Lack of pathogenic potential of peripheral α-synuclein aggregates from Parkinson’s disease patients
© The Author(s). 2018
Received: 24 January 2018
Accepted: 26 January 2018
Published: 8 February 2018
In Parkinson’s disease (PD) there is widespread accumulation in the brain of abnormal α-synuclein aggregates forming intraneuronal Lewy bodies (LB). It is now well established that LB-type α-synuclein aggregates also occur in the peripheral autonomic nervous system in PD, from where it has been speculated they may progressively spread to the central nervous system through synaptically-connected brain networks and reach the substantia nigra to trigger herein dopaminergic dysfunction/degeneration and subsequent parkinsonism. Supporting a pathogenic role for α-synuclein aggregates we have previously shown that LB purified from postmortem PD brains promote α-synuclein pathology and dopaminergic neurodegeneration when intracerebrally inoculated into wild-type mice. However, the pathogenic capacity of PD-derived peripheral α-synuclein aggregates remains unknown. Here we addressed this question using purified LB-type α-synuclein aggregates from postmortem PD stellate ganglia (SG), a paravertebral sympathetic ganglion that exhibits consistent and conspicuous Lewy pathology in all PD patients. In contrast to our previous findings using nigral LB extracts, intracerebral inoculation of SG-derived LB into mice did not trigger long-term nigrostriatal neurodegeneration nor α-synuclein pathology. The differential pathogenic capacities of central- and peripheral-derived α-synuclein aggregates appear independent of the absolute amount and basic biochemical properties of α-synuclein within these aggregates and may rely instead on differences in α-synuclein conformation and/or yet unrecognized brain region-specific intrinsic factors. Our results argue against a putative pathogenic capacity of peripheral α-synuclein aggregates to promote α-synuclein pathology in the brain, propagate between neuronal networks or induce neurodegeneration.
Brain pathology in Parkinson’s disease (PD) is characterized by prominent loss of dopaminergic neurons from the substantia nigra pars compacta (SNpc) and the presence in affected brain regions of intraneuronal Lewy bodies (LB) and Lewy neurites (LN) consisting mostly of aggregated α-synuclein. At late stages of the disease, deposits of aggregated α-synuclein are widely spread in the central nervous system (CNS). In addition, it is now well established that LB-type α-synuclein aggregates also occur in the peripheral autonomic nervous system (pANS) [1, 8, 25]. It has been proposed that Lewy pathology may actually start in the periphery and progressively spread to the CNS through synaptically-connected brain networks, driving neuronal dysfunction and death along the way . Supporting this concept, most PD patients experience a premotor period several years before the emergence of parkinsonism characterized by a variety of non-motor symptoms driven by neuronal damage in extranigral regions of the central and peripheral autonomic nervous system . Furthermore, α-synuclein brain pathology can be triggered in experimental animals, including rodents and non-human primates, by intracerebral, intramuscular, intragastrical or intravenous inoculation of recombinant α-synuclein fibrils or brain homogenates from affected mice and patients [5, 11, 13, 14, 16, 18–21]. Indeed, we have previously shown that intranigral inoculation of α-synuclein-containing LB extracts purified from the SNpc of PD brains promotes widespread α-synuclein pathology and dopaminergic neurodegeneration in recipient mice . However, the pathogenic capacity of PD-derived peripheral α-synuclein aggregates remains unknown.
Here we assessed for the first time the in vivo pathogenic potential of peripheral α-synuclein aggregates by inoculating α-synuclein-containing LB extracts purified from postmortem stellate ganglia (SG) of PD patients into the SNpc of wild-type mice. SG is a paravertebral sympathetic ganglion that exhibits consistent and conspicuous Lewy pathology in PD patients, but not in control subjects [7, 8, 17]. In fact, SG is one of the few peripheral tissues that invariably exhibits α-synuclein pathology in PD and the one with the highest burden of pathological α-synuclein . In particular, abundant LB and LN-like structures immunoreactive for α-synuclein, phosphorylated α-synuclein and ubiquitin are seen in the SG of PD patients, with a highest density at the periphery of the ganglia . Lewy pathology in the SG is accompanied by neurodegenerative changes in this ganglion, such as enlarged neurons with reduced tyrosine hydroxylase (TH) immunoreactivity, loss of pigmented and non-pigmented neurons with increased cellularity and formation of Nageotte nodules, as well as frequent microglia/macrophage activation . For the above reasons, here we have chosen the SG to purify PD-linked peripheral α-synuclein aggregates to assess their pathogenic potential in vivo.
