Abstract
Post-translational modifications of alpha-synuclein (aSyn), in particular phosphorylation at Serine 129 (Ser129-p) and truncation of its C-terminus (CTT), have been implicated in Parkinson’s disease (PD) pathophysiology. Although great interest has emerged for these species as potential biomarkers and therapeutic targets in PD, little is known about their (sub)cellular manifestation in the human brain. In this study, we mapped distribution patterns of Ser129-p and CTT aSyn in neurons with and without Lewy pathology. The combination of highly selective antibodies with multicolor STED microscopy allowed detailed phenotyping of subcellular neuropathology in PD. Nigral Lewy Bodies revealed an onion skin-like 3D architecture, with a framework of Ser129-p aSyn and neurofilaments encapsulating CTT and membrane-associated aSyn epitopes. Based on the identification of subcellular pathological phenotypes in this study we present a novel hypothesis for maturation stages of Lewy pathology and provide evidence for a key role for Ser129-p aSyn in Lewy inclusion formation.
Introduction
The presence of cellular inclusions – termed Lewy Bodies (LBs) and Lewy Neurites (LNs) – in predilected brain regions pathologically defines Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). LBs and LNs are defined as eosinophilic inclusions with different morphologies, typically dependent on brain region (brainstem, limbic or cortical) 1, 2. To this end, the mechanism determining the formation and morphology of these inclusions remains elusive. LBs and LNs are strongly immunoreactive for alpha-synuclein (aSyn), which is one of their major components 3. aSyn is a 14kDa protein ubiquitously and highly expressed in neurons under physiological conditions. Its enrichment in presynaptic terminals has been established 4-7, while more recent studies have reported additional intraneuronal localizations for aSyn, including mitochondria, endoplasmatic reticulum (ER) and Golgi 7. The primary sequence of aSyn contains 140 amino acids, and is composed of three distinct domains. An important role has been proposed for the lipophilic N-terminus (NT) and nonamyloid component domain (NAC domain) in the interaction of aSyn with membranes as the first 93 residues can adopt an alpha-helical conformation in the presence of lipids, and they bear the locations for the currently known point mutations in aSyn that lead to familial forms of PD 7, 8. Residues 96-140 encompass the negatively charged, acidic C-terminus (CT) of aSyn for which important roles have been proposed in the interaction of aSyn with other proteins or metal ions 9. The CT further harbors the majority of sites where aSyn can be post-translationally modified (PTM) 10.
The list of aSyn PTMs detected in the human brain has grown extensively in recent years, which highlights the physicochemical and structural flexibility of aSyn 11, 12. Some of these PTMs have been implicated in PD pathology - in particular the phosphorylation at Serine 129 (Ser129-p) and truncations of the C-terminus (CTT). Ser129-p aSyn and different CTT fragments of aSyn, with cleavage sites for instance at residues 119 (119CTT) and 122 (122CTT), were identified in pathology-associated fractions of the DLB brain using mass spectrometry and immuno-based biochemical assays 13, 14. Immunohistochemical analyses in postmortem brain tissue of DLB patients and also aSyn transgenic mouse brains point to a potential role of 122CTT in axonal and synaptic degeneration 15. Although Ser129-p and CTTaSyn can be detected in small amounts under physiological circumstances 16, these PTMs are enriched under pathological conditions 13, 14, 17-19. Experimental studies have suggested an important role for CTT in aSyn aggregation, as enhanced fibril formation was reported for this PTM with recombinant aSyn in vitro 19-23. However, the exact role of CT modification by either phosphorylation or truncation in aSyn aggregation and toxicity, remains subject of active debate 24.
A great interest has emerged for PTM aSyn as a potential biomarker and therapeutic target for PD 25, 26, and led to the development of new research tools, including antibodies specifically directed against Ser129-p and CTT aSyn. Although such antibodies were reported to show immunoreactivity in LBs and LNs 13, 14, 27, only little is known about their detailed immunoreactivity patterns in the human brain under physiological and pathological conditions. More information on their subcellular distribution is crucial for a better understanding of the physiological and pathological relevance of Ser129-p and CTT aSyn, and therefore highly relevant for ongoing and upcoming immunovaccination therapies targeting different aSyn species.
In this study, we aimed to define spatial distribution and (sub)cellular manifestation of Ser129-p and CTT aSyn in human brain tissue with and without Lewy pathology. For this purpose, we mapped immunoreactivity patterns of highly selective antibodies directed against Ser129-p and CTT aSyn in postmortem brain tissue of PD patients and non-neurological subjects using high-resolution 3D confocal scanning laser microscopy (CSLM) and stimulated emission depletion (STED) microscopy. Our results provide novel insights into antibody-labeled subcellular pathology in PD, demonstrating a systematic onion-like architecture of nigral LBs with differential affinities for different aSyn epitopes. Ser129-p aSyn at the periphery of these onion-skin type LBs was associated with a cage-like framework of intermediate neurofilaments. Label-free coherent anti-Stokes Raman scattering microscopy revealed the presence of increased lipid and protein contents in the center of a-Syn immunopositive inclusions, suggesting encapsulation of these components in the core of LBs. Cytoplasmic CTT reactivity was associated with mitochondria, while Ser129-p aSyn was structured in a filamentous network in diseased neurons in the PD brain, possibly representing an early stage of Lewy inclusion formation. Based on the identification of subcellular pathological phenotypes in this study, we present an hypothesis for maturation stages of Lewy pathology and provide evidence for a key role for Ser129-p aSyn aSyn in Lewy inclusion formation.
