Rigorous evaluation of pathological α-synucleinopathy transmissibility between animals in an experimental model

The conformational templating (molecular prion-like) corruption of endogenous α-synuclein using recombinant α-synuclein pre-formed fibrils (PFF) or patient derived tissue extracts to template endogenous protein into pathological conformations has been shown by several groups using multiple model systems [1]. These studies indicate that α-synuclein pathology can spread within animal subjects to cause progressive neurodegenerative diseases [2]. Bona Fide prion strains can spread to naïve animals by environmental contact with surfaces contaminated by bodily excretions indicative of the epidemiolocal infectivity of the misfolded prion protein [3]. Reports of peripheral sources of pathological α-synuclein in the skin, kidney, and gastrointestinal system in patients and experimental models raise serious safety concerns about whether α-synuclein can act as an epidemiological infective prion in addition to its molecular prion properties. This prompted us to directly test the inter-subject transmissibility of pathological α-synuclein [4, 5]. We designed a simple yet robust experiment to test whether exposure to urine, feces, shared housing, fight wounds, or contact with transgenic animals harboring high pathological α-synuclein burden could cause pathological α-synuclein to transmit to non-injected, naive cage mates. TgM83 mice overexpress human mutant (A53T) α-synuclein and show a predictable, progressive accumulation of pathological α-synuclein in the central nervous system (CNS) following intramuscular (IM) injection of PFFs into the hindlimb [6–8]. Behaviorally, IM PFF inoculated TgM83+/− mice display a predictable and progressive overt motor phenotype leading to paralysis, and eventually must be humanely euthanized [6]. To test the transmissibility of pathology between experimental animals, in cages of 5 TgM83+/− mice, 2 mice were inoculated bilaterally with 10 µg PFFs per side while the other 3 were co-housed in a naïve state (Fig. 1A). Quality control and production methods for PFFs has been extensively reported previously [6–7]. As expected, TgM83+/− injected with PFFs developed a motor phenotype and survived on average 135 days post injection (Fig. 1B). The naïve mice were aged 1 year post co-housing (on average, 135 days with injected animals and an additional 230 days after injected group was humanely euthanized) and no animal from the naïve group developed a motor phenotype, indicating a paucity of pathogenic transmission (Fig. 1B). Co-housing was stopped at 1 year post-inoculation, and ~ 15 months of age to prevent the confound of any potential self-aggregation of α-synuclein in these mice [8]. To investigate if pathological α-synuclein was transmitted, the CNS tissue from all mice were assessed by immunohistochemistry for pathological α-synuclein inclusion pathology. TgM83+/− mice injected with PFF developed robust accumulation of α-synuclein inclusion pathology with the expected distribution in the CNS [6] as shown in the brainstem, spinal cord, and the periaqueductal gray (PAG) with pSer129 α-synuclein antibodies 81 A and EP1536Y, a well-established surrogate marker of pathological α-synuclein (Fig. 1C). Staining with a total α-synuclein antibody (94-3A10) also revealed α-synuclein pathology in the PFF-injected mice (Fig. 1C). No α-synuclein inclusion pathology was observed with 81 A, EP1536Y or 94-3A10 antibody staining in the CNS of the naïve co-housed TgM83+/− mice, while normal neuropil distribution of α-synuclein was observed with antibody 94-3A10 (Fig. 1D), and additionally shown in higher magnification images (Supplemental Fig. 1). These data demonstrate that even in a highly susceptible animal model there is not evidence for inter-subject transmission of pathological α-synuclein, thus misfolded α-synuclein is far less host-recipient transmissible than pathologic prion strains such as Scrapie. With our experimental design, we cannot distinguish between the lack of sufficient seeding competent α-synuclein species being shed from the PFF-injected animals or that naïve co-housed mice are resistant to transmissibility of pathological α-synuclein through environmental contact. However, in either case, this highlights that α-synuclein displays robust molecular prion-like activity, but not the epidemiological infectivity of prion proteins. This is supported by evidence showing inefficient seeding of pathological α-synuclein from oral, but not intramuscular, intravenous, or intraperitoneal, routes of exposure even with high amounts of PFF used [9]. Furthermore, these data indicate there should be minimal risk to laboratory or husbandry staff in handling and caring for α-synuclein PFF-injected animals from simple environmental exposure to the bedding and animals directly.

Fig. 1.

Experimental Assessment of Pathological α-Synuclein Transmission. A Experimental design to test if animals with high burden of pathological α-synuclein can transmit pathology to naïve mice. 3 cages of 5 female (15 animals total) TgM83+/− were used. In each cage 2 TgM83+/− mice were injected bilaterally in hindlimb muscles with 10 µg of human recombinant α-synuclein pre-formed fibrils (PFF, 20 µg total). Two treated mice were then co-housed with 3 naïve mice. Cages of standard wood/corn cob bedding with no enrichment were changed under standard conditions for ventilated cages once every weeks. Co-housing continued until the inoculated mice were humanely euthanized due to paralysis. The average time naïve mice were co-housed with inoculated mice was 135 days. Naïve mice were further aged until 1 year post co-housing (on average another 230 days post death of inoculate animals, and ~ 15 months total age) with inoculated animals before being euthanized for immunohistochemistry. B Kaplan Meier Survival Plots of naïve and PFF-inoculated TgM83+/− mice. As expected, TgM83+/- PFF injected mice developed a motor phenotype and survived ~ 135 days post injection. None of the co-housed naïve mice developed a motor phenotype. One mouse was euthanized at 343 days post co-housing due to perianal soiling possibly due to fighting which would have been fighting exposure to only naïve co-housed mice at this point in the experiment All other mice were euthanized for analysis at 363 days post initial co-housing. C Immunohistochemical analysis of injected TgM83+/−, performed as previously described [6], for pathological α-synuclein using 81 A and EP1536Y pSer129 α-synuclein antibodies, a standard marker for pathological α-synuclein, as well as total α-synuclein (94-3A10). All 6 TgM83+/− PFF injected mice, showed α-synuclein inclusion pathology in the CNS as expected [6] in the brainstem, spinal cord, and periaqueductal gray (PAG). D Staining as in (C) from naïve TgM83+/− mice co-housed with inoculated mice. All 9 naïve mice, including the mouse humanely euthanized due to perianal soiling, showed no α-synuclein inclusion pathology. Scale bar = 120 μm

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

AS, MM and BG performed, collected, and analyzed experiments and data. MM, TG and DB provided feedback and analysis of results. MM and BG wrote the first draft of the manuscript. All authors edited and approved the final manuscript.

Funding

The work was supported by grants R01NS100876 and RF1NS129567 to BIG, as well as support from the Norman Fixel Institute for Neurological Disorders, the University of Florida McKnight Brain Institute, and the Harry T. Mangurian Foundation.

Acknowledgements: None.

Data availability

All study materials, images, and data are available from the co-corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

All animal studies necessary for this study were approved before starting by the University of Florida Institutional Animal Care and Use Committee protocol 201307843.

Competing interests

The authors declare no competing interests.