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By Ying Xue Xie, Nima N. Naseri, Jasmine Fels, Parinati Kharel, Yoonmi Na, Diane Lane, Jacqueline Burré, and Manu Sharma

Excerpt from the article published in Nature Communications 13, 4918, 22 August 2022, DOI: https://doi.org/10.1038/s41467-022-32625-1

Editor’s Highlights

• The release of pathogenic aggregates of the neuronal protein α-Synuclein (αSyn) into the extracellular space instigate the neuron-to-neuron transmission and spread of αSyn pathology in synucleinopathies including Parkinson’s disease.
• Multiple cellular pathways have linked PD and αSyn aggregation with lysosomal storage disorders.
• The aggregates/fibrils of αSyn could form inside the acidified lumen of lysosomes, e.g., from monomers taken up via chaperone-mediated autophagy or macroautophagy of synaptic vesicles
• The accumulation of pathogenic αSyn species in neuronal lysosomes, and pinpoint to lysosomal exocytosis as a pathway of release for these pathogenic αSyn species into the extracellular milieu.
• This pathway, at first sight, appears to have relevance to the release of undegraded aggregates of other proteins as well that transmit pathology from cell-to-cell.

Abstract

Considerable evidence supports the release of pathogenic aggregates of the neuronal protein α-Synuclein (αSyn) into the extracellular space. While this release is proposed to instigate the neuron-to-neuron transmission and spread of αSyn pathology in synucleinopathies including Parkinson’s disease, the molecular-cellular mechanism(s) remain unclear. To study this, we generated a new mouse model to specifically immunoisolate neuronal lysosomes, and established a long-term culture model where αSyn aggregates are produced within neurons without the addition of exogenous fibrils. We show that neuronally generated pathogenic species of αSyn accumulate within neuronal lysosomes in mouse brains and primary neurons. We then find that neurons release these pathogenic αSyn species via SNARE-dependent lysosomal exocytosis. The released aggregates are non-membrane enveloped and seeding-competent. Additionally, we find that this release is dependent on neuronal activity and cytosolic Ca²⁺. These results propose lysosomal exocytosis as a central mechanism for the release of aggregated and degradation-resistant proteins from neurons.

Introduction

Cytoplasmic aggregates of the synaptic protein α-synuclein (αSyn) are a characteristic feature of multiple neurodegenerative diseases termed “synucleinopathies”, including Parkinson disease (PD). Over the course of these age-linked diseases, αSyn assembles into amyloid-type β-sheet-rich aggregates, depositing as Lewy bodies and/or Lewy neurites within neurons^1,2,3. The neuro-anatomical propagation of αSyn pathology is highly stereotyped, and has been defined well-enough to be used in staging PD—originating in the medulla and olfactory bulb, then advancing to the midbrain and basal forebrain, followed by “spread” to the neocortex^4,5. This observation, combined with detection of extracellular αSyn in human cerebrospinal fluid⁶, led to the original conjecture that pathogenic species of αSyn may be transmitted neuron-to-neuron via prion-like permissive templating. Subsequently, in PD patients who had received fetal neuron transplants, appearance of Lewy pathology in the non-diseased grafted neurons also pointed to the transmission of αSyn pathology from the surrounding PD-affected neurons^7,8. These observations were followed by animal studies which confirmed either neuron-to-neuron transmission of αSyn pathology⁹, or induction of intraneuronal αSyn pathology by an inoculum of extracellular αSyn fibrils^10,11.

However, unclarity persists in how the cytosolic aggregates are conveyed into the extracellular milieu: Proposed pathway(s) include secretion of αSyn monomers, oligomers and/or larger aggregates inside extracellular vesicles—such as exosomes^12,13,14, or microvesicles¹⁵—which have been categorized collectively as “unconventional secretion”. Other proposed mechanisms include synaptic vesicle release¹⁶, or exocytosis of other types of vesicles^17,18,19, exophagy²⁰, membrane translocation²¹, or trafficking of lysosome-associated aggregates through tunneling nanotubes^22,23. Yet, these studies have not identified a neuronal organelle that first accumulates and then secretes the pathogenic αSyn aggregates generated within the neurons, resulting in the current mechanistic gap.