Results
Morphological features of aSyn-positive inclusions as labeled by antibodies against different epitopes
In accordance with existing literature 13, 14, 27, immunohistochemical (IHC) stainings using antibodies (specified in Table S1) against truncated aSyn species (119 and 122 CTT aSyn) and Ser129-p aSyn labeled pathology-associated morphologies in post-mortem brain tissue from clinically diagnosed and neuropathologically confirmed advanced PD patients (Braak 5/6; Table S2), including LBs and LNs. These neuronal inclusions were also detected using antibodies directed against epitopes within specific domains (CT, NT and NAC domain), while this group of antibodies also revealed synaptic-like staining profiles, particularly in the hippocampus and transentorhinal cortex. Representative images of neuronal aSyn-positive inclusions labeled with antibodies against different epitopes, taken in the substantia nigra (SN), hippocampus and transentorhinal cortex of PD patients, are shown in Figure S1, together with KM-51, an antibody commonly used for neuropathological diagnosis 28.
We defined two main types of intracytoplasmic aSyn-immunopositive inclusion bodies based on the observed IHC labeling profiles. Many of the spherical LBs in the SN displayed immunoreactivity in targetoid appearance, i.e. with a strong immunopositive ring surrounding a central – weakly or unstained - core (Fig. S1A). Inspection in adjacent brain sections of the same patients suggested that this morphological appearance of LBs was most clearly visualized by antibodies against Ser129-p aSyn and CT aSyn (Fig. S1A, xiv, xxvi). Antibodies directed against other aSyn epitopes generally revealed less contrast between core and immunoreactive ring. In some LBs, an area of weaker immunoreactivity surrounding the strongest immunopositive portion of the LB could also be observed (f.i. Fig. S1A, xviii). The targetoid appearance of LBs has been described with many antibodies against aSyn in literature, and a subset of these morphologies were shown to represent the eosinophilic ‘classical LBs’ unambiguously identified by hematoxylin and eosin stainings 2. Peripheral immunoreactivity surrounding a weakly stained central core could also be observed in a subset of the dystrophic LNs in the SN (Fig. S1B).
Other intracytoplasmic aSyn positive inclusions showed diffuse and uniform labeling throughout the structure. This IHC pattern was generally observed for limbic and cortical LBs in the hippocampal CA2 region and transentorhinal cortex, respectively, but also for certain inclusions in the SN 1, 2. The shapes of this latter group of inclusion bodies showed substantial heterogeneity, including both globular structures (f.i. Fig. S1A, xxiii), as well as irregularly-shaped expansive-appearing cytoplasmic inclusions (f.i. Fig. S1A, i&iii).
Onion skin-like distribution of aSyn epitopes in Lewy inclusions
Triple labeling immunofluorescence experiments demonstrated that Ser129-p aSyn, 119CTT and 122CTT only partially co-localized in nigral LBs, using different combinations of antibodies (Table S2). In particular, Ser129-p immunopositive rings in nigral LBs localized consistently more towards their periphery as compared to staining patterns for 119CTT or 122CTT aSyn (Fig. 1A, C, Video S1,2). Interestingly, different localization patterns were also observed between antibodies against NAC, NT and CT of aSyn, with CT aSyn immunoreactivity surrounding the other domains (Fig. 1B, D, Video S3). A combination of these antibodies in different multiple labeling protocols revealed a gradual distribution of immunoreactivities in nigral LBs (Fig. 2A). This lamellar organization of different concentric rings together revealed an onion skin-like 3D morphology of LBs (Fig 2E, Video S4), with pronounced reactivity for CT and Ser129-p aSyn at the periphery, while antibodies against CTT aSyn, NT and NAC aSyn localized more towards the core of these structures (Fig. 2AC). The same build-up was observed using a set of different antibodies directed against similar epitopes (Fig. S2). The consistency of this observation was semi-quantitatively examined in 29 LBs in SN sections of 8 PD patients. For each LB, relative signal intensities (as % of peak intensity) were plotted per channel over a normalized LB diameter. The resulting average line profile revealed a clear separation of peak intensity localizations for the different aSyn epitopes, revealing that this gradual distribution was consistent (p <0.0001; Fig. 2F; Table S3), while 3D CSLM analyses further showed that the lamellar build-up was present throughout the entire structure (Fig. 2E, Video S4). Converging DAPI reactivity was consistently observed at the core of these inclusions, although this signal was generally weaker than its staining intensity in neighboring cell nuclei (Fig. 2B). Numerous dystrophic LNs in the SN were observed to contain compositions similar to these LBs (Fig. 2D).
Distribution patterns for different aSyn epitopes were also analyzed in Ser129-p positive inclusion bodies in which no ring-like appearance was observed, in the same sections (in case of SN) and in other sections of the same patients (hippocampus/transentorhinal cortex). These inclusion bodies were homogeneously labeled for Ser129-p aSyn, except for typical ‘cavities’ lacking Ser129-p reactivity (Fig. S3A,D). Although such inclusions were unambiguously stained for Ser129-p aSyn, antibodies against other aSyn epitopes (particularly 122CTT, NAC and NT aSyn) generally revealed weak signal intensities that were distributed diffusely throughout the inclusion, while immunoreactivity for DAPI was barely increased compared to the surrounding. Although these inclusions appeared relatively unstructured – e.g. no systematic distribution patterns for aSyn epitopes were observed - their outline was usually sharply demarcated by Ser129-p aSyn immunoreactivity (f.i. Fig. S3A, Video S5). Taken together, our results show morphology-dependent distribution patterns for aSyn epitopes in Ser129-p aSyn-positive inclusions, and provide evidence for a structured lamellar and onion skin-like 3D organization of certain midbrain LBs and LNs.