Purification of LB from human SG
Human sample information
Age at death (years)
aSyn in SG
PD stage 5
PD stage 5
PD stage 4
AD VI C+ TDP amygdala
AD IV C + thalamic infarct
Filter retardation assay
This technique was performed as previously described . Briefly, after heating at 100 °C for 5 min, samples (70 mg) were diluted in 200 μl of migration buffer (25 mM Tris-HClBase, 200 mM glycine, SDS 1%) and filtered through a cellulose acetate membrane (Schleicher & Schuell; 0.2 μm pore size) using a Minifold-1 Dot-Blot System (Schleicher & Schuell). Membranes were saturated in 5% dried skimmed milk in phosphate-buffereld saline (PBS) and probed with human α-synuclein antibody (see below for details). Appropriate secondary antibodies coupled to peroxidase were revealed using a Super Signal West Pico Chemiluminescent kit (Pierce). Chemiluminescence images were acquired using the ImageQuant RT ECL Imager (GE Healthcare).
SG-derived fractions were resolved by SDS-PAGE on 15% polyacrylamide gels and electrotransferred onto nitrocellulose membranes (GE Healthcare, #10401196), then blocked in 5% non-fat milk powder in tris-buffered saline (TBS) containing 0.1% tween-20 (TBS-T) for 1 h at room temperature (RT) and incubated overnight at 4 °C with a mouse anti-human α-synuclein antibody (1/500 in 4% bovine serum albumin/TBS-T, Thermo Scientific, #MS1572). Incubation with the anti-mouse secondary antibody coupled to horseradish peroxidase (1:2000 in 5% non-fat milk powder/TBS-T; Amersham Biosciences, #NXA931) was performed at RT for 1 h, followed by repeated washings with TBS-T. Immunoreactive bands were visualized using SuperSignal Femto Chemiluminescent Substrate (Pierce, #34096) according to the manufacturer’s instructions on an ImageQuant RT ECL imaging system (GE Healthcare).
Enzyme-linked immunosorbent assay
Total protein concentrations in the different sucrose extracts were determined using the bicinchoninic acid assay (#23227; Thermo Scientific, Waltham, MA). Samples were bath sonicated for 5 min, diluted to 20 μg/ml of total protein and analyzed in triplicates for total α-synuclein protein levels with a specific enzyme-linked immunosorbent assay (ELISA) kit against human α-synuclein (Life Technologies, #KHB0061) according to the manufacturer’s instructions.
For stereotactic inoculations, LB-containing SG fractions from the three PD patients were first mixed together in the same proportion (PD #1, fractions 17 and 18; PD #2, fraction 18; PD #3, fractions 16 and 17; see Fig. 1). Control animals were inoculated with a mixture of equivalent fractions obtained from SG of two age-matched human control subjects lacking α-synuclein pathology (Ctrl #1, fraction 17; Ctrl #2, fractions 16 and 17; see Fig. 1). An additional control group was injected with the corresponding buffer (vehicle) obtained from a sucrose gradient purification performed without the addition of any human tissue sample. In all cases, samples were bath-sonicated for 5 min prior to the in vivo inoculations. Wild-type C57BL/6 mice (3 months old) received 2 μl of either LB-SG fractions, control-derived fractions, or appropriate buffer (vehicle) by stereotactic delivery to the region immediately above the right substantia nigra (− 2.9 mm AP, 1.3 mm L and − 4.5 mm DV) at a flow rate of 0.4 μl/min. Animals were euthanized at either 24 h or 6 months after injection and the position of the 30-gauge needle was determined histologically.
Cylinder behavioral test
Mice were tested for left and right forepaw use with the cylinder test one week before surgery (to establish the basal conditions for each animal) and at 6 months post-surgery. For the performance of the cylinder test, mice were first allowed to habituate to the experimental room for at least one hour before each test. Then, mice were put in a glass cylinder and the total number of left and right forepaw touches performed within 5 min was counted. Data are presented as the percentage of contralateral forepaw use with respect to total forepaw use (contralateral + ipsilateral). All behavioral tests were performed during the light cycle by an investigator blinded to the experimental groups.