Interestingly, PD is associated with mutations in the glucocerebrosidase gene, which cause Gaucher disease, the most common lysosomal storage disorder^24,25. In addition, multiple cellular pathways have linked PD and αSyn aggregation with lysosomal storage disorders^26,27.

Here, we show that neuronally-produced pathogenic αSyn species accumulate within the neuronal lysosomes in mouse brains and in primary neurons, and that these seeding-competent species are then released into the extracellular space via SNARE-dependent lysosomal exocytosis.

Results

Pathogenic species of αSyn accumulate within the lysosomes of transgenic αSyn^A53T mouse brains

Rapid neurodegeneration and synapse loss in cysteine string protein-α knockout mice (CSPα^−/−) is rescued by transgenic expression of human αSyn carrying the familial PD mutation A53T (Tg-αSyn^A53T)²⁸. However, beyond 5 months, the rescued Tg-αSyn^A53T/CSPα^−/− mice begin exhibiting neurodegeneration due to the transgenic overexpression of αSyn^A53T. Curiously, we found that Tg-αSyn^A53T/CSPα^−/− mice have accelerated accumulation of the pathogenic versions/species of αSyn compared to Tg-αSyn^A53T/CSPα^+/− mice, detectable by antibodies against phospho-Ser129 αSyn, filamentous αSyn, and amyloid-type folds (Supplementary Fig. S1). Meanwhile, there was no change in monomeric αSyn levels (Supplementary Fig. S1). CSPα^+/− mice have normal CSPα function²⁹. This suggested that brains of Tg-αSyn^A53T/CSPα^−/− mice are collecting αSyn aggregates faster in the absence of CSPα.

Loss-of-function mutations in CSPα cause the lysosomal storage disorder adult-onset neuronal ceroid lipofuscinosis (ANCL) or Kufs disease^30,31,32,33. Specifically, loss of CSPα function leads to progressive accumulation of lysosomes containing undegraded material such as lipofuscin/residual bodies in ANCL patient brains³⁰, in CSPα^−/− mouse brains²⁹, in Drosophila models carrying the ANCL mutations³⁴, and in ANCL patient-derived induced neurons³⁵. Tg-αSyn^A53T/CSPα^−/− mice also accrue lysosomal proteins such as LAMP1 and cathepsin-L, as well as ATP5G—a mitochondrial protein characteristically stored in lysosomes of CSPα loss-of-function models and ANCL patient neurons (Supplementary Fig. S1). The enhanced accumulation of lysosomal contents in Tg-αSyn^A53T/CSPα^−/− brains, together with the exaggerated buildup of αSyn aggregates in these brains, plus the association of lysosomal dysfunction and lysosomal storage disorders with PD^24,25,26,27, led us to investigate whether neuronally-produced αSyn aggregates deposit within the lysosomes.

We next aimed to determine whether αSyn aggregates accumulate within lysosomes, even in the absence of CSPα^-/--driven lysosomal pathology. Thus, we tested whether aged Tg-αSyn^A53T mice, which display severe αSyn-driven pathology, have αSyn aggregates within their lysosomes.

We found pathogenic αSyn species, but not monomeric αSyn, in dextranosomes (heavy lysosomes loaded with dextran to enhance isolation-purity) isolated from the brains of 6-month-old Tg-αSyn^A53T mice (Fig. 1). These lysosomal fractions precisely coincided with pathogenic αSyn species and had highly enriched lysosomal proteins as well as cathepsin-D activity, with negligible contamination from other organelle protein-markers or mitochondrial and peroxisomal enzyme activities (Fig. 1). This experiment indicates that isolated intact lysosomes from aged Tg-αSyn^A53T mice contain pathogenic αSyn species.

