In order to further characterize M83 disease, we inoculated hemizygous and/or homozygous M83 neonates with two different brain extracts: one from sick M83 mice inoculated with brain extracts from sick M83 mice, and the other derived from a human [multiple system atrophy (MSA)] source passaged in M83 mice, either by the intraperitoneal (IP) or intracerebral (IC) routes. We found that the α‐syn P levels depend on the mouse genotype and that M83 passage of the human MSA brain extract induces lower immunoreactivity detected by ELISA tests at first passages.

Brain lesions in patients with Parkinson's disease (PD), called Lewy bodies and Lewy neurites, are mainly composed of aggregates of α‐synuclein (α‐syn) protein. Mutations of the gene encoding α‐syn, such as the A53T mutation, have been reported in inherited PD cases. Homozygous M83 transgenic mice expressing human A53T α‐syn (Giasson et al . 2002 ) spontaneously develop severe motor clinical signs at 8–16 months of age (Giasson et al . 2002 ), associated with the cerebral accumulation of pathological Ser129‐phosphorylated form of α‐syn (α‐syn P ), heavily phosphorylated at serine 129. Hemizygous M83 mice can also develop the disease, but only after 22 months (Giasson et al . 2002 ). A ‘prion‐like’ mechanism of α‐syn P spreading has been described in this transgenic model (Luk et al . 2012 ; Mougenot et al . 2012 ). Intracerebral inoculations of brain extracts from sick M83 mice, or of recombinant α‐syn fibrils, dramatically accelerate the disease, whereas brain extracts from asymptomatic M83 mice do not (Luk et al . 2012 ; Mougenot et al . 2012 ; Watts et al . 2013 ). Recent studies suggested that peripheral inoculations of preformed α‐syn fibrils via the intramuscular or intraperitoneal routes can also accelerate M83 disease (Sacino et al . 2014 ; Breid et al . 2016 ). The hypothesis of the present study is that M83 disease can be accelerated by the intraperitoneal inoculation of sick M83 brain homogenates and that the accumulation of α‐syn P in the central nervous system may depend on the initial level of expression of the normal α‐syn protein in the mouse brain. M83 disease has been described in detail using immunohistochemistry (Giasson et al . 2002 ). Here, ELISA tests were used to quantify α‐syn P , and a novel ELISA test was developed (Betemps et al . 2014 , 2015 ) with increased sensitivity that allows to detect α‐syn P even in cerebral regions with few α‐syn inclusions.

Survival time was defined as the time from birth to death; intracerebral or intraperitoneal inoculations of brain extracts were performed the day of birth. In order to focus our analysis on M83 disease, and on the actual behavioral description (Giasson et al . 2002 ), we right‐censored mice that were (i) found dead without any identified symptoms in the previous days and testing negative in the ELISA analysis (or not analyzed by ELISA), (ii) killed for reasons other than M83 disease and testing negative in the ELISA, or (iii) still alive at the end of the experiment (18 months). Survival time was adjusted with Cox regression models. The proportional hazards assumption of the Cox model was checked by examining the weighted residuals. Survival time distributions between groups were compared using Tukey's multiple tests. For the statistical analysis of the ELISA data, comparisons of the optical density (OD) means between small groups (when at least one of the two groups compared contains fewer than 6 animals) were performed with Bayesian regression models using Gibbs sampling and the Metropolis algorithm to generate a Markov chain by sampling from full conditional distributions. Flat priors were used for the mean and the variance in both groups. Three chains, with 10,000 iterations each, were run. Concerning the others groups (Fig. 4 , the first and second passages of M83, and the first and second passages of MSA), mixed‐effect regressions were used to model the OD differences between groups. In each model, a random effect was used to reflect the variability of the repetitions for the mouse and a fixed effect to represent the difference between groups. Validation of the models was based on examination of the residuals. When residuals of the models gave a clear indication of heteroscedasticity, variance matrixes were appropriately parameterized. Differences in OD were considered significant for an associated p ‐value lower than 0.05. Statistical analyses were carried out with R software (Team 2015 ), and with the survival package (Therneau.T 2015 ) and the R2WinBUGS package (Sturtz et al . 2005 ).