Twenty-four hours or six months after stereotactic inoculations, mice were euthanized by perfusion with 4% PFA and their brains were processed for immunohistochemical analyses. Immunostaining was performed on 20 μm-thick free-floating sections incubated with different primary antibodies (see below) for 24 h at 4 °C. Biotinylated secondary antibodies, followed by signal amplification using the avidin-biotin complex (ABC) method, were used. Immunostaining was revealed using 3,3′-diaminobenzidine tetrahydrochloride (DAB, Sigma Aldrich, #D5905-50TAB). When mouse secondary antibodies were required, a Vector M.O.M Immunodetection kit (Vector Laboratories, #BMK2202) was used, according to the manufacturer’s instructions. The following antibodies were used: Tyrosine Hydroxylase (1/1000, Calbiochem, #657012), human α-synuclein Ab-2 (clone syn211, 1/250, Thermo Scientific, #MS-1572), α-synuclein (1/1000, BD Transduction Laboratories, #610786), phosphorylated α-synuclein (phospho S129, 1/750, Abcam, #2014–1), Ionized calcium binding adaptor molecule 1 (Iba1, 1/1000, Wako Pure Chemical Industries, #019–19,741) and Glial Fibrillary Acid Protein (GFAP, 1/1000, Sigma-Aldrich, #G3893).
Nigrostriatal integrity: the total number of TH-positive SNpc neurons was assessed by stereology with the optical fractionator method in regularly spaced (every sixth) 20 μm-thick sections spanning the entire SNpc using StereoInvestigator software (MBF Bioscience). Striatal TH innervation was assessed by optical densitometry in regularly spaced 20 μm-thick sections corresponding to different striatal anatomical levels using Sigma Scan.
α-Synuclein pathology: (1) the total number of α-synuclein-positive neurons in the SNpc was assessed by stereology, as above; (2) the total number of hyperphosphorylated α-synuclein (pSyn)-positive neurons in the SNpc was manually assessed under light microscopy in four sections spanning the entire SN; (3) pSyn at regional level was assessed by optical densitometry using the ImageJ software in pSyn-immunostained sections scanned with an Epson Perfection V750 PRO scanner (Long Beach, CA); (4) intracellular α-synuclein levels were assessed by optical densitometry at cellular level using Sigma Scan in transmitted-light microscopy images of α-synuclein-positive cells from the SNpc (~ 650 neurons per group, randomly selected among the different animals).
Neuroinflammation: for inflammatory reaction assessment, microglia and astrocyte density were estimated by OD at regional level in Iba1- and GFAP-immunostained sections, respectively, using Sigma Scan software. All analyses were performed blinded to the researcher.
Proteinase K digestion
Twenty-micrometer-thick sections were washed with TBS and incubated in proteinase K (PK; Invitrogen, Carlsbad, CA; 1 μg/ml in TBS) at RT for 10 min. The sections were then washed in TBS and immunostained for α-synuclein as indicated above.
All values are expressed as the mean ± standard error of the mean (SEM). Statistical comparisons between control-injected and LB-SG-injected animals were performed with SigmaStat software (v4, Systat Software Inc., USA) using two-tailed t-test, Mann-Whitney rank sum test or one-way ANOVA, as appropriate. Selection of the pertinent statistical test for each experiment was determined after formally testing for normality. In all analyses, the null hypothesis was rejected at the 0.05 level.