Pathogenic species of αSyn accumulate within the lumen of neuronal lysosomes

The lysosomes studied above are a mixture of lysosomes from all brain cells. To isolate and study lysosomes specifically from neurons, we generated transgenic mice expressing ^HALAMP1^Myc—late-endosomal/lysosomal protein LAMP1 epitope-tagged on its luminal domain with tandem 2xHA, and on its cytoplasmic tail with 2xmyc—driven by the neuron-specific synapsin-I promoter (Fig. 2a–d and Supplementary Fig. S2a–d), enabling affinity-isolation of neuronal late-endosomes/lysosomes from mouse brains. The Tg-^HALAMP1^Mycmice were then crossed to the Tg-αSyn^A53T mice to generate Tg-αSyn^A53T/Tg-^HALAMP1^Mycprogeny (Supplementary Fig. S3a–g). Immunoisolation experiments from aged Tg-αSyn^A53T/Tg-^HALAMP1^Myc mouse brains indicated that pathogenic versions of αSyn co-isolate with neuronal late-endosomes/lysosomes (Fig. 3a). We found that ~10% of the SDS-resistant αSyn aggregates, and ~20% of the more mature αSyn aggregates (filamentous and amyloid-type) reside within neuronal late-endosomes/lysosomes (Fig. 3a). No monomeric αSyn resided in neuronal late-endosomes/lysosomes (Fig. 3a). Together with the dextranosome-isolation experiments (Fig. 1), this result indicates that a non-trivial portion of pathogenic αSyn species in the brain is found within neuronal lysosomes.

Electron microscopy showed at the ultrastructural level that αSyn aggregates were localized within the lysosomal lumen of cortical neurons in 6-month-old Tg-αSyn^A53T mouse brains (Fig. 3b). This was further corroborated by limited proteolysis of immunoisolated lysosomes from 6-month-old αSyn^A53T/Tg-^HALAMP1^Myc mice, where αSyn aggregates were resistant to proteinase K digestion, only in the absence of a detergent, similar to the lysosome-luminal enzyme cathepsin-L, and in contrast to the lysosomal vATPase subunits that are exposed on the cytosolic side of lysosomal membrane (Fig. 3c). Altogether, these experiments localize αSyn aggregates inside the lysosomal lumen.

To understand the fate of these lysosomal αSyn aggregates, we developed a neuronal model which accumulates pathogenic αSyn species. Compared to Tg-αSyn^A53T mice, homozygous Tg-αSyn^A53T mice (Tg^x2-αSyn^A53T), which express twice the amount of transgenic αSyn^A53T, have shorter lifespans, earlier onset of neuromuscular pathology, and begin accumulating pathogenic αSyn species earlier—by only 6 weeks of age (Supplementary Fig. S4a–f). Accordingly, long-lived primary neurons from Tg^x2-αSyn^A53T mice also accrued pathogenic αSyn species by 6 weeks, including aggregates detectable by antibodies against αSyn fibrils and amyloid-type conformations (Supplementary Fig. S5a, b), which were Triton X-100 insoluble, extractable in SDS, and disrupted by urea (Supplementary Fig. S5c).

Importantly, we further verified the accumulation of these pathogenic αSyn aggregates in lysosomes using a second independent immunoisolation approach. Isolation of lysosomes from Tg^x2-αSyn^A53T neurons using the HA-tagged lysosomal protein TMEM192 (previously characterized in^36,37) captured αSyn aggregates within neuronal lysosomes at DIV49, but not earlier at DIV35 and DIV21 (Supplementary Fig. S6). No signal for these pathological αSyn species was detected in the lysosomes isolated from DIV49 αSyn knockout (αSyn^−/−) neurons.