Proteins were separated in 15% sodium dodecyl sulfate‐polyacrylamide gels and electroblotted onto 0.45 μm polyvinylidene fluoride membranes (Bio‐Rad). The membranes were incubated with 0.4% (Fig. 2 ) or 4% paraformaldehyde, and 0.01% glutaraldehyde (Fig. S2 ) in phosphate‐buffered saline for 30 min at 22°C under agitation, and were saturated 1 h with 5% nonfat dry milk in 0.1% PBST (Blotting‐Grade Blocker, Bio‐Rad). Rabbit antibody against pSer129 α‐syn (Abcam Cat# ab51253 RRID:AB_869973) at 1 : 1000 dilution was used to detect α‐syn P in the insoluble fractions. C20‐R antibody (1 : 10 000) (Santa Cruz Biotechnology Cat# sc‐7011‐R RRID:AB_2192953, Santa Cruz, CA, USA) against total α‐syn, and anti‐glyceraldehyde‐3‐phosphate dehydrogenase antibody (1 : 5000) (Millipore Cat# MAB374 RRID:AB_2107445, ref. MAB374, Millipore, Temecula, CA, USA) as a loading control, were used for western blot analyses of the supernatants. The membranes were then incubated for 1 h with anti‐rabbit antibody (Thermo Fisher Scientific Cat# 32460 RRID:AB_1185567, Rockford, IL, USA) (1 : 1000) or anti‐mouse antibody (Thermo Fisher Scientific Cat# 32430 RRID:AB_1185566). The immunocomplexes were revealed with chemiluminescent reagents (Supersignal WestDura, ref. 34076, Pierce, Interchim, Montluçon, France), and analyzed using the VersaDoc system and Quantity One software (Bio‐Rad).

Levels of α‐syn P were measured by ELISA that specifically identifies α‐syn P in sick M83 mice, with modification of a published protocol (Betemps et al . 2014 , 2015 ). Plates (MaxiSorp ™ , Thermo Scientific Nunc, Roskilde, Denmark) were left uncoated or coated with capture antibodies AS11 [produced in our laboratory (Mougenot et al . 2010 )] or Syn303 (BioLegend Cat# 824301 RRID:AB_2564879 Ozyme, St‐Quentin‐en‐Yvelines, France) (Giasson et al . 2002 ; Mishizen‐Eberz et al . 2003 ) diluted to 1 μg/mL, and incubated overnight at 4°C. These antibodies recognize the epitopes with amino‐acid residues 118–125 and 2–4, respectively. Plates were washed five times with 300 μL of phosphate buffer saline with 0.05% Tween20 (PBST) and then saturated with 200 μL of Superblock T20 (Thermo Scientific, Rockford, IL, USA) for 1 h at 25°C, under agitation (150 rpm). The clarified homogenates were diluted in PBST and 1% bovine serum albumin (BSA) to obtain a final concentration of 0.2% homogenate. The results were then obtained with 200 μg brain equivalents. After washing the plates five times with 300 μL of PBST, 100 μL of these homogenates were loaded into each well and incubated at 25°C under agitation for 2 h. The plates were washed five times with PBST and α‐syn P was detected with a rabbit polyclonal antibody against PSer129 α‐syn (Abcam Cat# ab59264 RRID:AB_2270761, Cambridge, UK) diluted to 1 : 3000 in PBST 1% BSA; plates were incubated for 1 h at 25°C under agitation. The plates were washed another five times with PBST, and anti‐rabbit IgG HRP conjugate (SouthernBiotech Cat# 4010‐05 RRID:AB_2632593, Birmingham, AL, USA) was added at 1 : 2000 (with AS11 and Syn303 capture antibodies) or at 1 : 8000 (without capture antibody) dilution in PBST‐1% BSA; plates were incubated for 1 h at 25°C. After washing the plates five times with PBST, 100 μL of 3,3′,5,5′‐tetramethylbenzidine solution (ref. T0440, Sigma, Saint‐Quentin‐Fallavier, France) were added to each well and plates were incubated for 15 min with shaking. The reaction was stopped with 100 μL of 1 N HCl, and the absorbance was measured at 450 nm with the microplate reader Model 680 (Bio‐Rad, Marnes‐La‐Coquette, France). Thresholds were calculated as the average of three repeated ELISA tests on two negative half‐brains from young, asymptomatic M83 mice of 2 and 5 months of age, plus three times the SD.

