Determinants of seeding and spreading of α-synuclein pathology in the brain

Abstract

In Parkinson's disease (PD), fibrillar forms of α-synuclein are hypothesized to propagate through synaptically coupled networks, causing Lewy pathology (LP) and neurodegeneration. To more rigorously characterize the determinants of spreading, preformed α-synuclein fibrils were injected into the mouse pedunculopontine nucleus (PPN), a brain region that manifests LP in PD patients and the distribution of developing α-synuclein pathology compared to that ascertained by anterograde and retrograde connectomic mapping. Within the PPN, α-synuclein pathology was cell-specific, being robust in PD-vulnerable cholinergic neurons but not in neighboring noncholinergic neurons. While nearly all neurons projecting to PPN cholinergics manifested α-synuclein pathology, the kinetics, magnitude, and persistence of the propagated pathology were unrelated to the strength of those connections. Thus, neuronal phenotype governs the somatodendritic uptake of pathological α-synuclein, and while the afferent connectome restricts the subsequent spreading of pathology, its magnitude and persistence is not a strict function of the strength of coupling.

Fig. 1. Induction of local p-aSYN pathology within the PPN.

(A) Schematic showing PFF injections and pathology assessment. WT, wild type. (B) Distribution of biotin-tagged aSYN PFFs within the PPN at 1 wpi. PAG, Periaqueductal gray. (C) Representative image showing PFF-induced aSYN aggregation detected by p-aSYN. (D) p-aSYN–positive inclusions in ChAT-positive neurons. (E) Representative images after PK or control treatment. (F) Quantification of aggregate location at 1, 6, and 12 wpi on the injected side (Student’s t test with post hoc correction for three t tests). (G) Ratio of aggregate containing cholinergic PPN neurons on the injected side [one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test]. (H) Count of non-CNs (Kruskal-Wallis test followed by Dunn’s multiple comparisons test). (I) Representative images depicting ChAT- and NeuN-positive neurons. (J) Count of ChAT-positive PPN neurons (Kruskal-Wallis test followed by Dunn’s multiple comparisons test). (K) Graph showing percentage of IbA1-positive pixels at the injection site for PFF and monomer groups (one-way ANOVA followed by Tukey’s post hoc test). (L) Microglia (IbA1) within the PPN region. Quantification includes n = 3 for 1 wpi, n = 4 for 6 wpi, and n = 5 for 12 wpi. Scale bars, 250 μm (A), 100 μm (C), 25 μm (D, E, and L), and 500 μm (I). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 2. Presynaptic inputs to PPN CNs revealed by rabies-mediated retrograde tracing.

(A) Schematic showing strategy of trans-synaptic tracing. (B) Injected PPN region stained for ChAT, RG, and mCherry. Starter cells (arrowheads) costain for RG, mCherry (indicator for TVA expression), and eGFP (indicator for rabies). (C) Number of traced eGFP-positive input neurons per animal. (D) Calculation of the convergence index revealed 32 presynaptic input neurons per starter cell. (E) Monosynaptic input neurons in selected brain areas. (F) Quantification of presynaptic neurons in gross anatomical regions normalized against the total number of input neurons throughout the respective brain. (G) Fraction of total input neurons for basal ganglia structures and other brain regions accounting for at least 1% of total input. (H) PPN CNs receive significantly more synaptic input from midbrain and motor-related medullar brain regions than from basal ganglia structures (one-way ANOVA followed by Tukey’s multiple comparisons test). (I) Heatmaps of inputs to cholinergic PPN neurons accounting for more than 0.1% of all inputs. Color scale represents the percentage of total inputs. Red circle indicates the location of starter cells. Quantification includes n = 4. Scale bars, 100 μm (B; overview), 50 μm (B; high magnification), and 250 μm (E). ***P < 0.001. All abbreviations used can be found in table S2 in the Supplementary Materials.

Fig. 3. Efferent projections of PPN CNs revealed by Cre-dependent eGFP overexpression.

