Localized Induction of Wild-Type and Mutant Alpha-Synuclein Aggregation Reveals Propagation along Neuroanatomical Tracts

Abstract

Misfolded alpha-synuclein (αS) may exhibit a number of characteristics similar to those of the prion protein, including the apparent ability to spread along neuroanatomical connections. The demonstration for this mechanism of spread is largely based on the intracerebral injections of preaggregated αS seeds in mice, in which it cannot be excluded that diffuse, surgical perturbations and hematogenous spread also contribute to the propagation of pathology. For this reason, we have utilized the sciatic nerve as a route of injection to force the inoculum into the lumbar spinal cord and induce a localized site for the onset of αS inclusion pathology. Our results demonstrate that mouse αS fibrils (fibs) injected unilaterally in the sciatic nerve are efficient in inducing pathology and the onset of paralytic symptoms in both the M83 and M20 lines of αS transgenic mice. In addition, a spatiotemporal study of these injections revealed a predictable spread of pathology to brain regions whose axons synapse directly on ventral motor neurons in the spinal cord, strongly supporting axonal transport as a mechanism of spread of the αS inducing, or seeding, factor. We also revealed a relatively decreased efficiency for human αS fibs containing the E46K mutation to induce disease via this injection paradigm, supportive of recent studies demonstrating a diminished ability of this mutant αS to undergo aggregate induction. These results further demonstrate prion-like properties for αS by the ability for a progression and spread of αS inclusion pathology along neuroanatomical connections.IMPORTANCE The accumulation of alpha-synuclein (αS) inclusions is a hallmark feature of Parkinson's disease (PD) and PD-related diseases. Recently, a number of studies have demonstrated similarities between the prion protein and αS, including its ability to spread along neuroanatomical tracts throughout the central nervous system (CNS). However, there are caveats in each of these studies in which the injection routes used had the potential to result in a widespread dissemination of the αS-containing inocula, making it difficult to precisely define the mechanisms of spread. In this study, we assessed the spread of pathology following a localized induction of αS inclusions in the lumbar spinal cord following a unilateral injection in the sciatic nerve. Using this paradigm, we demonstrated the ability for αS inclusion spread and/or induction along neuroanatomical tracts within the CNS of two αS-overexpressing mouse models.

Keywords: alpha-synuclein; axonal transport; prion; prions; propagation; sciatic nerve; α-synuclein.

FIG 1

Induction of disease in the M83^+/− αS transgenic mouse model following unilateral sciatic nerve injections. Kaplan-Meier curves reveal the transmissibility of the indicated recombinant proteins following unilateral sciatic nerve injection in the M83^+/− line of αS transgenic mice. Mice were injected with mouse WT αS fibs (mfib αS), E46K human αS fibs (hfib αS E46K), human Δ71–82 αS, recombinant WT SOD1 fibrils, or PBS.

FIG 2

Induction and spread of αS pathology in the M83^+/− mouse model following injection with mouse αS fibs. Mice were injected unilaterally in the sciatic nerve with mouse WT αS fibs (mfib αS) and harvested at 1 month (a) or 2 months (b) p.i. or when the mice reached the end stage of disease (c). (d) Mice were also injected with human αS fibs containing the E46K mutation (hfib αS E46K) and harvested at the end stage of disease or, for those with no symptoms, at 12 months p.i. Representative images show αS pathology in both the ipsilateral (ipsi-) and contralateral (contra-) DRG and several CNS regions, depicted by cartoons with black dots representing the specific locations the images were taken (n ≥ 8 animals per cohort). Tissue sections were stained with an antibody to αS phosphorylated at Ser129 (2G12) and counterstained with hematoxylin. Scale bars = 50 μm. Sp.C., spinal cord.

FIG 3

αS inclusion pathology in M83^+/− injected mice. M83^+/− mice were injected unilaterally in the sciatic nerve with the indicated homogenates and harvested at the end stage of disease (mfib αS and hfib αS E46K) or at 6 months p.i. (PBS, human Δ71–82 αS, and WT SOD1 fibs). Spinal cords were stained with a conformation-specific antibody against αS, Syn 506 (a), and an antibody specific for cytoplasmic Lewy body inclusions, p62/Sqstm1 (b).

FIG 4

Absence of αS inclusion pathology in M83^+/− mice injected with control inocula. Mice were injected unilaterally in the sciatic nerve with PBS (a), soluble human αS containing Δ71–82 (b), or WT SOD1 fibs (c) and aged to 6 months p.i. prior to harvesting tissue (n = 6 animals per cohort). Representative images show αS pathology in both the ipsilateral and contralateral DRG and several CNS regions, depicted by cartoons with black dots representing the specific locations the images were taken. Tissue sections were stained with an antibody to αS phosphorylated at Ser129 (2G12) and counterstained with hematoxylin. Scale bars = 50 μm.

