Peripheral administration (oral, intranasal, intraperitoneal, intravenous) of assembled A53T α-synuclein induced synucleinopathy in heterozygous mice transgenic for human mutant A53T α-synuclein (line M83). The same was the case when cerebellar extracts from a case of multiple system atrophy with type II α-synuclein filaments were administered intraperitoneally, intravenously or intramuscularly. We observed abundant immunoreactivity for pS129 α-synuclein in nerve cells and severe motor impairment, resulting in hindlimb paralysis and shortened lifespan. Filaments immunoreactive for pS129 α-synuclein were in evidence. A 70% loss of motor neurons was present five months after an intraperitoneal injection of assembled A53T α-synuclein or cerebellar extract with type II α-synuclein filaments from an individual with a neuropathologically confirmed diagnosis of multiple system atrophy. Microglial cells changed from a predominantly ramified to a dystrophic appearance. Taken together, these findings establish a close relationship between the formation of α-synuclein inclusions in nerve cells and neurodegeneration, accompanied by a shift in microglial cell morphology. Propagation of α-synuclein inclusions depended on the characteristics of both seeds and transgenically expressed protein.
Jennifer A. Macdonald and John L. Chen contributed equally
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corrected publication 2021
The ordered assembly of a small number of proteins into pathological amyloid filaments defines most human neurodegenerative diseases, including Alzheimer’s (AD) and Parkinson’s (PD) [
A link between α-synuclein assembly and disease was established by the findings that missense mutations in
Accumulating evidence indicates that assembled α-synuclein propagates in the nervous system in a manner akin to prions.
The dual-hit hypothesis of PD posits that the filamentous assembly of α-synuclein begins in the nose and the digestive tract, following entry of a pathogen through the nasal cavity, which can reach the gut following swallowing [
Here we investigated the effects of oral, nasal, intravenous, intraperitoneal and intramuscular administration of α-synuclein seeds on assembly, neurodegeneration and neuroinflammation. Following the oral and nasal administrations, implicated in the dual-hit hypothesis, we characterised for the first time the initial sites affected by α-synuclein pathology and their similarity to those first implicated in prion transmission [
Human A53T α-synuclein or human A53T α-synuclein lacking residues 71–82 (Δ71–82), which shows a greatly reduced ability to assemble into filaments [
Experiments used 3-month-old heterozygous M83 mice. For nasal administration, mice received 50 μl of 400 μM assembled A53T α-synuclein (0.28 mg) over both nostrils daily for 28 days. Oral administration was achieved by daily gavage using 20-gauge plastic tubes (Instech Laboratories) of 200 μl of 400 μM assembled A53T α-synuclein (1.1 mg) for 28 days. Intravenous injection consisted of daily injections of 1 mg assembled A53T α-synuclein into the tail vein for 4 days. In some experiments, 100 μg, 10 μg or 1 μg were injected. For intraperitoneal injection, 200 μl of 400 μM assembled A53T α-synuclein (1.1 mg) or assembled Δ71–82 A53T α-synuclein was administered. PBS was used as control. Motor impairment was assessed by Rotarod, using acceleration from 4 to 40 rpm over 5 min. The time was recorded when mice fell from the rod or when they rotated passively for two consecutive revolutions. Severe motor impairment consisted of abnormal gait, abnormal posture when lifted by the tail, hindlimb paralysis and an abnormal righting reflex.
Cerebellum from a 68-year-old male with a neuropathologically confirmed diagnosis of MSA was homogenised in PBS at 100 mg/ml or 200 mg/ml. At autopsy, numerous α-synuclein-positive inclusions were present in motor cortex, striatum, substantia nigra, pontine nuclei, inferior olive and cerebellum. They were glial cytoplasmic inclusions and, for some regions, also neuronal cytoplasmic and intranuclear inclusions. Homogenates were sonicated using an XL2020 ultrasonic processor (Misonix) at output level 2 (ON, 0.9 s, OFF 0.1 s, for a total of 5 s). Following a 5 min centrifugation at 3,000 g, the supernatants were aliquoted and stored at -80° C until use. Injections were given intravenously, intramuscularly or intraperitoneally. As the control, cerebellum from a 68-year-old male without synucleinopathy was used at 200 mg/ml.