Mouse brains were dissected just after death. Then, α‐syn was extracted from brain or spinal cord samples as described in a previous study (Betemps et al . 2014 ). The samples were crushed in a volume of high salt buffer (50 mM Tris‐HCl, pH 7.5, 750 mM NaCl, 5 mM EDTA, 1 mM dithiothreitol, 1% phosphatase and protease inhibitor cocktails), using a mechanical homogenizer (grinding balls, Precellys 24, Bertin Technologies, Montigny‐le‐Bretonneux‎, France) to obtain a 10% homogenate (w/v). To detect and quantify α‐syn P by ELISA, the homogenates were clarified by centrifugation at 1000 g (5 min at +4°C). An additional extraction step on the total homogenates was necessary to detect α‐syn P by western blot. For this supplemental extraction, 100 μL of homogenate were incubated on ice in N ‐lauroylsarcosyl (ref 61747, Sigma, Saint‐Quentin‐Fallavier, France) at a final concentration of 10% before being ultracentrifuged at 435 000 g (1 h at +4°C) over a 10% sucrose cushion. Pellets were suspended with 20 μL of TD4215 denaturing buffer (4% sodium dodecyl sulfate, 2% β‐mercaptoethanol, 192 mM glycine, 25 mM Tris, 5% sucrose) and denatured at 100°C for 5 min. To detect total α‐syn or glyceraldehyde‐3‐phosphate dehydrogenase by western blot, clarified homogenates were used.

The initial MSA inoculum was prepared from a sample of substantia nigra . The patient was first diagnosed with Parkinson's disease at 42 years of age, with akinesia, ataxia, memory impairment, dysarthria symptoms, and then diagnosed with MSA because of pontocerebellar atrophy detected by medical imaging at 52 years of age. He died at age 54. The human biological sample and associated data were obtained from the Cardiobiotec Biobank (CRB‐HCL Hospices Civils de Lyon BB‐0033‐00046). This tissue sample was obtained according to French legislation (specifically, written informed consent was obtained from patients for all samples). Cardiobiotec is authorized by the French Ministry of Social Affairs and Health (DC2015‐2566), with transfer authorization (AC 2015‐2576). The Biobank database is registered with the French Data Protection Authority (CNIL).

Clinical monitoring of the mice took place three times a week to detect any symptom of M83 disease. First, we observed the spontaneous mobility of the mice: persistent immobility or slow ambulation are the first indications pointing to disease onset. Balance disorders and the partial paralysis of a hind limb are the behavioral hallmarks of the onset of M83 disease. Balance disorders can be highlighted by observing the spontaneous activity of the mice, revealing falling after rearing up, or can be detected by gently pushing the mouse on its side. Another typical symptom is the freezing of a hind limb during spontaneous walking, lasting a few seconds. The hind limb paralysis is also evaluated by observing the ability of the mouse to right itself when placed on its side. A mouse is considered as ‘sick’ when two independent observers highlight at least one of these last two symptoms. When the mouse develops these first symptoms or any intercurrent disease not associated with M83 disease, we proceed to killing by lethal intraperitoneal injection of pentobarbital. After death, the mice were genotyped using The Jackson Laboratory quantitative PCR protocol on a Roche Light Cycler 480.

