) small EVs enriched for CD63 and CD81. Given our proteomic observations suggesting that autophagic cargo are expelled from LRRK2 neurons and previous literature that has found CD63 expression largely restricted to exosomes, we propose that these two EV populations (large EVs and small EVs) represent the SLAM-EVs and exosomes, respectively.

To provide additional insight into the nature and function of the two populations of secreted vesicles detected, we used immunoblotting to compare cargos associated with either SLAM-EVs or exosomes. Given the size and density of autophagosomes (50), we first asked whether known autophagosome cargos were enriched in our P20 fraction. Previously, our lab defined the contents of neuronal autophagosomes under basal conditions and found that both synaptic proteins, including Synapsin-1, and mitochondria enriched in the nucleoid marker Transcription Factor A, Mitochondrial (TFAM) are targeted to autophagosomes under basal conditions (41). More recently, we found that several cargos are increased in autophagosomes isolated from the brains of mice expressing mutant LRRK2, including the RNA-binding protein, Heterogeneous nuclear ribonucleoprotein K (hnRNPK) (43). hnRNPK has been shown to interact with LC3 and is secreted within EVs released via LC-3- Dependent Extracellular vesicle Loading and Secretion (LDELS) (26). Consistent with our model that hyperactive LRRK2 activity induces the release of secreted autophagic vesicles, we saw significant increases in the release of Synapsin-1 (1.8-fold increase), TOMM40 (3.6-fold increase) TFAM (3.2-fold increase), and hnRNPK (5.9-fold increase) in the LRRK2 P20 pellet as compared to controls when measured by immunoblotting (Fig. 3 E–H). In contrast, these proteins were minimally detected in the P100 fractions from either LRRK2^G2019S or wild type neurons.

Exosomes originate in the MVB, where different contents, e.g., ESCRT proteins and RNAs, are loaded into small vesicles before being released into the extracellular space (51). We tracked the expression of the ESCRT protein and canonical EV cargo, TSG-101, and observed a 3.5-fold increase in the small EV/P100 fraction in LRRK2^G2019S mutants (Fig. 3I). In contrast, we observed no significant change in enrichment of TSG-101 in the P20 fraction corresponding to large EVs. Exosomes are very small and thus contain limited cargo, often enriched for miRNA and mRNA (51, 52). Our smRNA transcriptomic analysis confirmed that miRNAs were released by both wild type and LRRK2^G2019S neurons. To determine whether RNAs were contained within secreted exosomes, we asked whether P100 fractions isolated from wild type or LRRK2^G2019S neurons contained RNA. We used the RNA specific dye, SYTO RNASelect (Thermo), to measure relative levels in fluorescence across our different conditions and observed that RNA could be readily detected in both wild type and LRRK2^G2019S P100 fractions (SI Appendix, Fig. S2C↗), suggesting that RNAs are likely cargos within released exosomes.

Our initial strategy to isolate large EVs/P20 and small EVs/P100 via ultracentrifugation allowed for a crude enrichment of secreted autophagic vesicles and exosomes, respectively, but contain a diverse range of vesicles and secreted organelles. To confirm that the predicted autophagic cargos (Synapsin-1, TOMM40, TFAM) were in the same vesicle population as secreted LC3, we next performed a buoyant linear density gradient to further subfractionate our two vesicle populations. Isolated large EVs/P20 or small EVs/P100 were bottom loaded onto a linear iodixanol gradient followed by an overnight spin at 180,000× g spin to separate vesicles by density (Fig. 3J). 12 individual fractions were collected and then concentrated by dialysis before immunoblotting. We observed that LC3 was detected in fractions 3-9 (density range: 1.020ρ to 1.158ρ), confirming that LC3 containing vesicles were buoyant and likely diverse (Fig. 3K). We next looked at the predicted autophagosome cargos and found that both Synapsin-1 (density range:1.067ρ to 1.123ρ) and mitochondrial markers, TOMM40 and TFAM (density range: 1.123ρ to 1.158ρ), were detected in overlapping fractions (Fig. 3K). Interestingly, TOMM40 and TFAM were detected in a more limited density range, suggesting SLAM-EVs containing mitochondria may be more dense. These results suggest a diverse class of LC3 containing vesicles that range in density, likely due to the enclosed cargos. To confirm we are isolating exosomes, we also performed a buoyant linear density of our P100 fraction and observed the majority of CD63 and CD81 signal at 1.083ρ to 1.123ρ, consistent with previous reports (53) (SI Appendix, Fig. S3B↗). Thus, we identified two vesicle populations, secreted autophagic vesicles, or SLAM-EVs, and exosomes, both of which are upregulated in LRRK2^G2019S neurons (Fig. 3L).