To detect assembled α-synuclein by immunohistochemistry, we used two anti-pS129 α-synuclein antibodies (clone 64, Wako and EP1536Y, Abcam) and two anti- α-synuclein antibodies (1903, Abcam and LB509, Covance). Phosphorylation-dependent antibodies were used at 1:1,000 and phosphorylation-independent antibodies at 1:10,000. To assess microglia in the context of α-synuclein inclusions, anti-ionized calcium binding adaptor molecule 1 (Iba1) (019–9741, 1:500, Wako), a panel of epitope-specific anti-α-synuclein antibodies (α-Syn34-45, 1:200, BioLegend; α-Syn80-96, 1:100, BioLegend; α-Syn117-122, 1:100, BioLegend) and an antibody specific for α-synuclein phosphorylated at S129 (EP1536Y, 1:750 or ab184674 1:500, Abcam) were used. For negative-stain immunoelectron microscopy, we used ab59264 and PER4 at 1:100 or ab51253 at 1:50 [
Mice were perfused transcardially with 4% paraformaldehyde in 0.1 M PBS, pH 7.4. Brains and spinal cords were dissected and post-fixed overnight at 4°C, followed by cryo-protection in 20% sucrose in PBS for a minimum of 24 h. Coronal brain sections (40 μm) were cut using a VT1000 S vibratome (Leica). Transverse spinal cord sections (30 μm) were cut on a Leica SM2400 microtome (Leica Microsystems). Sections were stored at 4° C in PBS containing 0.1% sodium azide. Endogenous peroxidase activity was quenched by incubation in 0.3% H2O2 for 30 min. Following a brief wash in PBS + 0.1% Triton-X100 (PBST), the sections were incubated in blocking buffer (PBST + 5% normal goat serum) for 15 min. This was followed by an overnight incubation at room temperature with primary antibody in blocking buffer. After three rinses with PBST, the sections were incubated with biotin-conjugated secondary antibodies for 1 h at room temperature. Following a further three rinses with PBST, the avidin–biotin-conjugated complex was applied at room temperature for 30 min. The signal was visualised with the Vector VIP substrate kit (Vector Laboratories). Tissue sections were mounted on frosted end glass slides (Thermo Scientific) and coverslipped.
Free-floating brain sections were incubated for 1 h in 5% normal goat serum in PBST. For epitope-specific α-synuclein antibodies, tissue sections were first treated with 80% formic acid for 1 min at room temperature prior to blocking. Formic acid was not used for antibodies specific for α-synuclein phosphorylated at S129. Sections were incubated in primary antibodies overnight at 4° C. Secondary antibodies conjugated to Alexa Fluor 488 or 594 were then added and incubated for 2 h at room temperature. Prior to labelling with DAPI, some sections were incubated with 3 μM pentameric formyl thiophene acetic acid (pFTAA) for 30 min, followed by 3 washes in PBST. Autofluorescence was quenched by immersing sections for 30 s in TrueBlack Lipofuscin Autofluorescence Quencher (Biotium) solution, followed by 3 washes in distilled water. Sections were then mounted on SuperFrost slides (Thermo Fisher) and coverslipped with Vectashield Antifade mounting medium (Vector Labs). Images were acquired on an LSM 780 confocal microscope (Zeiss).
Brains and spinal cords were homogenised at 10 ml/g in 10 mM Tris–HCl, pH 7.4, 800 mM NaCl, 10% sucrose, 1 mM EGTA and 1 mM PMSF with proteinase inhibitors. The sarkosyl-insoluble fraction was prepared as described [
Immunoelectron microscopy was done as described [
α-Synuclein filaments were extracted from the cerebellum of a neuropathologically confirmed case of MSA, as described [
Stereology was carried out as described [
Photographs were taken using an Olympus BX41 microscope equipped with a Nikon digital Sight DS-2Mv digital camera under a × 20 objective. Each picture amounted to 1,600 × 1,200 pixels. Immunoreactivity for pS129- α-synuclein was quantitated using the green channel of ImageJ. The threshold was set to 110 in greyscale. The circularity was 10-infinity. For assessment of microglial morphology, the investigator annotated Iba1-immunoreactive microglia based on morphology from photographs taken under a × 20 objective. The investigator was blinded with respect to the nature of the groups.
Analyses were carried out using GraphPad Prism 7 software. They included log-rank tests for survival data and unpaired one-tailed t-tests for rotarod data and electron micrograph measurements. The significance level was set at
Oral administration consisted in daily gavaging of heterozygous mice transgenic for human A53T α-synuclein (line M83) with 200 μl of 400 μM assembled A53T α-synuclein for 28 days. Nasal administration involved daily administration of 50 μl of 400 μM assembled A53T α-synuclein for 28 days. We used an antibody specific for α-synuclein phosphorylated at S129 to show widespread staining throughout brain and spinal cord 5 months after oral and nasal administration of assembled A53T α-synuclein (Fig. Synucleinopathy in M83± mice following oral (
Following the administration of assembled A53T α-synuclein by either the oral or the nasal route, M83± mice developed severe motor abnormalities characterised by abnormal posture and gait, hindlimb dysfunction, inability to right and, eventually, paralysis. Mean time from aggregate delivery to end stage disease was 195 ± 6 days (n = 10) after oral (Fig.