Centralization of lipids and proteins in nigral LBs
As we observed different localization for antibodies recognizing membrane-associated aSyn domains (NT/NAC domain) and CT recognizing antibodies, we explored whether this differential epitope availability could be explained by the distribution patterns of proteins and lipids in LBs. For this purpose, we used a label-free optical imaging technique, CARS microscopy, in combination with CSLM. In total, 57 Ser129-p aSyn-positive inclusions were scanned in SN sections of 5 PD patients (Table S1). Results revealed substantial heterogeneity in protein and lipid distributions in the Ser129-p immunopositive inclusions, both within and between patients (Fig. 3D). We identified inclusions with different chemical compositions: part of the inclusion bodies showed high protein and lipid content as compared to the direct environment (35%), others contained higher protein content but no increased lipid content (30%), and finally inclusions were observed without detectable increases for either lipid or protein content (35%) (Fig. 3A-C). In 14 out of 20 inclusions with increased lipid content, lipids clustered at their central portion, while a similar pattern was observed for proteins (28 out of 37 inclusions). In all inclusions with centralized lipids, an increased protein content was also detected in the core (14 out of 14 inclusions). Overall, our results show that in the subset of aSyn-positive inclusion bodies that shows increased protein and lipid content compared to the surrounding, these components are generally found centralized in the structure, suggesting an encapsulation of highly aggregated material in LBs.
Ser129-p aSyn and cytoskeletal proteins form filamentous cage at the periphery of LBs
The previously described results together suggest a structured, lamellar architecture of LBs. As cytoskeletal proteins are a major constituent in organizing cellular organelles and substructures, we therefore explored the distribution of intermediate neurofilaments and beta-tubulin in the described onion-skin type LBs, and their association with Ser129-p aSyn immunoreactivity patterns. Although LBs are defined in brightfield microscopy as spherical smooth-edged inclusions, detailed STED imaging revealed that the outline of many LBs revealed a corona of radiating immunoreactivity, best – but not exclusively - visualized using antibodies against Ser129-p aSyn (Fig. S4). The radiating Ser129-p immunoreactivity patterns resembled the shape and orientation of filaments (Fig. S4, Fig. 4) described in early electron microscopy (EM) studies of LBs 29, 30.
Co-staining of Ser129-p aSyn with markers for intermediate neurofilament and beta-tubulin showed immunoreactivity for cytoskeletal markers at the periphery of nigral LBs (Fig. 4). Together with Ser129-p aSyn, these markers visualized a filamentous ‘cage-like’ framework (Fig. 4D). Beta-tubulin reactivity was also present at the periphery of LBs, where it overlapped substantially with Ser129-p aSyn. Moreover, beta-tubulin also revealed filamentous structures with radiating orientation, although even more localized towards the periphery of the LB (Fig. 4B). Neurofilament reactivity showed a different distribution, as it visualized two rings: one ring localized at the central portion of the Ser129-p aSyn immunopositive band in LBs, while another ring surrounded the Ser129-p/beta tubulin signals. Interestingly, these rings were connected by radiating filamentous structures, giving rise to a structure resembling a wheel (Fig. 4D). The structure of this filamentous framework and its positioning around the Ser129-p immunopositive band could be best observed in 3D reconstructions (Video S6).
Interestingly, in other cytoplasmic inclusion morphologies or in dystrophic LNs, this arrangement of cytoskeletal components was not observed, although weak and diffuse immunoreactivity of cytoskeletal markers was generally present. This suggests that the organization of Ser129-p aSyn and cytoskeletal markers was typical for cytoplasmic inclusions, in particular for the before-mentioned ‘onion skin-type’ LBs (Fig. S5 A,C).
Different subcellular manifestations for Ser129-p and CTT aSyn
Apart from their localization in aSyn-immunopositive inclusion bodies, Ser129-p and 122CTT aSyn antibodies also revealed cytoplasmic immunoreactivity in neurons outside of these structures. Immunoreactivity for 119CTT was specific for pathological inclusions with limited immunoreactivity in the cytoplasm. The appearance of cytoplasmic reactivity was different for Ser129-p and 122CTT aSyn (Fig. 5A). In particular, 122CTT aSyn revealed many immunoreactive punctae throughout the neuronal cytoplasm, while Ser129-p aSyn immunoreactivity visualized an intracytoplasmic network of thin fibers. Limited co-localization was observed between these signals (Fig 5A).
122CTT aSyn immunopositive punctae are associated with mitochondria
122CTT immunopositive punctae were not only observed in PD patients, but also in brain tissue sections of donors without Lewy pathology (Fig. S6A). The pattern appeared more pronounced in the hippocampus and transentorhinal cortex compared to the SN (Fig. 5B). We observed the punctate reactivity pattern of 122CTT aSyn using different antibodies against this epitope (syn105 and asyn-134), while this pattern was not observed for 119CTT aSyn. These 122CTT aSyn-reactive punctae were more pronounced in the cytoplasm of neurons compared to the environment. Analysis with subcellular markers revealed that a subset of the 122CTT-reactive punctae in the cytoplasm of neurons co-localized with mitochondrial morphologies immunoreactive for Porin/VDAC reactivity, which is a marker for the outer membrane of mitochondria (Fig. 5B). This association between CTT aSyn and mitochondria was also observed in 3D (Video S7). Together, our findings suggest widespread 122CTT aSyn expression in the brain of donors with and without PD, particularly in the neuronal cytoplasm where 122CTT aSyn is possibly associated with mitochondria.