We then applied two independent proximity-labeling techniques to specifically mark and track the pathogenic αSyn species located within neuronal lysosomes. First, we targeted Apex-2 to the lysosomal lumens of Tg^x2-αSyn^A53T primary neurons. We lentivirally expressed a synapsin-1 promoter-driven construct, comprised of Apex-2 fused to the luminal domain of a truncated version of LAMP1 (^Apex-2LAMP1) (Supplementary Fig. S7a–c), which proximity-labels the luminal contents of neuronal late-endosomes/lysosomes with biotin. In these Tg^x2-αSyn^A53T neurons expressing ^Apex-2LAMP1, we found that pathogenic αSyn species as well as lysosomal proteins (typified by cathepsin-L) were biotinylated (Fig. 3d). Second, we used an in situ proximity ligation assay, and found that filamentous αSyn aggregates co-localized with cathepsin-D, i.e., within the lysosomal lumen of Tg^x2-αSyn^A53T primary neurons (Fig. 3e). Together, these results show that pathogenic αSyn species accumulate within the lumen of neuronal lysosomes.

Pathogenic αSyn species are secreted from neurons via SNARE-dependent lysosomal exocytosis

Release of αSyn from neurons, particularly the seeding-competent pathogenic versions of αSyn, is considered a key step in the spatial progression of synucleinopathies³⁸, and αSyn has been documented in extracellular fluids of PD patients⁶. We also found pathogenic species of αSyn in the cerebrospinal fluid of aged Tg-αSyn^A53T mice (Fig. 4a). Importantly, this extracellular pool of αSyn aggregates is not enveloped in membranes, as they were not protected from proteinase K digestion (Fig. 4a), and their proteolytic susceptibility was unaffected by the presence of detergent, akin to the lysosomal luminal protein cathepsin-L and neuroserpin—a non-lysosomal secreted protein (Fig. 4a). This was contrary to the well-protected contents of exosomes, typified by TSG101, which becomes susceptible to proteolysis only in the presence of detergent (Fig. 4a).

The pathogenic αSyn species were also detected in the medium of Tg^x2-αSyn^A53T neurons collected at DIV49, but were not yet present at DIV35 or in the media of DIV49 αSyn^−/− and WT neurons (Supplementary Fig. S5d). This extracellular pool of αSyn aggregates, similar to that in cerebrospinal fluid, was not protected from proteinase K digestion (Fig. 4b), indicating that it is not membrane-enveloped. Additionally, proteolytic susceptibility of these αSyn aggregates, as well as that of cathepsin-L and neuroserpin were unaffected by the presence of detergent (Fig. 4b). In contrast, membrane-enveloped exosomal contents were well-protected, typified by TSG101, which became susceptible to proteolysis only in the presence of detergent (Fig. 4b). Notably, we found no evidence of neuronal death as a potential source of extracellular αSyn aggregates (Supplementary Fig. S5e, f).

Accumulation of the pathogenic αSyn species inside lysosomes, together with their un-enveloped presence in the extracellular space, prompted us to investigate whether lysosomes are directly releasing the αSyn aggregates contained inside them.

We thus collected the medium from Tg^x2-αSyn^A53T primary neurons, in which the lysosomal contents had been biotinylated via neuron-specific expression of ^Apex-2LAMP1 (as in Fig. 3band Supplementary Fig. S7). We found that lysosomal contents, typified by cathepsin-L, as well as pathogenic αSyn species, could be precipitated from the medium of Tg^x2-αSyn^A53Tneurons via their biotin-modification (Fig. 5a). In contrast, exosomal contents, typified by TSG101, were not biotinylated/precipitated. This result points to lysosomal exocytosis—SNARE-dependent fusion of lysosomes with the plasma membrane—as the likely mechanism for the exit of αSyn aggregates from neurons.

SNARE-dependent lysosomal exocytosis is triggered by cytosolic calcium³⁹, which in turn is modulated by neuronal activity and/or exchange with organellar calcium stores. We first pharmacologically modulated neuronal activity in Tg^x2-αSyn^A53T neurons, by using TTX or CNQX/APV to inhibit activity, and K⁺ or bicuculline methiodide to enhance activity. Release of αSyn aggregates as well as that of cathepsin-L directly correlated with neuronal activity (Supplementary Fig. S8a). Next, we inhibited the release of calcium from the endoplasmic reticulum using dantrolene/2-APB, or inhibited the influx of extracellular calcium using YM-58483, in Tg^x2-αSyn^A53T neurons (Supplementary Fig. S8b). Both strategies reduced the release of αSyn aggregates and cathepsin-L into the medium (Supplementary Fig. S8b). Neither the manipulation of neuronal activity nor the reduction of cytoplasmic calcium caused neuron death (Supplementary Fig. S8c, d). These pharmacological experiments further suggest that release of pathogenic αSyn species from neurons is mediated via calcium-regulated SNARE-dependent lysosomal exocytosis.