The animals were housed per group in enriched cages in a temperature‐controlled room on a 12 h light/dark cycle, and received water and food ad libitum, in our approved facilities (No. C69 387 0801) for breeding and experimental studies, in accordance with EEC Directive 86/609/EEC and French decree No. 2013‐118. The experimental studies described in this article were performed in containment level 3 facilities. Monitoring of the M83 mice in the experimental protocols is followed up by the Comité d’éthique CE2A – 16, ComEth ANSES/ENVA/UPEC.

Mice were obtained by crossing hemizygous M83 mice, produced by breeding a homozygous M83 male with a B6;C3H female. The neonates (wild‐type, hemizygous and homozygous; the genotype was unknown until death) were inoculated at birth either in the cerebral parenchyma or intraperitoneally with brain homogenates of sick M83 mice, showing the typical clinical signs of M83 disease, and containing insoluble Ser129‐phosphorylated α‐synuclein (α‐syn P ) detected by western blot and ELISA analyses (data not shown) (Betemps et al . 2014 ). The history of serial passages, and details regarding the mice from which the brain homogenates were prepared with each passage, are summarized in Table S1 . All homogenates were prepared from half sagittally sectioned brains. In the case of inoculations originating from a source of MSA, only homozygous M83 mice were inoculated, as neonates obtained by crossing homozygous M83 mice.

Impact of the type of inoculum on levels of pathological Ser129‐phosphorylated α‐synuclein (α‐syn) detected by ELISA tests in sick homozygous M83 mice. Levels of α‐synwere quantified in samples from the spinal cord of sick M83 mice by ELISA testing without capture antibody (left panel) or with AS11 capture antibody (right panel). The first passage was performed by intracerebral inoculation with a homogenized sample offrom the brain of a patient with MSA or a brain homogenate from sick M83 mice in young adult M83 mice (more details in Materials and methods ). The second passage was performed by intracerebral inoculation of brain homogenates from sick M83 mice from the first passage in young adult M83 mice. The third passage involved intracerebral or intraperitoneal inoculation of brain homogenates from sick M83 mice from the second passage in M83 neonates. Levels of α‐synwere significantly higher in mice inoculated with the M83 inoculum than in mice inoculated with the MSA inoculum, concerning both the first and the second passages. Results were obtained from equivalent quantities of brain samples. Bars correspond to mean ± SD. Statistical analysis was performed using mixed‐effect regression or using a Bayesian regression model (see Materials and methods ). Thresholds were calculated as the average of three repeated ELISA tests on two negative half‐brains from young, asymptomatic M83 mice of 2 and 5 months of age, plus three times the SD. Results were obtained with 200 μg brain equivalents. Three replicate ELISAs were performed for each sample. ***0.001; **0.01; *0.05; #non‐significant.

The ELISA analyses also detected some differences when comparing M83 homozygous mice inoculated with M83 sensu stricto or derived from a MSA source (Fig. 4 ). We analyzed α‐syn P accumulation in the spinal cord, which is known to contain abundant inclusions in sick M83 mice (Giasson et al . 2002 ), first comparing accumulation at the first and second passages of M83 sensu stricto to the same passages of MSA, that were performed by IC inoculations in adults. Unexpectedly, ELISA tests detected significantly higher α‐syn P immunoreactivity in mice inoculated with M83 sensu stricto , than in those derived from the MSA source (at first passage, p < 0.0001 in ELISA tests performed either in the absence of any capture antibody or with capture by the AS11 antibody; at second passage, p = 0.0003 and p = 0.0041 using these two ELISA tests, respectively). At the third passage that was performed by IC inoculations in neonates, ELISA tests also showed lower α‐syn P immunoreactivity in mice derived from the MSA source than in those inoculated with M83 sensu stricto , but this difference was nevertheless not statistically significant.