Staining for pS129 α-synuclein following oral administration of seeds was present after 2 months in the nucleus of the solitary tract, dorsal motor nucleus of the vagus nerve and intermediolateral spinal cord (Fig. Staining for pS129 α-synuclein (Wako pSyn#64) following oral administration of assembled A53T α-synuclein to M83± mice.
M83± mice received 1 mg assembled A53T α-synuclein intravenously on 4 consecutive days. They developed synucleinopathy and progressive motor dysfunction and were culled after 156 ± 5 days (n = 4) (Fig. Synucleinopathy in M83± mice following intravenous injection of assembled A53T α-synuclein.
M83± mice received an intraperitoneal injection of 200 μl of 400 μM assembled A53T α-synuclein. As shown in Fig. Staining of lumbar spinal cord from M83± mice for pS129 α-synuclein (Abcam, EP1536Y) following intraperitoneal injection of assembled A53T α-synuclein. Staining was in evidence 3 and 5 months, but not 1 month, after injection. No specific staining was seen after 5 months in uninjected, PBS-injected or M83± mice injected with assembled Δ71–82 A53T α-synuclein. The dashed line delineates the ventral horn. Scale bars, 100 μm
Mice injected with assembled A53T α-synuclein developed progressive motor impairment and were culled after 155 ± 13 days (n = 13). Controls received an intraperitoneal injection of 200 μl PBS and were culled 165 days later (n = 8). There was no staining for pS129 α-synuclein, nor were there motor symptoms. The effects of assembled A53T α-synuclein on the number of spinal cord motor neurons were compared with those of assembled Δ71–82 A53T alpha-synuclein and PBS (n = 5) (Fig. Motor neuron numbers in lumbar spinal cord of M83± mice following intraperitoneal injection of PBS, assembled Δ71–82 A53T α-synuclein and assembled A53T α-synuclein. The number of motor neurons of PBS-injected mice is taken as 100%. Two-way ANOVA F(8,52) = 24.64, followed by Dunnett’s multiple comparisons test. **
Cerebellum from a 68-year-old male who had died with a neuropathologically confirmed diagnosis of MSA was used. Numerous α-synuclein-positive glial and neuronal inclusions were present in cerebellar white matter (Fig. Type II α-synuclein filaments extracted from MSA cerebellum. Cryo-EM data collection and processing Magnification 105,000 Voltage (kV) 300 Electron exposure (e–/Å2) 45.0 Defocus range (μm) −1.8 to −2.4 Pixel size (Å) 1.145 Symmetry imposed None Initial particle images (no.) 39,798 Final particle images (no.) 36,247 Map resolution (Å) 3.27 FSC threshold 0.143 Helical twist (°) −1.36 Helical rise (Å) 4.70
M83± mice were injected intravenously, intraperitoneally and intramuscularly with MSA cerebellar extracts. They developed progressive motor impairment and were culled when exhibiting hindlimb paralysis. Staining for pS129 α-synuclein was present in brain and spinal cord of all cases with a distribution and in amounts similar to those of M83+/+ mice with hindlimb paralysis.
For intravenous injection, mice received a daily injection of 100 μl of 100 mg/ml tissue over 4 consecutive days (equivalent to 40 mg tissue). They were culled after 225 ± 19 days (n = 8). For intramuscular injection, mice received a single bilateral injection of 50, 100 or 200 μl of 200 mg/ml tissue (equivalent to 10, 20 or 40 mg tissue) into
Ten M83± mice were injected intraperitoneally with 100 μl of 200 mg/ml MSA and control cerebellar extracts. Following the injection of MSA extracts, abundant α-synuclein inclusions developed in the central nervous system (Fig. Synucleinopathy in M83± mice following intraperitoneal injection of cerebellar extracts from an individual with neuropathologically confirmed MSA.
Microglia and nerve cells with α-synuclein inclusions were labelled in brainstem (pons region) from 20-month-old M83+/+ mice. Using a panel of epitope-specific α-synuclein antibodies, as well as pFTAA, microglial cells were juxtaposed to nerve cells with α-synuclein inclusions, often with their processes wrapped around these cells. pFTAA-labelled nerve cells were also immunoreactive for pS129 α-synuclein (Fig. Microglia and α-synuclein inclusions in 20-month-old M83 +/+ mice.