(A) Schematic showing the strategy of cholinergic output tracing. (B) Injected PPN region stained for ChAT, eGFP, and 4′,6-diamidino-2-phenylindole (DAPI). Output tracing starter cells (arrowheads) are indicated by eGFP expression. (C) Distribution of starter cells between cholinergic PPN, cholinergic LDT, and non-CNs (one-way ANOVA followed by Tukey’s post hoc test). (D and E) Quantification of axonal density (positive pixel count normalized to the total area, D) and total positive pixel count (E) in 10 predetermined brain regions (three sections per brain region and animal). (F) Representative images of eGFP-positive axonal projections from PPN CNs. (G) Heatmaps of eGFP-positive axonal density in coronal brain sections. Color scale represents average axonal density assessed semiquantitatively with a grading system from 1 to 4. Red circle indicates the location of starter cells. Quantification includes n = 4. Scale bars, 100 μm (B; overview), 25 μm (B; high magnification), and 100 μm (F). ***P < 0.001. All abbreviations used can be found in table S2 in the Supplementary Materials.

Fig. 4. Comparison of systemic p-aSYN pathology induced by aSYN PFF injection.

(A) Strategy for PFF injections and assessment of developing brain-wide pathology. (B and C) p-aSYN soma and neurite pathology scores at 1, 6, and 12 wpi of 29 preselected brain regions. (D to F) Caudo-rostral distribution of brain regions relative to the injection site (PPN, 0) showing p-aSYN soma pathology at 1 (D), 6 (E), or 12 (F) wpi. (G and H, left column) Images depicting p-aSYN pathology in LDT or VTA at 6 and 12 wpi. (G and H, middle column) p-aSYN soma pathology was significantly decreased at 12 wpi compared to 6 wpi in LDT or VTA (Mann-Whitney test). (G and H, right column) This decrease was not associated with loss of NeuN-positive cells (Kruskal-Wallis followed by Dunn’s multiple comparisons test). (I and J) Heatmaps depicting brain-wide p-aSYN soma pathology at 6 and 12 wpi. Color scale represents average p-aSYN soma pathology assessed semiquantitatively with a grading system from 1 to 4. Quantification includes n = 3 for 1 wpi, n = 4 for 6 wpi, and n = 5 for 12 wpi. Scale bars, 250 μm (G and H). *P < 0.05.

Fig. 5. Comparison of tracing results and brain-wide p-aSYN pathology.

(A and B) Correlating the 50 highest input (A) or output regions (B) for PPN CNs with their respective p-aSYN soma or neurite pathology revealed no correlation between the degree of synaptic input/degree of PPN cholinergic axonal output density and p-aSYN pathology at 1, 6, and 12 wpi (nonparametric Spearman’s rank correlation test). (C) In contrast, p-aSYN soma pathology correlated strongly with p-aSYN neuritic pathology at 1, 6, and 12 wpi (nonparametric Spearman’s rank correlation test). (D) Ratio of input neurons or p-aSYN soma pathology at 6 and 12 wpi in four predetermined brain regions that are either medium (~4% of total input) or high (~8% of total input) inputs of PPN CNs (unpaired t tests). (E) Representative images of PPN input tracing (left, eGFP-labeled neurons), PPN output tracing (middle, eGFP-labeled axons), and p-aSYN pathology (right) of the central amygdala (CEAl) and the substantia nigra at 12 wpi. Quantification includes n = 4 for output and input tracing, n = 3 for PFF 1 wpi, n = 4 for PFF 6 wpi, and n = 5 for PFF 12 wpi. Scale bar, 250 μm (E). *P < 0.05, **P < 0.01, and ****P < 0.0001. BSTov, bed nuclei of the stria terminalis, oval nucleus; GRN, gigantocellular reticular nucleus.

Fig. 6. Synaptic connectivity alone does not predict the spreading or persistence of p-aSYN pathology.

(A to C) Plots of the cholinergic PPN input connectome depicting the 40 strongest input structures and brain regions exhibiting p-aSYN pathology after 1 wpi with their corresponding p-aSYN soma pathology for 1, 6, and 12 wpi. Brain nuclei are represented as circles distributed along the rostrocaudal axis. The diameter of each circle is proportional to the projection strength, whereas the color of the circles corresponds to the strength of pathology. Brain nuclei on the left side show no p-aSYN pathology, while nuclei on the right side exhibit p-aSYN pathology at 1, 6, or 12 wpi.