Phosphorylated exogenous alpha-synuclein fibrils exacerbate pathology and induce neuronal dysfunction in mice

Approximately 90% of alpha-synuclein (α-Synuclein) deposited in Lewy bodies is phosphorylated at serine 129 suggesting that the accumulation of phosphorylated α-Synuclein is critical in the pathogenesis of Parkinson’s disease. However, in vivo experiments addressing the role of phosphorylated α-Synuclein in the progression of Parkinson’s disease have produced equivocal data. To clarify a role of Ser129 phosphorylation of α-Synuclein in pathology progression we performed stereotaxic injections targeting the mouse striatum with three fibrilar α-Synuclein types: wt-fibrils, phosphorylated S129 fibrils and, phosphorylation incompetent, S129A fibrils. Brain inoculation of all three fibrilar types caused seeding of the endogenous α-Synuclein. However, phosphorylated fibrils triggered the formation of more α-Synuclein inclusions in the Substantia Nigra pars compacta (SNpc), exacerbated pathology in the cortex and caused dopaminergic neuronal loss and fine motor impairment as early as 60 days post injection. Phosphorylated fibril injections also induced early changes in the innate immune response including alterations in macrophage recruitment and IL-10 release. Our study further shows that S129 phosphorylation facilitated α-Synuclein fibril uptake by neurons. Our results highlight the role of phosphorylated fibrilar α-Synuclein in pathology progression in vivo and suggest that targeting phosphorylated α-Synuclein assemblies might be important for delaying inclusion formation.


Phosphorylated exogenous alpha-synuclein fibrils exacerbate pathology and induce neuronal dysfunction in mice
Pathological accumulations stained positive with the rodent specific α-Synuclein antibody (D37A6) following PK treatment at 37 0 C for 30min and 1h. Note that under such treatment TH signal is lost. These PK-resistant accumulations are not revealed in untreated sections possibly because the antigen binding sites are not exposed for immunolabelling with the D37A6 antibody. Scale bar represents 25μm presence was more abundant compared to wt-and S129A-PFF. β-actin was used as a loading control. b Mouse primary cortical cultures were treated with half the concentration (0,2μg/10 5 cells) of P-PFF compared to the other types for 24 hours.
Following Triton-X extraction, internalized PFF were visualized with the human specific α-Synuclein antibody 4B12. Even at lower concentration P-PFF exhibited increased ability of uptake by neurons. β-actin was used as a loading control. c Seeding of endogenous α-Synuclein following treatment of mouse cortical cultures with PFF.6-day-old mouse cortical cultures were treated with P-, wt-, and S129A-PFF (0,4μg/10 5 cells) for 5 days. Seeding of the endogenous α-Synuclein, as depicted by foci formation, was visualized by immunocytochemistry using the rodent specific antibody (D37A6). Exogenously added human PFF were detected by co-staining with the human specific antibody LB509. TO-PRO-3 (blue) was used as a cell nuclear marker. Scale bar represents 10μm.

Supplementary Figure S13
Supplementary Figure S13 a Western blotting analysis for P-, S129A-and (mock-P) S129A-PFF. 1μg of fibrils were analyzed in a 10% SDS-PAGE gel using the C20 antibody (α-Synuclein monomer in higher exposure is shown in cropped gel/blot). No signal was observed for the (mock-P) and the S129A-PFF with the α-Synuclein (phospho Ser) antibody. Human PLK2 (Snk) was not detected in any of the fibrilar material treated with the enzyme. Lysate from SH-SY5Y cells, overexpressing human wt α-Synuclein (wt-ASYN) 3 , was used as a positive control. b Mouse primary cultures (6div) were treated with P-PFF, S129A-PFF and (mock-P) S129A-PFF for 24 hours. Following Triton-X extraction, internalized fibrils were visualized with the human specific α-Synuclein antibody 4B12. Treatments with either the mutant S129A-or its phosphorylated equivalent (mock-P) S129A-PFF resulted in a similar pattern of uptake. In contrast, increased internalization capacity was observed for the P-PFF. β-actin was used as a loading control. c Immunocytochemistry with the rodent specific α-Synuclein antibody (D37A6) in primary mouse cortical cultures treated with P-PFF, S129A-PFF and (mock-P) S129A-PFF for 5 days. Both mutant S129A-PFF and (mock-P) S129A-PFF appeared similarly effective to seed the endogenous α-

Behavioral analysis
Mice were habituated to the testing room 30 min before tests. Each mouse was handled for 2-3 minutes daily for one week prior to behavioral testing.

Open field
Locomotor activity was assessed as an index of gross motor function in a transparent, acrylic open field arena (The activity of each mouse was tracked by an overhead camera (Panasonic WV-BP332) and analyzed using specialized video tracking software (Ethovision XT 8.5, Noldus, The Netherlands). Locomotor activity was evaluated, as measured by the total distance travelled (cm) in 10 minutes.

Rotarod
Motor coordination and balance were assessed on an accelerating Rotarod (UGO BASILE). Each mouse was placed on the rotating rod with a diameter of 7 cm and after at least one minute of walking face forward on the rotating rod at the speed of 4 rpm (revolutions per minute), 3x5 min trials with accelerating speed from 4 to 40 rpm were conducted with 45 min resting intervals in between. Latency to fall or passively rotate was measured and the mean latency for all 3 trials was used for analysis.

Challenging beam traversal
The challenging beam was used to examine possible deficits in fine motor coordination as previously described 6 . Each mouse was trained to traverse a plexiglass beam consisting of four sections (25 cm each, 1 m total length) of different width (3.5 cm to 0.5 cm by 1 cm increments). After 2 days of training trials a wire mesh (1 cm x 2 cm rectangles) was placed approximately 1 cm above the beam and animals were video-recorded while traversing the grid-surfaced beam for a total of five trials. Errors per step (number of limb slips through grid surface per step) were measured .The mean errors per step for the four best trials were analyzed.

Pole test
The pole test is typically used to assess fine motor coordination and balance in basal ganglia-related movement disorders in mice 6,7 . Each mouse was placed head-up on top of a vertical wooden pole (50 cm height, 1 cm in diameter) placed in its home cage as previously described 6 . The animals received two days of training, each consisting of 5 trials. On the test day, time to orient downward (t-turn, sec) and time to descend the pole (t-total, sec) were calculated. Mean time of 5 trials was analyzed for each animal.

Grip strength
A digital grip strength meter (Ahlborn, Almemo-2450) was used to measure grip strength (g) of fore-and hindlimbs. The average of three trials was calculated.