Antibodies against Ser129-p aSyn reveal intracytoplasmic network
The Ser129-p aSyn immunopositive fibrous network was frequently visible in large neuromelanin-containing neurons in the SN (Fig. 5C). Most often, the network was observed in many neurons containing an expansive-appearing inclusion uniformly stained for Ser129-p aSyn, and in a smaller fraction of neurons containing onion-skin type LBs. However, this network was also observed in a subset of neuromelanin-containing neurons without apparent inclusion in PD patients (f.i. Fig. 6A, 1). Other neurons without inclusion in donors with PD did not reveal a Ser129-p aSyn immunopositive network, while it was not observed in donors without Lewy pathology (Figure S6B). The Ser129-p aSyn reactive fibers did not co-localize with markers for intermediate neurofilaments, beta-tubulin (Fig. 5C) or endoplasmatic reticulum (ER; Fig. S5), indicating that the alignment of Ser129-p positive fibers is not explained by localization of Ser129-p aSyn to these intracytoplasmic networks. The observed diameter of the structures in the network was generally 70-80 nm and limited by the resolution of the applied scan settings of the STED microscope. Together, these observations suggest that the Ser129-p aSyn immunopositive filamentous network is a pathological feature, possibly representing an early stage of Lewy pathology.
Intraneuronal reactivity patterns of Ser129-p aSyn allows identification of pathological subcellular phenotypes
By comparing cytoplasmic Ser129-p aSyn-immunoreactivity patterns with different specific antibodies within and between patients, we were able to identify several commonly observed reactivity patterns in melanin-containing neurons of brains with Lewy pathology. Representative examples for these subcellular pathological phenotypes are summarized in Fig. 6A, as visualized by CSLM 3D reconstructions of large z-stacks of neuromelanin-containing nigral neurons in adjacent brain sections of the same patient. Commonly observed cellular phenotypes in the SN of PD patients based on Ser129-p aSyn immunoreactivity included: 1) neurons with Ser129-p aSyn immunopositive cytoplasmic network but without apparent inclusion; 2/3) neurons with network and smaller inclusions with uniform Ser129-p aSyn labeling; 4) neurons revealing a Ser129-p aSyn immunopositive network and (a combination of) and uniformly stained and onion skin-like inclusions; 5) neurons with a Ser129-p aSyn immunopositive network and onion skin-type inclusions – often containing radiating filaments; 6) neurons without a network, but with onion skin-type inclusion. These different faces of Ser129-p aSyn immunoreactivity possibly reflect different stages of LB formation (Figure 6B, as discussed later in the text), and may suggest a role for Ser129-p aSyn at different stages of Lewy inclusion formation.
Discussion
aSyn has been established as a major component of Lewy pathology, but the subcellular distribution of specific pathology-associated forms of this protein – both within and outside the inclusions - is unclear. Detailed insight into the aSyn-based architecture of neuronal inclusions may allow for a better understaining of Lewy inclusion formation, which is a key event in PD pathophysiology. In the present study we explored the subcellular distribution patterns for CTT and Ser129-p forms of aSyn by means of specific antibody multiple labeling immunostainings in brain sections of PD patients in combination with high-resolution 3D CSLM and STED microscopy. This allowed an unprecedented detailed view on antibody-labeled subcellular pathological phenotypes, revealing several novel aspects of Lewy pathology.
First, we observed a Ser129-p-immunopositive cytoplasmic network in PD patients, using different antibodies directed against this epitope. Diffuse or granular cytoplasmic immunoreactivity has been described as a specific feature of certain antibodies against aSyn in different studies using light microscopy 2, 28, including antibodies with a proposed preferential affinity for disease-associated aSyn 31, 32. However, to the best of our knowledge this study is the first to visualize the alignment of filamentous Ser129-p immunopositive structures in an intracytoplasmic network. This network was most often observed in the vicinity of an inclusion, although 3D revealed that this network could also be observed in neurons without apparent inclusions, indicating that this feature could represent an early phenotype of LB formation. Interesting in this perspective are the results of a previous study, which found that increased expression levels for Ser129-p aSyn in soluble fractions of cingulate and temporal cortices – as measured by western blotting – preceded the presence of histologically identified Lewy inclusions 33. These findings suggest a role of Ser129-p aSyn already at early stages of LB formation - thereby contradicting theories that Ser129-p aSyn occurs after LB formation 24 - which may have important implications for the interpretation of results of biomarker studies measuring Ser129-p aSyn in body fluids or peripheral tissues. The notion of a role of Ser129-p aSyn early in inclusion formation is supported by the experimental findings that inclusion formation after administration of recombinant (full-length and CTT) aSyn pre-formed fibrils is induced by recruitment of soluble endogenous aSyn and its intracellular phosphorylation at Ser129 34. Possible roles of Ser129-p at this stage could be a stabilizing effect on aggregating proteins 35 - Ser129-p was demonstrated to inhibit aSyn fibrillogenesis in vitro 24, 36 - while Ser129-p aSyn has also been suggested to serve as an activator of autophagic instruments 37, 38.
Although the different localization of the Syn105 and 11A5 antibodies in LBs and dystrophic LNs, directed against 122CTT and Ser129-p aSyn, respectively, has been described before 27, we now confirm this finding using different antibodies against similar epitopes, and for different CTT aSyn (calpain-cleaved at res. 119 and 122) species. We further observed a separation between antibodies against membrane-associated aSyn domains (NT/NAC domains) of aSyn and antibodies recognizing its CT in LBs. Integrating antibodies against these aSyn PTMs and domains in multiple labeling protocols showed a 3D onion skin-like orchestration of LBs, with concentric lamellar bands with preferential affinities for different aSyn antibodies. The consistency of this lamination was revealed by semi-quantitative analysis in a series of LBs and different PD patients. A multilamellar appearance of LBs has been described in different studies that focused on the ultrastructure of LBs using EM techniques 29, 30, while lamination patterns in LBs and dystrophic LNs were also suggested in studies using light microscopy 27, 39, 40. Interestingly, in the present study this lamellar phenotype was visualized by the gradual distribution of immunoreactivities for antibodies directed against different epitopes on one single protein, aSyn.