Distribution of pathological Ser129‐phosphorylated α‐synuclein (α‐syn P ) detected by ELISA in the central nervous system of homozygous (Ho) or hemizygous (He) sick M83 mice inoculated intracerebrally (IC) or intraperitoneally (IP). The following neuroanatomical regions were analyzed from sick HoIC ( n = 4, mean ± SD) (a), HoIP ( n = 4, mean ± SD) (b), HeIC ( n = 3 mean ± SD) (c), and HeIP ( n = 4 mean ± SD) (d) mice: OB: olfactory bulb, Cx: cerebral cortex, St: striatum, Hi: hippocampus, Me: mesencephalon, Cb: cerebellum, BS: brainstem, CSC: cervical spinal cord, TSC: thoracic spinal cord, LSC: lumbar spinal cord. Two capture antibodies against α‐syn were used: AS11 and Syn303. The highest immunoreactivities were observed with the Syn303 antibody and α‐syn P was similarly identified in the same cerebral regions and in the spinal cord in each group. Results were obtained with 200 μg brain equivalents. Thresholds were calculated as the average of three repeated ELISA tests on two negative half‐brains from young, asymptomatic M83 mice of 2 and 5 months of age, plus three times the SD. Three replicate ELISAs were performed for each sample.

Irrespective of the inoculum source, the distribution of α‐syn P was similar in the central nervous system (CNS) of sick Ho and He mice, whether IP‐ or IC‐inoculated, mainly accumulating in caudal brain regions (brainstem and mesencephalon) and in the spinal cord (Fig. 3 ), as previously described in sick M83 mice (Betemps et al . 2014 ). The highest ELISA immunoreactivities were identified using the Syn303 capture antibody, which also revealed detectable α‐syn P in the cerebellum, cerebral cortex and striatum (Fig. S2 ). Immunohistochemical analyses have already shown that these regions harbor α‐syn P deposits in sick M83 mice (Luk et al . 2012 ; Betemps et al . 2014 ).

Biochemical analysis of pathological Ser129‐phosphorylated α‐synuclein (α‐syn P ) in the nervous system of inoculated or non‐inoculated (aging) mice by ELISA and western blot. (a) Levels of pathological Ser129‐phosphorylated α‐synuclein (α‐syn P ) detected by an antibody against Ser129‐phosphorylated α‐syn were measured using ELISA in extracts from the brainstem or, for non‐inoculated hemizygous (He) M83 mice (Aging He), from the spinal cord. The left panel shows results obtained without capture antibodies, and the right panel shows results obtained with the AS11 capture antibody. Levels of α‐syn P were significantly higher in HoIC than in HeIC mice (*mean difference: 1.25 and 95% credible interval [0.89–1.61], according to Bayesian regression models) but no significant difference was found between the HoIP and HeIP groups (#), due in particular to an outlier HeIP mouse showing much higher α‐syn P levels in this group. Results were obtained with 200 μg brain equivalents. (b and c) Western blot analysis of brainstem extracts sampled from He or Ho IC‐ (b) or IP‐ (c) inoculated mice. Ser129‐phosphorylated α‐syn was identified in the sarkosyl‐insoluble fractions, whereas total α‐syn and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) were identified in clarified brain homogenates. Molecular weight markers (in kDa) are indicated to the right of the panels.