To test this hypothesis directly, we modulated the SNARE proteins responsible for lysosome-to-plasma membrane fusion. First, we identified VAMP7 as the prominent v-SNARE in immunoisolated lysosomes (Supplementary Fig. S9a). We then screened for the Qb-type t-SNAREs which interact and thus co-immunoprecipitate with VAMP7. SNAP23 was the clearest Qb/t-SNARE candidate (Supplementary Fig. S9a). Based on these results and prior studies validating them⁴⁰, we tested four strategies to disrupt lysosomal exocytosis: shRNA knockdown of either SNAP23 (SNAP23^KD) or VAMP7 (VAMP7^KD), and overexpression of dominant-negative fragments of either SNAP23 (SNAP23^DN) or VAMP7 (VAMP7^DN). In wild-type primary neurons, the release of cathepsin-L was used to signal lysosomal exocytosis, opposed to neuroserpin—a non-lysosomal secreted protein. VAMP7^KD and SNAP23^DN had no significant effect on lysosomal exocytosis, but VAMP7^DN severely reduced it by ~80% and SNAP23^KD partially reduced it by ~40%, with no effect on cell viability (Supplementary Fig. S9b–d). Therefore, we applied the latter two strategies to disrupt lysosomal exocytosis next.

Importantly, in Tg^x2-αSyn^A53T neurons, both VAMP7^DN and SNAP23^KD strategies reduced the release of pathogenic αSyn species into the medium, and the effect of SNAP23^KD was rescued by overexpression of a knockdown-resistant version of wild-type SNAP23 (Fig. 5b). Moreover, the release of pathogenic αSyn species was significantly correlated with the change in lysosomal exocytosis, measured as cathepsin-L released into the medium (Fig. 5b, c). No β-actin was detectable in the medium (Fig. 5b). These results suggest that SNARE-dependent exocytosis of lysosomes is essential and rate-determining for the release of pathogenic αSyn species from neurons.

αSyn species released via lysosomal exocytosis can seed aggregation of purified recombinant αSyn and of endogenous αSyn in neurons

Pathogenic spread requires that the released αSyn is able to template or “seed” the assembly of monomeric αSyn into amyloid-type aggregates.

To test whether the pathogenic αSyn species exocytosed from neurons are seeding-competent, we performed in vitro aggregation of purified recombinant myc-tagged wild-type αSyn (Supplementary Fig. S10a), in the presence of extracellular media collected from neuron cultures generated either from wild-type mice, or from Tg^x2-αSyn^A53T mice with or without lentiviral expression of VAMP7^DN fragment (Fig. 6a–c). Aggregation kinetics, measured by amyloid-binding fluorescent dye K114 (Fig. 6a) or Thioflavin-T (Fig. 6b), were enhanced by medium from Tg^x2-αSyn^A53T neurons, compared to the medium from wild-type neurons. Suppressing lysosomal exocytosis in the Tg^x2-αSyn^A53T neurons, via VAMP7^DNexpression, diminished the aggregation-promoting effect of the Tg^x2-αSyn^A53T medium (Fig. 6a, b). When myc-αSyn aggregation kinetics were measured by quantitative immunoblotting for either disappearance of myc-tagged αSyn (myc-αSyn) monomers or for the appearance of filamentous and amyloid-type aggregates (shown in αSyn^Fila and αSyn^Amylblots), we again found that compared to the wild-type neuron medium, aggregation was augmented by the Tg^x2-αSyn^A53T medium, and the expression of VAMP7^DN fragment diminished this effect (Fig. 6c). Notably, the effect of wild-type neuron medium on myc-αSyn aggregation rate was indistinguishable from the effect of unconditioned medium and αSyn^−/−neuron medium (Supplementary Fig. S10b). In all the assays, a lag-time of nearly 2 weeks is apparent before the appearance of amyloid signal with the wild-type medium (Fig. 6a–c). Importantly, this lag is shortened or eliminated by the secreted aggregates in Tg^x2-αSyn^A53Tmedium (Fig. 6a–c), with a reduced effect when lysosomal exocytosis is suppressed by VAMP7^DN fragment (Fig. 6a–c).