As we observed clustering of membrane-associated aSyn epitopes mainly at the core of onion skin-like LBs, we explored the distribution of proteins and lipids in LBs using CARS microscopy, a label-free imaging technique. Results showed that increased lipid content could be detected in a fraction of nigral Ser129-p positive inclusions. Unfortunately, due to the limited morphology of the fresh-frozen tissue sections after CARS imaging, a subclassification of LB morphologies by high-resolution CSLM imaging was not possible. However, in the majority of lipid-enriched inclusions, lipids were found to be centralized in the core of this structure together with proteins. In a previous study, we demonstrated the presence of lipid and membraneous structures in LBs using correlative light and electron microscopy (CLEM) and TEM, which was confirmed by CARS and Fourier transform infrared spectroscopy (FTIR) 41. Moreover, reactivity in the core of LBs has further been described for different lipophilic dyes 40-42. The functional relevance behind clustering of lipids in the center of LBs is not clear at this point. However, extensive experimental evidence has demonstrated the binding of aSyn to (bio)membranes, while the presence of lipid molecules was repeatedly reported to increase the aggregation rate of aSyn 43.
At the periphery of nigral onion skin-type LBs, radiating Ser129-p aSyn- and beta-tubulin-immunopositive filamentous structures could be observed, embedded in a cage-like framework of neurofilaments. The combination of CARS data and structural observations together raise the suggestion that aggregated proteins are encapsulated in the core of LBs by an organized framework of Ser129-p aSyn and cytoskeletal components. The displacement of intermediate filament proteins from their normal cellular distribution and the encapsulation of aggregated proteins by neurofilaments have been described as consistent features of intracellular aggresome formation 44, 45. Although the arrangement of cytoskeletal structures at the periphery of LBs has been reported before, this study provides important new detailed insights into the structure of and relation between different filamentous components in LBs. Thereby, our results support previous studies proposing that LBs share phenotypic features with aggresomes 46. Our observations indicate that the interplay of Ser129-p aSyn with cytoskeletal proteins may be an important step in the process of LB formation.
Among many features of Ser129-p Syn immunoreactivity patterns within and between PD patients, we identified commonly observed subcellular pathological phenotypes in neuromelanin-containing neurons in the SN of PD patients (Fig. 5A), which may reflect different maturation stages of LB pathology. Based on our high-resolution observations in different experimental setups, combined with CARS data, we propose a hypothetical sequence of events in the formation of LBs in the SN (Fig. 5B). We speculate that different subcellular phenotypes of aSyn pathology are tightly coupled to the progressive collapse of protein degradation systems 47. In healthy dopaminergic neurons, the basal proteolytic activity of the intracellular protein degradation systems – in particular the ubiquitin-proteasomal system (UPS) and chaperone-mediated autophagy (CMA)- are able to maintain protein homeostasis (Fig. 5B, step 1) 47. In situations of increased protein burden, these systems are overloaded, and superfluous aSyn could start to aggregate with itself, other proteins, membranes and/or organelles 41. Extensive phosphorylation of aSyn may take place at this point to stabilize the expanding mass of cellular debris and to activate the macrophagic machinery 38. The alignment of aggregated material for focused macroautophagic clearance may explain the observed Ser129-p aSyn-immunopositive intracytoplasmic network (step 2). When the increase in macroautophagic activity is insufficient to reduce protein burden, aggrephagy – a specialized form of autophagy 44, 45 - is initiated by the sequestration of aggregated material into an expansive inclusion (step 3), which will grow and occupy an increasing cytoplasmic surface. At the point the inclusion cannot be cleared by proteolytic systems, cytoskeletal systems - major constituents in organizing cellular organelles and substructures - may be recruited to the LB to actively restructure the inclusion into a compact and stable morphology (step 4, 5). Interestingly, the idea of a restructuring of LBs during their maturation in a compaction-like manner has been proposed before in literature 2. In the resulting onion skin-like morphology, highly aggregated proteins and lipids are centralized in the core of the structure, encapsulated by a ‘filamentous cage’ of Ser129-p aSyn and cytoskeletal components (step 5, 6). This hypothetical sequence of events in LB maturation could be further explored in future experimental studies, for instance in cellular and animal models of aSyn aggregation in PD.
In line with previous studies using antibodies against CTT aSyn species, we found this PTM to localize towards the core of LBs 16, 27, 48. CTT aSyn was repeatedly found to increase the propensity of aSyn to form amyloid aggregates in vitro 19-21, 23, and these observations together have led to the hypothesis that CTT aSyn plays a critical role in the initiation of protein aggregation 27. However, as LBs were demonstrated to contain a medley of fragmented membranes and organelles 41, including components that are able to cleave aSyn, for instance caspase-1 23, it cannot be ruled out that the enrichment of CTT aSyn in the core of LBs and LNs is the result of post-aggregation events.
Punctate cytoplasmic reactivity for 122CTT aSyn only showed limited co-localization with the Ser129-p immunoreactive network, and was independent of the presence of Lewy pathology (Fig. S6A). This PTM has been previously suggested to be a normal cellular process 19 and, indeed, presence of CTT aSyn was detected in brains of non-neurological control subjects by western blotting 16. Interestingly, a substantial part of the 122CTT aSyn immunoreactive punctae in the cytoplasm was observed to localize at the outer membrane of VDAC/Porin-reactive mitochondria in diseased neurons from PD patients, but also in healthy neurons from non-neurological control subjects. This could be placed in line with the findings of the previously mentioned study, in which a 15kDa band corresponding with CTT aSyn, was observed in fractions enriched for lysosomes and mitochondria derived from SH-SY5Y cells expressing human WT α-synuclein 16. Future experimental studies are necessary to explore the functional relevance of the co-occurence of 122CTT aSyn and mitochondria.