While the release of IL-1β, which resides between the interluminal space of the double membrane autophagosome, has been characterized (19), the fate of the inner vesicle upon this release is largely speculative. We propose that this inner vesicle is released extracellularly as a single-membrane vesicle we have termed a SLAM-EV. Consistent with this model, a recent study found that neuronal LC3 can transfer to nearby astrocytes in the absence of cell–cell contact, suggesting a secretory transfer mechanism (54). We therefore wanted to confirm that both the autophagy marker LC3 and known cargos of autophagy, e.g., the mitochondrial protein TOMM40, were indeed contained within these extracellular vesicles. We treated the large EVs/P20 fraction with Proteinase K (ProtK) to determine which proteins were membrane protected (Fig. 4A). Nearly all LC3II (95%) was still detectable following ProtK treatment, suggesting this marker is membrane-protected within an intact vesicle, consistent with a topology for SLAM-EVs in which this marker is enriched in the inner leaflet of a single-membrane vesicle (20) (Fig. 4 B and C). We next probed for known autophagic cargos (e.g., Synapsin-1, mitochondrial proteins). We observed a significant fraction of Synapsin-1 that was membrane protected (64%), consistent with the majority of this protein being vesicle enclosed within SLAM-EVs (Fig. 4 B and C). Interestingly, using markers for both outer (TOMM40) and inner mitochondrial proteins (HSP60), we observed a smaller protected fraction (30%), suggesting that there is a population of secreted mitochondria that are not vesicle protected and thus not ProtK-resistant (55) in addition to a membrane-protected subfraction. To further characterize SLAM-EVs, we performed transmission electron microscopy on the large EVs/P20 fraction. We identified single membrane vesicles of similar size to autophagosomes (~1 to 1.5 micron diameter). Within these vesicles, we could identify round, electron- dense structures reminiscent of mitochondrial fragments observed within autophagosomes isolated from the mouse brain (Fig. 4D) (41). Together, our results suggest a subset of released mitochondria are located within SLAM-EVs, representing a mechanism of mitochondrial release.

The R-SNARE protein, SEC22B, associates with autophagosomes to mediate fusion with the plasma membrane and is required for the extracellular release of IL-1β (50, 56). We therefore asked whether SEC22B was required for the release of SLAM-EVs in LRRK2^G2019S neurons. We used shRNA to knockdown Sec22B expression (65% knockdown) and then collected large EVs/P20. Upon Sec22B knockdown, we observed a significant loss of both LC3II (71% reduction) and Syn-1 (68% reduction), consistent with SEC22B being required for secretory autophagy (Fig. 4 E and F). Interestingly, we observed a significant, but smaller loss of secreted TOMM40 (39% reduction) upon Sec22B knockdown (Fig. 4 E and F). This finding complements our previous result that a subset of secreted mitochondria is contained within SLAM-EVs, while other mitochondria or mitochondrial fragments are secreted via a SEC22B-independent pathway.