In transgenic mice that had been inoculated at birth with a pooled brain extract from sick M83 mice, ELISA analyses identified α‐syn P in the brainstem of all examined homozygous mice (4/4 HoIC and 4/4 HoIP), and also of most hemizygous mice (14/16 HeIC and 10/14 HeIP) (Fig. 2 a, Table S2 ). This last result contrasts starkly with that obtained for non‐inoculated, aging hemizygous mice. Among the 34 hemizygous mice, we detected very low levels of α‐syn P , even with the AS11 capture antibody, in the spinal cord of only two mice (16 and 25 months old) in which balance disorder or paralysis was identified (Fig. 2 a). Interestingly, in the brainstem of HoIC inoculated mice, α‐syn P levels were significantly higher (~3x OD) than in HeIC mice (Fig. 2 a) (mean difference: 1.25 and 95% credible interval [0.89–1.61], according to Bayesian regression models). None of the non‐transgenic inoculated mice tested were positive in the ELISA. All HoIC MSA (4/4) and HoIP MSA (10/10) mice also showed α‐syn P positivity in their brainstem and their spinal cord by ELISA (Fig. 4 ). Following western blot analyses of sarkosyl‐insoluble fractions (Fig. 2 b and c), both sick homozygous and hemizygous IC‐ or IP‐inoculated mice showed the typical α‐syn P pattern (Betemps et al . 2014 ).

Survival of M83 mice after intracerebral (IC) or intraperitoneal (IP) inoculation of brain homogenates from diseased M83 mice. (a) M83 disease‐associated survival of mice after IC or IP inoculation as neonates of brain homogenates from sick M83 mice. Blue and red lines correspond to IC and IP mice inoculated as neonates, respectively. Solid and dashed lines represent homozygous (Ho) and hemizygous (He) mice, respectively, and dotted lines (which overlap in the figure) show non‐transgenic mice. We ended the experiment at 18 months, when the last surviving and still healthy, hemizygous or non‐transgenic, inoculated mice were killed (Table S2 ). (b) M83 disease‐associated survival of M83 mice IC‐ or IP‐inoculated with several passages of sick M83 mice inoculated with human brain MSA homogenate. Blue and red lines correspond to IC and IP mice inoculated as neonates, respectively (third passage). Black and brown dotted‐dashed lines correspond to first and second IC MSA passages in adult M83 mice, respectively. Crosses refer to mice for which typical M83 disease was not identified.

Survival data of IC‐ or IP‐inoculated homozygous, hemizygous or non‐transgenic mice, determined using the Kaplan–Meier method, are shown in Fig. 1 . We defined a mouse as ‘sick’ when we identified balance disorders or partial paralysis of the hind limb, the first symptoms of M83 disease (Giasson et al . 2002 ) (see Materials and methods ). Detailed information on survival data or clinical signs and biochemical assays are given for individual mice in Table S2 . Homozygous M83 mice IC inoculated at birth (HoIC), which all developed clinical disease before the age of 8 months, showed significantly shorter survival periods than homozygous M83 mice IP inoculated at birth (HoIP) ( p < 0.01) which developed clinical disease within 4–14 months after inoculation (and for which 3/7 mice developed the symptoms before 8 months). All the homozygous M83 mice also developed the clinical disease within 7 months after IC inoculation of a brain extract from a second passage of multiple system atrophy in M83 mice (HoIC MSA). This was significantly earlier than the IP‐inoculated homozygous mice (HoIP MSA) ( p < 0.05) that developed M83 disease within 4–13 months after the inoculation (7/10 mice developed M83 disease earlier than 8 months) (Fig. 1 b). In a non‐inoculated cohort of 34 hemizygous mice observed up to 16 months, signs of hind limb paralysis were only found in a single mouse (16 months old), and balance disorders were observed in another single mouse (25 months old). In contrast, 16/20 IC‐ and 10/19 IP‐inoculated hemizygous mice (HeIC and HeIP) developed typical M83 clinical symptoms before 18 months (chosen as the end‐point of the experiment), as illustrated in an IP‐inoculated hemizygous 10.8‐month‐old mouse shown in the Supplemental Movie. Survival periods (Fig. 1 a) of HeIP and HeIC mice were not significantly different ( p = 0.13). HoIP and HoIC mice had significantly shorter survival periods than HeIP and HeIC, respectively ( p < 0.001 for both). None of the inoculated wild‐type mice developed any neurological signs by 18 months of age.