Different states of microglia can be defined morphologically [ Quantitation of lumbar spinal cord microglial cells of different morphologies in mice injected intraperitoneally with assembled A53T α-synuclein or with MSA brain extract. One month after injection of assembled A53T α-synuclein or control cerebellar extracts, most microglial cells were ramified. At 3 months after injection of assembled A53T α-synuclein, there was an increase in dystrophic microglia. At 5 months after injection of assembled A53T α-synuclein and in end-stage mice injected with MSA cerebellar extracts, dystrophic microglia predominated and ramified microglia were almost absent
We show that oral, nasal, intravenous and intraperitoneal administration of assembled A53T α-synuclein induced synucleinopathy in M83± mice. The same was true when cerebellar extracts from a case of MSA with type II α-synuclein filaments were injected intravenously, intramuscularly or intraperitoneally. Synucleinopathy was defined by the presence of abundant immunoreactivity for pS129 α-synuclein in nerve cells and the development of motor impairment, resulting in hindlimb paralysis. Intraperitoneal injection of assembled A53T α-synuclein or cerebellar MSA extracts resulted in a reduction in spinal cord motor neurons.
Following oral and nasal administration of assembled A53T α-synuclein, mice developed hindlimb paralysis. Brains and spinal cords showed abundant pS129 α-synuclein staining and α-synuclein filaments. After oral administration, staining was observed first in the solitary tract nucleus, the dorsal motor nucleus of the vagus nerve and the spinal intermediolateral nucleus, followed by other brain and spinal cord regions. These findings are consistent with early autonomic nervous system involvement, followed by spreading to other regions of the central nervous system. They suggest that oral ingestion of α-synuclein filaments is sufficient for them to cross the epithelial lining of the gastrointestinal tract, before they are taken up by nerve cells in the enteric plexus and reach the brainstem by retrograde trans-synaptic transport along the vagus nerve, consistent with previous findings [
In addition, our findings indicate that white matter tracts in the brain and spinal cord are affected following the oral administration of assembled α-synuclein, consistent with recent observations that α-synuclein pathology accumulates in afferent sensory tracts, causing degeneration of myelinated fibres, and affects oligodendroglia [
Unlike α-synuclein from wild-type mice, transgenic protein from M83± mice has been reported to be detergent-insoluble [
Nasal administration of assembled A53T α-synuclein also led to abundant inclusions and severe motor impairment in M83± mice. The injection of assembled mouse α-synuclein into the olfactory bulb of wild-type mice has been shown to lead to deficits in olfactory function and the spreading of α-synuclein inclusions to other brain regions [
Transport through the blood, followed by crossing of the blood–brain barrier, may be the main route by which assembled α-synuclein reaches the central nervous system following intravenous and intraperitoneal injections, which also gave rise to abundant α-synuclein inclusions and motor impairment. Five months after injection, 70% of spinal cord motor neurons had been lost and abundant α-synuclein inclusions were present, consistent with evidence indicating that filamentous α-synuclein inclusions precede neurodegeneration [
We showed earlier that short filaments of α-synuclein form the majority of seed-competent species in M83+/+ brains [
When injected intracerebrally into M83± mice, MSA extracts were more potent than PD extracts in inducing synucleinopathy [
Neuroinflammation is a common pathological characteristic of major neurodegenerative diseases [
Upon peripheral administration of assembled A53T α-synuclein or cerebellar homogenates from a case of MSA, we observed a close relationship between the formation of α-synuclein inclusions in nerve cells and neurodegeneration, accompanied by a shift in microglial cell morphology.
We thank Drs B. Falcon, M. Jucker and S.H.W. Scheres for their help.
MG designed experiments and drafted the manuscript; MMS expressed, purified and assembled A53T α-synuclein; RB administered α-synuclein seeds peripherally; JC performed immunohistochemistry, Western blotting, rotarod testing and survival analysis; MS extracted MSA filaments and performed cryo-EM; IL performed and JM analysed immunofluorescence; JM performed and AG assisted with immunohistochemistry and unbiased stereology; ZJ, TW and JLH identified patients and characterised the MSA brain. All authors read and approved the final manuscript
This work was supported by the UK Medical Research Council (MC_U105184291 to MG).
Data and materials are available from the corresponding author upon request.
Experiments were conducted in accordance with the UK Animals (Scientific Procedures) Act of 1986, with local ethical approval (MRC Laboratory of Molecular Biology Animal Welfare and Ethical Review Body Committee). The studies carried out at the UCL Queen Square Institute of Neurology and the Edinburgh Brain Bank were approved through the ethical review processes at each institution.
The authors declare that they have no competing interest.
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