ATG8 family members (LC3/GABARAP) are stably associated with both the inner and outer membrane of mature autophagosomes. In canonical secretory autophagy, the outer LC3/GABARAP membrane fuses with the plasma membrane to release both lumen contents and the inner autophagosome vesicle (57). If autophagosome content is secreted in this manner from the cortical neurons examined here, LC3 should be detected at the cell surface upon vesicle fusion. We transfected wildtype and LRRK2^G2019S neurons with fluorescently labeled LC3 and tracked expression at the cell surface using live TIRF microscopy. Consistent with secretory autophagy events, we observed frequent LC3 bursts at the cell surface in both control and LRRK2^G2019S neurons (Fig. 4G). We quantitated presumed secretory autophagy events by identifying stationary bursts of fluorescence that decayed over 10 to 120 s, similar to other SNARE mediated temporal dynamics (58) (Fig. 4I). We detected significantly more LC3 release events in LRRK2^G2019S neurons (Fig. 4H and Movies S1↗ and S2↗), with no change in the duration of events (SI Appendix, Fig. S4B↗). Interestingly, we noted that LC3 secretion events were spatially enriched within a somatodendritic compartment which often extended into a single tapered process, consistent with the primary dendrite (Fig. 4G and SI Appendix, Fig. S4A↗). Morphological characterization confirmed that in ~70% of neurons imaged, LC3 secretion events could be observed at the presumed primary dendrite. We transfected neurons with the AIS marker, Ankyrin G (AnkG), and confirmed that the LC3 release events were excluded from the axon (SI Appendix, Fig. S4A↗). Thus, LC3 secretion events are spatially restricted to the somatodendritic compartment. This observation mirrors previous observations from live cell imaging indicating that once axonal autophagosomes enter the soma, they are constrained to the somal and dendritic regions (5). Here, we find that in LRRK2^G2019S neurons there is an upregulation in the release of inner autophagic vesicles as SLAM-EVs that contain diverse contents, including synaptic proteins and mitochondria, at these sites (Fig. 4J).

Our immunoblotting results suggest that small EVs containing both CD81 and CD63 are also secreted from LRRK2^G2019S neurons. As immunoblotting relies on the pooling of EVs to look for global changes, we next used ExoView (Spectradyne) to track tetraspanin expression at the level of individual EVs. We captured EVs from both wild type and LRRK2^G2019S neurons using an anti-CD81 antibody followed by subsequent antibody detection against CD81, CD9, and CD63 (Fig. 5A). Consistent with our earlier observations, more CD81+ EVs were released from LRRK2^G2019S neurons compared to wild type neurons (Fig. 5B). Additionally, we detected more CD81+/CD63+, and CD81+/CD9+ dual-labeled vesicles captured from LRRK2^G2019S EVs (Fig. 5B). These observations corroborated our immunoblotting results, where we observed substantial CD81 and CD63 release from LRRK2^G2019S neurons, consistent with the release of exosomes.

CD63 is a commonly used marker of exosomes (51, 59). Given our immunoblot results that CD63 is significantly enriched in the small EV/P100 fraction from LRRK2^G2019S neurons, we sought to better characterize the secretion of CD63 positive EVs using live cell imaging. We expressed the pH sensitive fluorophore, pHluorin, tagged to CD63 (CD63-pHluorin) in primary cortical neurons isolated from wild type or LRRK2^G2019S mice. The modified GFP signal of CD63pHluorin is quenched in acidic environments such as the MVB, and is visible upon fusion with the extracellular membrane for EV release (Fig. 5C). We measured CD63-pHluorin signal in control and LRRK2^G2019S neurons using TIRF microscopy. New fusion events could be identified by tracking sudden bursts of fluorescence that persisted 10 s to 150 s (Fig. 5 D–G and SI Appendix, Fig. S4C↗). We observed a 2.2-fold increase in fusion events in LRRK2^G2019S neurons compared to wild type neurons suggesting that exosomal secretion is upregulated in LRRK2^G2019S neurons (Fig. 5D and Movies S3↗ and S4↗). Additionally, this upregulation of CD63 secretion events is dependent on LRRK2 hyperactivity as pretreatment of neurons with MLI-2, a selective LRRK2 kinase inhibitor (60), blocked the observed increase of CD63 fusion events (Fig. 5D). In contrast to the LC3 secretion events described above, CD63-positive release events were primarily observed on the cell soma with no clear spatial clustering near or at the primary dendrite, although occasionally fusion events could be observed on neurites. We confirmed that these events represented exosomes as application of the exosome inhibitor, GW4869, significantly reduced CD63-pHluorin events (Fig. 5H). GW4869 blocks the release of exosomes via inhibition of Neutral Sphingomyelinase, a critical component for the budding of intraluminal vesicles into the lumen of the MVB (61).