The immunoreactivity of aSyn antibodies often surrounded an unstained central core, which showed converging immunoreactivity for DAPI - a dye that binds to T-A-rich regions of DNA 49. This feature has already been reported before 50. Although at this point the relevance of this observation is not clear, it was speculated that this may be the result of mitochondrial DNA incorporated in LBs 50. Alternatively, DAPI may interact with certain aggregated proteins or lipids in the center of LBs. The limited immunoreactivity in the core of LBs may be the result of limited accessibility of antigen in this densely packed domain 40 or of masking or destruction of epitopes by its strong chemical environment. Importantly, LBs were shown to contain many (>300) proteins as identified by proteomics, which are predominantly centralized at the core of LBs based on our CARS results.
In summary, the present study provides a STED perspective on the architecture of Lewy pathology. Our results reveal a structured lamellar distribution of different forms of aSyn in nigral LBs and LNs. Our data suggest that LBs are actively regulated, structured encapsulations of aggregated proteins and lipids by a cage-like filamentous framework of Ser129-p aSyn and cytoskeletal components. Analysis of subcellular reactivity patterns led to the identification of different pathology-associated, Ser129-p aSyn-immunopositive subcellular phenotypes, suggesting a central role for Ser129-p aSyn in Lewy inclusion formation. The applied combination of well-characterized highly specific antibodies and super-resolution microscopy techniques in this study allowed an unprecedented detailed phenotyping of antibody-labeled subcellular pathology, which opens exciting opportunities for better characterization and understanding of PD pathophysiology.
Material and methods
Postmortem human brain tissue
Postmortem human brain tissue from clinically diagnosed and neuropathologically verified donors with advanced PD as well as non-demented controls was collected by the Netherlands Brain Bank (www.brainbank.nl). In compliance with all local ethical and legal guidelines, informed consent for brain autopsy and the use of brain tissue and clinical information for scientific research was given by either the donor or the next of kin. Brains were dissected in compliance with standard operating protocols of the Netherlands Brain Bank and Brain Net Europe. The details of all donors included in this study are listed in Table S1. Most of these PD donors developed symptoms of dementia during their disease course (Table S1), while all had extensive α-synuclein pathology throughout the brain (Braak aSyn stage 5/6). Formalin-fixed paraffin-embedded (FFPE) tissue blocks of the substantia nigra (SN) and hippocampus - also containing part of the parahippocampal gyrus- from 9 donors with advanced PD and 6 non-demented controls (details in Table S1) were cut into 10 and 20 µm thick sections, which were utilized for immunohistochemistry and multiple labeling experiments. In addition, fresh-frozen tissue blocks of the SN from 5 patients with advanced PD were cut into 10 µm for CARS microscopy (Table S1).
Generation and initial characterization of antibodies towards specific epitopes of aSyn
A detailed overview of all utilized antibodies and their epitopes in this study is provided in Table S2. Generation and characterization of antibody 11A5 and syn105 was previously described 14, 15, 51. Additional novel antibodies were generated by immunizing rabbits either with E. coli derived recombinant full length aggregated aSyn (asyn-055 and asyn-059) or KLH-conjugated peptides representing the C-terminus of 119CTT, 122CTT aSyn, or aSyn derived peptide phosphorylated at Ser129, respectively (for asyn-131, asyn-134, asyn-142). After screening of serum titers, standard B cell cloning was performed to generate rabbit monoclonal antibodies (mAbs). Recombinant mAbs were screened for binding to the peptides representing the aa1-60, 61-95, and aa96-140 by ELISA, respectively (for asyn-055 and asyn-059), or the C-terminus of aSyn119CTT, aSyn122CTT or phosphorylated at Ser129, respectively (for asyn-131, asyn-134, asyn-142), by ELISA and surface plasmon resonance (SPR). For asyn-055 and asyn-059, counter-screen by ELISA was performed with beta- and gamma-synuclein. For asyn-131, and asyn-134 ELISA- or SPR-based counter-screenings using C-terminal elongated peptides were performed to identify mAbs highly specific for the C-termini of 119CTT or 122CTT aSyn. ELISA- or SPR based counter-screenings using the corresponding non-phosphorylated peptide were performed to identify asyn-142 as highly specific for aSyn phosphorylated at Ser129. All animal experiments followed highest animal welfare standards and were performed according to approved ethics protocols.
Antibodies included in the multiple labeling experiments were first optimized for immunohistochemistry. In the multiple labeling for the studied aSyn epitope, immunoreactivity patterns were validated using at least one different antibody raised against a similar epitope, with exception of the antibodies against 119CTT and the N-terminus of aSyn, as no other available antibody against a similar epitope could be integrated in the multiple labeling protocol.
Immunohistochemistry
Protocols for the antibodies against aSyn were optimized for light microscopy to characterize their immunoreactivity in human postmortem formalin-fixed paraffin-embedded brain tissue. All IHC protocols could be optimized without antigen retrieval procedure and without addition of Triton. The EnvisionTM+ kit (DAKO, Santa Clara, USA) was used as a high-sensitivity visualization system, with 3,3’-diaminobenzidine (DAB; 1:50 diluted in substrate buffer; DAKO) as the visible chromogen. Stained sections were analyzed using a Leica DM5000 B photo microscope (Leica Microsystems, Heidelberg, Germany). All brightfield images included in Figure S1 were acquired using a HC PL APO 63×1.40 oil objective using a Leica DFC450 digital camera (Leica Microsystems).