Purification of SG LB from PD patients
Lack of nigrostriatal degeneration in mice inoculated with SG-derived LB extracts
Lack of α-synuclein pathology in mice inoculated with SG-derived LB extracts
Because we previously found that inoculation of SNpc-derived LB extracts from PD patients to mice triggered the pathological conversion and intracellular accumulation of endogenous murine α-synuclein , we next assessed whether injection of LB-SG fractions resulted in a similar pathogenic effect. Using an antibody that recognizes murine α-synuclein, we assessed first by stereology the number of α-synuclein-immunopositive neurons in the SNpc of LB-SG-injected mice at 6 months post-inoculation. No differences were found in the number of SNpc α-synuclein-positive neurons between LB-SG- and control-injected animals (Fig. 3b). Similarly, no differences were found in the intracellular levels of α-synuclein, assessed by optical densitometry, between LB-SG- and control-injected animals at 6 months (Fig. 3c). In the absence of changes in α-synuclein levels, we next assessed whether endogenous α-synuclein in LB-SG-injected mice might have adopted a pathological insoluble/aggregated beta-sheet conformation resistant to proteinase K (PK) digestion, as we have previously seen for SNpc-derived LB extracts . Ruling out this possibility, no differences were found in the susceptibility of α-synuclein to PK digestion (1μg/ml for 10 min) between LB-SG- and control-injected animals at 6 months (Fig. 3d). Further excluding a pathological conversion of α-synuclein in LB-SG-injected mice, no changes in hyperphosphorylated pathological forms of α-synuclein were found between LB-SG- and control-injected animals at 6 months (Fig. 3e-f). LB-SG- and control-injected animals exhibited equivalent levels of pSyn, both at the site of injection (i.e. SNpc, Fig. 3e) and in distant anatomically connected brain regions (i.e. striatum and neocortical areas, Fig. 3f). Overall, these results indicate that, in contrast to nigral LB extracts , peripheral LB-SG extracts are not able to trigger long-term α-synuclein pathology in mice.
This study assessed the pathogenic potential of peripheral α-synuclein aggregates from PD patients. In contrast to our previous observation using nigral-derived LB extracts , peripheral α-synuclein aggregates from SG did not produce any pathologic effect when inoculated into the brain of recipient wild-type mice, up to 6 months post-injection. While exogenous human α-synuclein from SNpc- and SG-derived LB extracts were both quickly internalized by host murine SNpc neurons, only animals receiving SNpc-derived LB extracts exhibited PD-like pathology . Indeed, in previous experiments, animals injected with SNpc-derived LB extracts exhibited nigrostriatal neurodegeneration and widespread α-synuclein pathology by 4 months post-inoculation , while none of which was observed in LB-SG-injected mice at 6 months. The differences in pathogenicity between SNpc- and SG-derived extracts cannot be attributed to methodological issues, as both studies were performed in an identical manner, including the intracerebral site of injection (i.e. SNpc) . Both SNpc and SG LB fractions were purified using the same sucrose gradient fractionation method and the amount of total α-synuclein recovered in these fractions was comparable (~ 15 ng/mg vs ~ 17 ng/mg, respectively, as quantified by ELISA). In addition, the distribution pattern of α-synuclein aggregates, revealed by filter retardation assay, was equivalent between nigral and SG LB fractions, corresponding in both studies to recovered fractions #16–18. The differential pathogenicity did not result either from the presence of a putative pathogenic factor other than α-synuclein specifically within nigral-derived LB fractions, since the presence of α-synuclein in these fractions was an absolute requirement for their pathogenic effect . The differential pathogenicity between nigral and SG LB fractions might be linked to differences in α-synuclein conformation within these fractions and/or to yet unrecognized region-specific intrinsic factors. Supporting this concept, crosslinking experiments have shown that endogenous α-synuclein species are different between human brain and human small intestine . Along this line, a previous study in A53T α-synuclein-overexpressing transgenic mice reported that α-synuclein oligomers obtained from different CNS regions exhibited differential pathogenic capacities in vitro, in terms of promoting α-synuclein amyloid fibril formation and neurotoxicity, despite sharing similar biochemical properties . It has also been recently revealed the existence of different strains of α-synuclein able to adopt different structural conformations that cause distinct histopathological and behavioral phenotypes when injected into experimental animals . In this context, SG LB fractions might need further maturation (e.g. conformation changes, protein interactions, further processing and/or additional post-translational modifications beyond phosphorylation) to acquire pathogenic characteristics identical to those originating from SNpc. Alternatively, SG LB extracts might just be slower in triggering α-synuclein pathology and thus require longer incubation times than SNpc-derived α-synuclein aggregates to produce pathology. Further studies to identify the exact composition and structure of PD-linked α-synuclein aggregates from different areas of CNS and peripheral nervous system (PNS) should shed light on this matter.