In addition to the bursts of CD63-pHluorin signal that rapidly decayed, we also noted bursts in fluorescence that persisted for minutes, suggesting that CD63 could also be tethered to or integrated into the plasma membrane upon MVB fusion as well as released (SI Appendix, Fig. S5 A–E↗). We tracked these longer events and observed a fourfold increase in fusion events in LRRK2^G2019S neurons compared to wild type neurons; this difference was rescued by pretreatment of the neurons with MLI-2 (SI Appendix, Fig. S5F↗), indicating this phenotype was due to hyperactive LRRK2 activity. To determine whether human neurons expressing the LRRK2^R1441H mutation also upregulate the release of CD63+ exosomes, we performed live cell imaging of LRRK2^R1441H and isogenic control iPSC-derived neurons expressing CD63-pHluorin. Again, we observed significantly more CD63-pHluorin events in LRRK2^R1441H neurons compared to wild type neurons (SI Appendix, Fig. S5G↗). Thus, hyperactive LRRK2 promotes the secretion of CD63+ EVs in both mouse and human neurons.

While the majority of secreted LC3 was detected in our P20 fraction, we also could detect a significant increase in secreted LC3-II in the LRRK2^G2019S P100 fraction in a direct comparison (P = 0.0286). Several groups have demonstrated that LC3/ATG8 lipidation and the LC3-conjugation machinery are critical for several secretory autophagy pathways, including LDELS (26), secretory autophagy during lysosomal inhibition (SALI) (25), and apilimod-mediated secretion of exosomes (21). To determine whether the LRRK2-mediated secretion of exosomes was similarly dependent on LC3 conjugation, we knocked down Atg7 using siRNA (SI Appendix, Fig. S5H↗) and tracked exosome release using CD63-pHluorin in LRRK2^G2019S neurons. We determined that ATG7 activity was required for LRRK2 mediated exosome release as we observed a 60% reduction in the number of CD-63-pHluorin secretion events in neurons expressing the Atg7 siRNA (SI Appendix, Fig. S5I↗), suggesting that LC3 conjugation was required for the increased exosomal release induced by hyperactive LRRK2.

Does the release of exosomes impact neuronal survival? Recently, it has been observed that augmenting exosomal secretion can extend lifespan in several ALS models. To determine whether the observed exosomal release was beneficial to LRRK2^G2019S expressing neurons, we treated DIV7 primary neurons with increasing doses of the exosome inhibitor GW4869. Following 2 h of GW4869 exposure, we tracked levels of activated Caspase 3/7. In wild type neurons, short-term, low doses of GW4869 failed to increase the percent of cells with activated Caspase 3/7, suggesting these cells were largely insensitive to the inhibition of exosomal secretion (Fig. 5 I and J). In contrast, LRRK2^G2019S neurons exposed to 5 μM GW4869 exhibited a dramatic increase in Caspase 3/7 activation (13% at baseline vs. 53% at 5 μM) (Fig. 5 I and J). This observation suggests that LRRK2^G2019S neurons rely on exosomal secretion for survival and are thus more sensitive to exosomal inhibition. In contrast, blocking the release of microvesicles using the drug Y27632, a competitive inhibitor of Rho-associated protein kinases ROCK1 and ROCK2, which play a key role in actin filament organization during microvesicle shedding (61, 62). We found that Y27632 treatment had no impact on cell survival in control or LRRK2^G2019S neurons (SI Appendix, Fig. S6 A and B↗). Thus, we find that LRRK2 hyperactive activity promotes the compensatory release of exosomes in neurons, and this release promotes neuronal survival under stress.