Discussion

After both IC and IP inoculation at birth with a whole brain homogenate from sick M83 mice, M83 mice began to develop clinical symptoms before 8 months, the age from which homozygous M83 mice begin to spontaneously develop M83 disease, as described by Giasson. The disease was confirmed by the onset of the typical clinical signs and the accumulation of Ser129‐phosphorylated α‐synP in the CNS. This was observed not only following inoculation of brain homogenate from M83 mice previously inoculated with brain extracts of sick M83 mice, but also after serial passages in M83 mice from a MSA case, a human synucleinopathy that was recently shown to be readily transmissible to M83 mice, unlike Parkinson's disease (Prusiner et al. 2015). In prion diseases, the comparative efficiencies of neuroinvasion after challenge by different inoculation routes may vary between different prion strains: for instance, the 87V scrapie strain specifically showed poor efficiency in causing disease after intraperitoneal challenge, despite persistence of high levels of infection outside the CNS (Bruce 1985; Collis and Kimberlin 1985).

Concerning the inoculations performed with M83 sensu stricto (i.e. not derived from MSA), which were carried out in both homozygous and hemizygous mice, the development of the disease was particularly striking in hemizygous mice, since it was previously reported that they rarely develop clinical disease and only at an advanced age (Prusiner et al. 2015). This high resistance of hemizygous mice was clearly confirmed here using an original ELISA test specifically recognizing Ser129‐phosphorylated α‐synP in this model (Betemps et al. 2014), that otherwise failed to identify α‐synP in most hemizygous mice, even at an advanced age (16‐36 months). However, as previously reported with intracerebral inoculations in adults (Watts et al. 2013; Prusiner et al. 2015), hemizygous mice efficiently developed the disease after both IP and IC inoculations as neonates.

The sequential onset of disease in cohorts of homozygous and hemizygous mice clearly demonstrates the close relationship between disease progression and the level of human α‐syn expression, in both IC‐ and IP‐inoculated mice (see also Fig. S1b). Also, at least after IC inoculations, ELISA quantification showed higher levels of α‐synP in Ho mice than in He mice. In experimental models of prion diseases, progression of the disease in two distinct mechanistic phases has been described. In the first phase, fixed levels of infectivity are reached, irrespective of the expression level of the normal prion protein (PrPC); then in the second, proteinase K‐sensitive PrP isoforms, distinct from classical disease‐associated scrapie prion protein, accumulate at a rate proportional to PrPC concentration, until the onset of clinical signs (Sandberg et al. 2014). In human synucleinopathies, some rare genetic cases of Parkinson's disease are associated with increased α‐syn expression related to duplications or triplications of the α‐syn encoding SNCA gene. In addition, it was recently suggested that even a modest lifelong increase of SNCA expression may represent an increased risk of developing sporadic Parkinson's disease for carriers of a common risk‐associated variant in a non‐coding distal enhancer element identified by genome‐wide association studies (Soldner et al. 2016).

The nature of the α‐synP molecular species identified by ELISA remains to be determined, but it is only found in regions of the CNS affected by the pathological process, as defined by the presence of α‐synP as intracellular deposits, by immunohistochemistry, and/or in sarkosyl‐insoluble fractions, by western blot (Betemps et al. 2014, 2015). As previously described by immunohistochemistry (Luk et al. 2012; Betemps et al. 2014), α‐synP was also detected in this study in some rostral brain regions (cerebral cortex and striatum) thanks to the increased sensitivity of the ELISA test using novel capture antibodies in the N‐terminal (Syn303 – epitope 2–4) or C‐terminal (AS11 – epitope 118–125) regions of α‐syn (Betemps et al. 2015). These regions interestingly belong to less structured parts in amyloid fibrils of the protein (Riek and Eisenberg 2016). The strongest labeling of antibodies against surface‐exposed epitopes localized at the beginning of the N‐terminus (1–10) or at the C‐terminus (90–140) was recently confirmed by immunohistochemistry in another transgenic (human A30P α‐syn) mouse line and in brain tissues from human patients (Almandoz‐Gil et al. 2016). However, levels of α‐synP in rostral regions in M83 mice remain much lower than those in caudal brain regions (brainstem and mesencephalon) and in the spinal cord. Interestingly, it has been reported in sick M83 mice that α‐syn oligomers showed higher Syn303 immunoreactivity when derived from inclusion‐bearing brain regions than from inclusion‐free regions, although these oligomers shared similar basic biochemical properties and were present in similar amounts in both regions (Tsika et al. 2010).