Our results indicate that peripheral α-synuclein aggregates, in particular those derived from the SG, lack the capacity to promote α-synuclein pathology in the brain, propagate between neuronal networks or induce neurodegeneration. This observation argues against one of the currently prevalent pathogenic hypothesis of cell-to-cell transmission of α-synuclein from the periphery to the CNS . However, the interpretation of our results needs some caution. In our study, we have chosen the SG as peripheral tissue because the SG is the peripheral structure that exhibits the highest burden of α-synuclein pathology and it does so invariably in all PD patients, but not control subjects . However, it is possible that α-synuclein aggregates from other peripheral tissues might behave differently from a pathogenic point of view and, therefore, our results might not be generalized to all peripheral structures. This question could be addressed in subsequent studies by injecting α-synuclein aggregates derived from other peripheral regions. For instance, due to its accessibility, the gastrointestinal tract has been proposed as one of the potential earliest sites of α-synuclein pathology from where α-synuclein aggregates, initiated by exposure to a putative pathogen or infectious agent, could spread retrogradely to the brain through vagal nerve connections [3, 8–12, 23]. It appears, however, that α-synuclein can also be transported anterogradely from the brain to the stomach , raising the question as to whether peripheral α-synuclein aggregates really precede brain pathology in PD or may instead occur concomitantly or even follow CNS involvement .
While we found here that peripheral α-synuclein aggregates from SG are not pathogenic when injected into the brain, the presence of α-synuclein accumlation in peripheral tissue is often associated with peripheral autonomic denervation [8, 29], thereby suggesting a potential pathogenic effect of these aggregates in the periphery. Whether these pathological alterations in peripheral tissue are actually caused by the presence of α-synuclein remains to be determined. Because here we used postmortem SG samples derived from late PD stages, it is also possible that peripheral α-synuclein aggregates from earlier disease stages might exhibit different pathogenic capacity. This question could be eventually tested using peripheral α-synuclein aggregates from early manifest or even pre-motor PD subjects, such as those with idiopathic REM sleep behavior disorder [23, 28] or incidental Lewy body disease subjects [1, 6]. However, our previous results using SNpc-derived LB extracts demonstrated that samples derived from late stages of the disease can actually be pathogenic .
Overall, here we demonstrate that, in contrast to CNS-derived LB , peripheral LB-type α-synuclein aggregates from PD patients are not pathogenic when injected to mice. The differential pathogenic capacity of CNS- and PNS-derived α-synuclein aggregates appears to be independent of the absolute amount and basic biochemical properties of α-synuclein within these fractions and may rely instead on differences in α-synuclein conformation and/or yet unrecognized brain region-specific intrinsic factors. The identification of such factors should shed light on the physiopathological role of α-synuclein in PD and open new avenues for therapeutic intervention.
In addition, the Authors would like to acknowledge additional support from FIS-ISCIII-FEDER (PI15/01937 to J.B.). We are also grateful to Ronald Melki and Luc Bousset (Paris-Saclay Institute of Neuroscience, France) for helpful discussions.
This work was supported by funds from the Fondo de Investigación Sanitaria-Instituto de Salud Carlos III (FIS-ISCIII, Spain)-European Regional Development Fund (FEDER, E.U.) (PI13/01897, to M.V.), Ministry of Economy and Competitiveness (MINECO, Spain) (SAF2016–77541-R and RTC-2014-2812-1, to M.V.) and CIBERNED (to M.V.).
Availability of data and materials
All the data and material associated to this work will be available upon request according to the journal policies.
Conceptualization, MV and ET; Human tissue collection, EG; Lewy body purification and biochemical characterization, AR, ICC, AP; Stereotactic injections, JB; Histological analyses: AR, ICC, AP and MV; Writing-Original Draft, AR and MV; Writing-Review & Editing, AR, ICC, AP, JB, EG, ET and MV; Task supervision and funding acquisition, MV. All authors read and approved the final manuscript.
All animal experiments were conducted in accordance with guidelines established by the Ethical Committee for the Use of Laboratory Animals in Spain (53/2013) and the European Ethical Committee (2010/63/EU), and approved by the Vall d’Hebron Research Institute (VHIR) Ethical Experimentation Committee.
The authors declare that they have no competing interests.
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