Moreover, in Tg^x2-αSyn^A53T primary neurons, appearance of αSyn aggregates was accelerated by media from Tg^x2-αSyn^A53T neurons, but not from wild-type neuron media (Fig. 6d). Importantly, media from Tg^x2-αSyn^A53T neurons expressing the VAMP7^DN fragment—which inhibits lysosomal exocytosis, had a significant reduction in this effect (Fig. 6d).

These results substantiate that αSyn species exocytosed by lysosomes are capable of amyloid-nucleation via permissive templating, as well as spreading this seeding activity into live neurons.

Discussion

Release of αSyn aggregates from neurons is thought to transmit neuropathology to other yet-unaffected neurons. Here, we find that pathogenic αSyn species accumulate within neuronal lysosomes in mouse brains and in primary neurons, and are then released from neurons via SNARE-dependent lysosomal exocytosis (Fig. 7).

It is important to note that for αSyn pathology to transmit from neuron-to-neuron, the lysosomal exocytosis mechanism explains the following observations, which underscore that the extracellular pool of pathogenic αSyn is likely not membrane-enveloped: (a) the stark pathology and rapid propagation caused by inoculated pre-formed fibrils^{10,11,41,42,43,44}, (b) the efficacy of humoral and cellular immunotherapy against αSyn (reviewed in ref. 45), (c) the efficacy of passive immunization using antibodies against αSyn epitopes (reviewed in ref. 45), and (d) much of the extracellular αSyn has been found as non-enveloped^6,46,47, while a minority of αSyn is contained in extracellular vesicles or exosomes^12,18,20. However, other proposed means of αSyn aggregate-transmission might simultaneously contribute to pathological propagation in synucleinopathies³⁸.

Key gaps still remain in how the pathogenic αSyn species are targeted and trafficked to lysosomes, as well as in the fate of extracellular αSyn aggregates following their release from neurons:

The aggregates/fibrils of αSyn could form inside the acidified lumen of lysosomes⁴⁸, e.g., from monomers taken up via chaperone-mediated autophagy or macroautophagy of synaptic vesicles, especially if their degradation is delayed due to reduced or disrupted lysosomal activity⁴⁹. Yet, we found no presence of αSyn monomers within isolated lysosomes, possibly due to their rapid degradation by lysosomal proteases, making this possibility less likely. Macroautophagy of oligomers and/or larger aggregates from the cytoplasm, termed “aggrephagy”, is a more likely mechanism, following from the well-recorded ability of autophagosomes to encapsulate cytoplasmic structures including aggregates⁵⁰, and target them to lysosomes.

Once released from the neurons, the fate of extracellular/interstitial αSyn aggregates also remains unclear, and can follow multiple possible scenarios: uptake into microglia⁵¹, astrocytes⁵², and/or oligodendrocytes⁵³, and/or drainage via glymphatics^54,55 or blood flow^6,56. However, for the extracellular αSyn aggregates to cause the stereotypic “spread” of pathology via a prion-like etiology, other neurons will have to take up the released aggregates, and this process remains even less understood than the glial uptake or clearance scenarios.

The results presented here provide evidence of the accumulation of pathogenic αSyn species in neuronal lysosomes, and pinpoint to lysosomal exocytosis as a pathway of release for these pathogenic αSyn species into the extracellular milieu. This pathway, at first sight, appears to have relevance to the release of undegraded aggregates of other proteins as well that transmit pathology from cell-to-cell (reviewed in ref. 38), and follow-up studies will be needed to test this speculation.