In LRRK2^G2019S neurons, autophagic degradation is chronically strained (7, 16). Previous reports suggest that an acute prevention of autophagosome degradation via Bafilomycin A 1 (BafA1) (63, 64) can also initiate the upregulation of secretion in several different cell types (65, 66). To determine whether hyperactive LRRK2 was promoting secretion of EVs via a similar mechanism as BafA1, we tracked secretion in neurons treated for 2 h with increasing concentrations of BafA1 (0 nM, 50 nM, 500 nM). We observed significantly more particles secreted by BafA1 treated neurons as measured via NTA (SI Appendix, Fig. S6 A and B↗). We next isolated Large EVs/P20 and Small EVs/P100 released from DMSO and BafA1 treated neurons for immunoblot analysis (SI Appendix, Fig. S6C↗). We observed that BafA1 treatment significantly increased the release of CD81 (2×-fold increase) in small EVs (SI Appendix, Fig. S6D↗), but, in contrast to LRRK2^G2019S neurons, CD63 was not elevated following BafA1 (SI Appendix, Fig. S6E↗). BafA1 treated neurons did release more TSG101 selectively within large EVs (SI Appendix, Fig. S6F↗). In contrast to LRRK2^G2019S, BafA1 treatment resulted in no change in LC3II levels in any fraction (SI Appendix, Fig. S6G↗), suggesting BafA1 does not prompt the release of SLAM-EVs. These results reveal distinct differences between BafA1-mediated secretion and the enhanced secretion induced by mutant LRRK2 expression in neurons.

As we observed no change in CD63 levels and only moderately increased CD81 signal, we asked whether BafA1 treatment promoted the secretion of the tetraspanin, CD9. We tracked CD9-pHluorin fusion events in primary cortical neurons and observed significantly more fusion events in neurons following BafA1 incubation (SI Appendix, Fig. S6 H and I↗). In contrast, LRRK2^G2019S neurons exhibited no change in CD9-pHluorin events compared to wild type (SI Appendix, Fig. S6J↗). Thus, while we observe that hyperactive LRRK2 activity initiates the release of two EV classes, exosomes and SLAM-EVs, BafA1 mediates the release of a distinct EV population.

Our studies on neurons in vitro identified two secretory pathways that are upregulated in both primary mouse cortical neurons and human iPSC-derived glutamatergic neurons expressing PD-associated mutations that induce hyperactive LRRK2 activity. To determine whether each of these pathways were also upregulated in vivo, we compared levels of relevant vesicle markers in plasma isolated from 1 y old LRRK2^G2019S and wild type mice.

First, we asked whether overall secretion from neurons was elevated by measuring circulating levels of neuronal protein, BIII-tubulin (SI Appendix, Fig. S7 C↗). We detected significantly more BIII-tubulin in LRRK2^G2019S plasma compared to wild type plasma (Fig. 6A), demonstrating increased neuronal secretion in LRRK2^G2019S neurons in vivo. Consistent with a prior report, we also observed more TSG101 in plasma from LRRK2^G2019S mice, evidence that overall secretion is elevated by hyperactive LRRK2 activity (Fig. 6B). In primary cultured neurons, both CD81 and CD63 were secreted by LRRK2^G2019S neurons. In isolated plasma, we observed a significant increase in CD81 in LRRK2^G2019S plasma (Fig. 6C), but failed to detect any change in CD63 (SI Appendix, Fig. S7 C and D↗). Together, these observations are consistent with neuronal upregulation of secretion and an upregulation of exosomal release in LRRK2^G2019S animals. We next asked whether upregulation of SLAM-EVs could be detected in LRRK2^G2019S animals. We tracked circulating levels of the neuronal autophagy cargo Synapsin-1 and found significantly more circulating Synapsin-1 in LRRK2^G2019S plasma (Fig. 6D). Thus, it is likely that both the exosomal pathway and secretory autophagy pathway are upregulated in LRRK2^G2019S animals.

Primary cortical neurons were isolated and cultured as previously described (16). iPSC lines were differentiated into cortical- like neurons and as previously described (16, 33). Details regarding ultracentrifugation parameters, density linear gradient subfractionation, proteinase K treatment, proteomic characterization, and smRNA characterization are listed in the SI Appendix, Materials and Methods↗.

Electron microscopy was performed by the University of Pennsylvania EM core. All live-cell imaging experiments were captured with a Perkin-Elmer Ultra VIEW Vox microscope fitted with a Visitron Orbital Ring-TIRF arm. All analysis was done blinded using FIJI.