Development of triple and multiple labeling protocols
1. Immunoreactivity patterns of aSyn epitopes
Using immunofluorescent stainings, antibodies against different domains and PTMs of aSyn were co-visualized and their local distribution patterns were assessed in pathological structures and neurons. Triple labeling experiments, including DAPI and two antibodies against aSyn were performed to obtain insight into their distribution patterns. Moreover, to allow systematic comparison of distribution patterns of different aSyn epitopes, protocols were developed to visualize multiple (4 or 5) antibodies against aSyn in the same section.To validate findings from the initial multiple labeling experiments, different antibodies against similar epitopes were selected. This ‘validation set’ of antibodies was optimized for additional multiple labeling protocols. The sets of antibodies used in the different protocols are specified in Table S2. No antigen retrieval methods or permeabilization steps were applied in any of these experiments.
For each protocol, we made use of a combination of direct and indirect immunodetection methods. Several primary antibodies (specified in Table S2) were directly labeled with fluorochromes following standard protocols of different labeling kits (art. no. A20181, A20183, A20186, 21335 for labeling with Alexa 488, Alexa 546, Alexa 647, and biotin, respectively; Thermo Fisher Scientific, Waltham, USA). Each protocol started with an indirect immunolabeling using unlabeled primary antibodies raised in rabbit/mouse using their appropriate secondary antibodies (with different conjugates, specified in Table S2). Sections were then blocked for 1 hour in 5% normal rabbit serum and 5% normal mouse serum in PBS. After this, a biotinylated primary antibody (raised in mouse or rabbit) could be incubated, and visualized by fluophore-conjugated streptavidin. Then, sections were incubated in blocking solution (2% normal goat serum) containing the diluted directly labeled antibodies together with DAPI (1 µg/ml). Sections were mounted in Mowiol mounting solution using glass cover slips (Art. No.: 630-2746; Glaswarenfabrik Karl Hecht, Sondheim, Germany). Negative control stainings lacking primary antibodies were performed to control for background/autofluorescence levels and aspecific staining. Single stainings using a directly labeled antibody against Ser129-p aSyn were scanned to determine autofluorescence levels of the studied morphological structures (LBs, LNs), which was found negligible under the applied scan settings.
Association CTT and Ser129-p aSyn with subcellular markers
In order to study the association of immunoreactivity of CTT and p-Ser129 aSyn with subcellular structures, additional multiple labeling protocols were further designed. Apart from the described antibodies against aSyn, these protocols also included some commercial antibodies as markers for subcellular structures, including mitochondria, ER and cytoskeletal proteins (Table S2). In these protocols, heat-induced epitope retrieval using citrate buffer (pH 6) and a permeabilization step (1hr incubation in 0.1% Triton-x) was included. Negative control stainings lacking primary antibodies were included to control for background/autofluorescence levels and aspecific staining.
Confocal laser scanning microscopy and stimulated emission depletion microscopy
CSLM and STED microscopy were performed using a Leica TCS SP8 STED 3X microscope (Leica Microsystems). All images were acquired using a HC PL APO CS2 100× 1.4 NA oil objective lens, with the resolution set to a pixel size of 20 nm x 20 nm. All signals were detected using gated hybrid detectors in counting mode. Sections were sequentially scanned for each fluorophore, by irradiation with a pulsed white light laser at different wavelengths (indicated in Table S2). Stacks in the Z-direction were made for each image. To obtain CSLM images of the DAPI signal, sections were irradiated with a solid state laser at a wavelength of 405 nm. For STED imaging, a pulsed STED laser line at a wavelength of 775 nm was used to deplete Abberior (580, 635P), Alexa (594, 647) or Li-Cor (680 nm) fluorophores, while continuous wave (CW) STED lasers with wavelengths of 660 nm and 592 nm were used to deplete the Alexa 546 and Alexa 488 fluorophores, respectively. The DAPI signal was not depleted, so this channel was scanned at the same resolution as the CSLM images.
After scanning, deconvolution was performed using CMLE (for CSLM images) and GMLE algorithms in Huygens Professional (Scientific Volume Imaging; Huygens, The Netherlands) software. Images were adjusted for brightness/contrast in ImageJ (National Institute of Health, USA). 3D reconstructions were made using the LAS X 3D Visualization package (Leica Microsystems). Final figures were composed using Adobe Photoshop (CS6, Adobe Systems Incorporated).
Image processing and semi-quantitative analysis
Structures were classified and selected for inclusion in the analysis based on morphological grounds (specified in Results section). Additional criteria for inclusion were 1) the diameter of the structure (at least 5µm) and 2) the presence of specific signal for all channels (signal intensity of raw CSLM images substantially higher than autofluorescence or background levels under the applied scan settings). Based on this selection, distribution patterns of immunoreactivities could be analyzed on deconvolved CSLM images of 29 LBs in the SN of 8 patients with advanced PD (Table S1). Z-stacks were made for each structure, of which three frames in the central portion of the structure (Z length: 0.30µm; step size between frames: 0.15 µm) were selected to quantify the x-y distribution for different markers. For the analysis, a maximum Z-projection of these selected frames was first made in ImageJ. Subsequently, three 100 px (2µm) thick lines were drawn over the equatorial planes of the LBs 40 in ImageJ, along which signal intensities for each channel were measured. The intensity for each channel at each point was normalized to its maximum intensity in the same structure, while the position along the diameter was expressed as % diameter. Normalized values were used to generate average line profiles per morphological structure. The center of the LB was defined as the origin of the structure 40. The position in the LB with the maximum intensity was determined per channel. Absolute positions, with low values corresponding to positions close to the origin of the LB and high values to positions at their periphery, were compared between channels (Test: One way ANOVA; post-hoc: Bonferroni).