Overall, like following intracerebral inoculation in adult mice (Luk et al. 2012; Mougenot et al. 2012; Betemps et al. 2014, 2015), the α‐synP distribution in the CNS of M83 mice was similar after either IP or IC inoculations, in both Ho and He mice, to that initially described in advanced age M83 mice spontaneously developing the disease (Fig. S2) (Giasson et al. 2002; Luk et al. 2012; Mougenot et al. 2012). Interestingly, investigating an inoculum effect on α‐synP accumulation, ELISA quantification showed that the MSA inoculum resulted in lower immunoreactivity in the spinal cord of sick M83 mice both in the absence of capture antibody and after capture by the AS11 capture antibody, at least at the first and second passages. This lower detection may be because of lower levels of the ELISA‐recognized α‐synP molecular species, as we found when comparing HoIC and HeIC mice. This, however, suggests that the immunoreactivity detected by ELISA may not be directly related to the onset of paralysis that determines sacrifice of the mice and thus the survival duration in our study. It has previously been reported that aging M83 mice also develop non‐motor symptoms, like spatial memory deficits, with a synaptic dysfunction, before the onset of age‐related gross motor symptoms (Paumier et al. 2013), which have not been considered here. More generally, we do not know with certainty which species of α‐syn are the most toxic in synucleinopathies, even in the natural human diseases, although the potential important role of oligomeric species has been considered (Lashuel et al. 2013). These findings may also be because of a different form of α‐synP aggregate, exposing the recognized epitopes differently, since the origin of the inoculum is different and since α‐syn from MSA patients could represent a specific strain, according to Watts and Prusiner (Watts et al. 2013; Prusiner et al. 2015). To further investigate this hypothesis, a conformation‐dependent immunoassay for the analysis of α‐syn conformation, like that developed for prions (Safar et al. 1998), might enable to identify different conformations of the protein, evaluating its ratio in α‐helical and β‐sheet conformation.

The precise mechanisms and molecular components that trigger the acceleration of disease in M83 mice also remain to be clarified. Previous studies have shown that the acceleration of M83 disease after IC inoculation of M83 brain extracts in adult M83 mice depends on the quantity of α‐synP into the inoculum (Luk et al. 2012; Betemps et al. 2014) (Fig. S1a), as observed in Ho and He inoculated mice (Fig. S1b). IC inoculation in neonates has previously shown induction of α‐synP in M20 transgenic mice, over‐expressing the normal human protein, after injection of amyloidogenic, and to some extent non‐amyloidogenic, forms of recombinant α‐syn (Sacino et al. 2013). Regarding the results that were observed after intraperitoneal challenges, the specific pattern of α‐syn expression because of the prion promoter in M83 mice may contribute to intraperitoneal transmissibility. We also cannot fully rule out the hypothesis that the immaturity of the blood–brain barrier in neonates modulates the propagation of α‐synP aggregation. However, it was recently shown that recombinant α‐syn fibrils can cross the blood–brain barrier after intravenous inoculation in adult rats (Peelaerts et al. 2015). Moreover, two recent studies described the acceleration of synucleinopathy after IP inoculation of recombinant α‐syn fibrils in adult hemizygous M83 mice, clearly showing that α‐syn alone can accelerate M83 disease by this inoculation route (Breid et al. 2016; Ayers et al. 2017). Our data extend these observations, showing that extracts from a brain with synucleinopathy also accelerate M83 disease after IP inoculation.