Coherent anti-Stokes Raman scattering
The workflow used for CARS microscopy is outlined in Figure S7. The detection of the lipid and protein distribution was performed on native, dried samples 52, 53. A commercial setup (Leica TCS SP5 II CARS, Leica Microsystems) was used with an HCX IRAPO L25X/0.95W (Leica Microsystems) objective. For the lipid distribution intensity images were taken at 2850 cm-1 (Pump-wavelength 816 nm, Stokes-wavelength 1064 nm) and for the protein distribution intensity images at 2930 cm-1 (Pump-wavelength 810 nm, Stokes-wavelength 1064 nm). The laser power at the sample was 28 mW (Pump) and 21 mW (Stokes). Integration times of 34 s per image with a pixel dwell time of 32 µs, 1024×1024 pixels and a spatial resolution of 300 nm were used 54. After the label-free detection of the lipid and protein distribution, immunofluorescent stainings were performed on the same sections (Fig. S7). Tissue sections were fixed in 4% formaldehyde for 10 minutes and stained for aSyn, using two primary antibodies raised against aSyn (LB509; ab27766, Abcam, Cambridge, UK) and Ser129-p aSyn (ab59264, Abcam) and their appropriate secondary antibodies. After this, sections were incubated in Sudan Black for 30 min and mounted in Mowiol. For fluorescence detection, a commercial setup (Leica TCS SP5 II CARS, Leica Microsystems, Heidelberg, Germany) was used. Data evaluation was done in Matlab with the Image Processing and Statistics toolboxes (The Mathworks, Inc., Mass.,USA). First, large overview CARS-intensity and fluorescence images were manually overlaid by comparison of morphological features. The distribution of aSyn, proteins and lipids was identified by the overlay of both fluorescence images (Fig. S8). Therewith, autofluorescence of the surrounding tissue and the fluorescent signal of the aSyn-immunopositive inclusions could be separated. The inclusion bodies were manually identified based on morphology. Only inclusions with a diameter of 5-20 µm were included for analysis.
Image processing and analysis of CARS images
For an objective evaluation of CARS intensity of aSyn-immunopositive inclusion bodies, the mean CARS intensity of the direct surrounding, a donut with a width of 3.5 µm, was compared with the CARS intensity of the inclusion (Fig. S8A, light blue and yellow area). The areas of aSyn-immunopositivity were transferred into the CARS-intensity images. Areas with no intensity in the CARS intensity images (holes) were excluded by intensity thresholding. CARS-pixel-intensities higher than 1.4 times the mean CARS-intensity of the surrounding were defined as higher protein/lipid content, which was determined based on pilot measurements in a subset of (~40) aSyn-positive inclusions. The ratio between the CARS-pixel-intensities of the LB and the mean CARS intensity of the surrounding were calculated and the areas with higher protein/lipid content were marked in red (Fig. S8). Morphological filtering and image processing were performed in Matlab R2017a, MathWorks.
Author contributions
T.M. and C.M. performed immunohistochemistry and multiple labeling experiments. T.M., C.M., E.T., J.K. and W.vdB. performed STED imaging, as well as processing and analysis of images. D.N., D.P., S.EM. and K.G. performed experiments and data analysis for CARS microscopy. D.M. performed the labeling of antibodies for multiple labeling experiments and contributed to the experimental design. M.B., W.Z., R.B., O.M., K.K., S.H., M.H., T.K., M.R., S.D. selected and characterized the aSyn antibodies. T.M., W.vdB, J.G.and M.B. designed research, analyzed and interpreted the data, and contributed to writing the manuscript.
Competing financial interests
The authors declare no competing interests. D.M., O.M., K.K., S.H., M.H., T.K., M.R., S.D., and M.B. are full-time employees of Roche/F. Hoffmann–La Roche Ltd, and they may additionally hold Roche stock/stock options. W.Z. and R.B. are full-time employees of Prothena Biosciences Inc.
Corresponding author
Correspondence to: Wilma van de Berg (wdj.vandeberg{at}vumc.nl)
Legends for supplementary video files:
Video S1: 3D reconstruction based on deconvolved CSLM images revealing the distribution of Ser129-p. 119CTT and 122CTT aSyn in an onion-skin type LB in the SN of patient PD7.
Video S2: 3D reconstruction based on deconvolved CSLM images revealing the distribution of Ser129-p. 119CTT and 122CTT aSyn in another onion-skin type LB in the SN of another patient (PD6).
Video S3: 3D reconstruction based on deconvolved CSLM images revealing the distribution of antibodies directed against NT, NAC domain and CT in a onion-skin type LB in the SN of patient PD8.
Video S4: 3D reconstruction of an entire onion-skin type LB based on deconvolved CSLM images showing the distribution patterns of antibodies against different PTMs and domains of aSyn in the SN of patient PD1.
Video S5: 3D reconstruction based on deconvolved CSLM images of expansive-appearing inclusion body showing the distribution patterns of antibodies against different PTMs and domains of aSyn in the SN of patient PD8.
Video S6: 3D reconstruction based on deconvolved STED images showing the filamentous cage-like framework formed by Ser129-p aSyn and cytoskeletal components at the periphery of an onion-skin type LB in the SN of patient PD7.
Video S7: 3D reconstruction based on deconvolved STED images showing localization of a CTT-reactive punctae at the outer membrane of a mitochondrion immunopositive for VDAC/Porin in the hippocampus of patient PD4.
Acknowledgements
We are grateful to all individuals that donated their brain to the Netherlands Brain Bank (NBB; www.brainbank.nl). We thank the team of the NBB, in particular Michiel Kooreman, for their cooperation and their help in the selection of brain tissue. We thank the Advanced Optical Microscopy Core O|2 (www.ao2m.amsterdam) for support with STED imaging. Further, we thank Lidia Janota for performing immunofluorescent stainings of the tissue sections used for CARS microscopy.