FIG 5

Detection of αS inclusion pathology in the white matter of the spinal cord. Representative images show αS pathology in the white matter surrounding the ventral horn of the spinal cord in mice injected unilaterally in the sciatic nerve with PBS (a) or mfib αS (b and c) and harvested at the indicated time points p.i. Tissue sections were stained with an antibody to αS phosphorylated at Ser129 (2G12) and counterstained with hematoxylin. Scale bars = 50 μm.

FIG 6

Accumulation of gliosis in M83^+/− injected mice. M83^+/− mice were injected unilaterally in the sciatic nerve with control inocula (PBS and WT SOD1 fibs) and harvested at 6 months p.i. or with mouse WT αS fibs (mfib αS) and harvested at 1 month or 2 months p.i. or when the mice reached the end stage of disease. (a) Spinal cords were immunostained to visualize astrocytes using an antibody to GFAP and representative images were captured (n = 3 animals per cohort). (b) GFAP immunoreactivity in the cervical and lumbar segments of the spinal cord was quantified for each cohort (n = 3 animals per cohort; mean ± SD). ***, P ≤ 0.001. (c) Spinal cords were also immunostained for microglia using an antibody to cd11b, and representative images were captured (n = 3 animals per cohort). Scale bars = 100 μm.

FIG 7

Induction of disease in the M20^+/− αS transgenic mouse model following unilatereal sciatic nerve injections. Kaplan-Meier curves reveal the transmissibility of the indicated recombinant proteins following unilateral sciatic nerve injection in M20^+/− line of αS transgenic mice. Mice were injected with mouse WT αS fibs (mfib αS), human Δ71–82 αS, recombinant WT SOD1 fibrils, or PBS.

FIG 8

Induction and spread of αS pathology in the M20^+/− mouse model following injection with mouse αS fibs. Mice were injected unilaterally in the sciatic nerve with mouse WT αS fibs (mfib αS) and harvested at 2 months (a) or 4 months (b) p.i. or when the mice reached the end stage of disease (c) (n ≥ 8 animals per cohort). Representative images show αS pathology in both the ipsilateral and contralateral DRG and several CNS regions, depicted by cartoons with black dots representing the specific locations the images were taken. Tissue sections were stained with an antibody to αS phosphorylated at Ser129 (2G12) and counterstained with hematoxylin. Scale bars = 50 μm.

FIG 9

αS inclusion pathology in M20^+/− injected mice. M20^+/− mice were injected unilaterally in the sciatic nerve with the indicated homogenates and harvested at the end stage of disease (mfib αS) or at 12 months p.i. (PBS, Δ71–82 αS, and WT SOD1 fibs). Spinal cords were stained with a conformation-specific antibody against αS, Syn 506 (a), and an antibody specific for cytoplasmic Lewy body inclusions, p62/Sqstm1 (b).

FIG 10

Absence of αS pathology in M20^+/− mice injected with control inocula. Mice were injected unilaterally in the sciatic nerve with PBS (a), soluble human αS containing Δ71–82 (b), or WT SOD1 fibs (c) and aged to 12 months p.i. prior to harvesting tissue (n = 6 animals per cohort). Representative images show αS pathology in both the ipsilateral and contralateral DRG and several CNS regions, depicted by cartoons with black dots representing the specific locations the images were taken. Tissue sections were stained with an antibody to αS phosphorylated at Ser129 (2G12) and counterstained with hematoxylin. Scale bars = 50 μm.

FIG 11

Accumulation of gliosis in M20^+/− injected mice. M20^+/− mice were injected unilaterally in the sciatic nerve with control inocula (PBS and WT SOD1 fibs) and harvested at 12 months p.i. or with mouse WT αS fibs (mfib αS) and harvested at 2 months or 4 months p.i. or when the mice reached the end stage of disease. (a) Spinal cords were immunostained to visualize astrocytes using an antibody to GFAP and representative images were captured (n = 3 animals per cohort). (b) GFAP immunoreactivity in the cervical and lumbar segments of the spinal cord was quantified for each cohort (n = 3 animals per cohort; mean ± SD). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (c) Spinal cords were also immunostained for microglia using an antibody to cd11b and representative images were captured (n = 3 animals per cohort). Scale bars = 100 μm.

FIG 12

Inability of mouse αS fibs to induce αS pathology in WT mice. WT mice were injected unilaterally in the sciatic nerve with mouse WT αS fibs (mfib αS) (a) or PBS (b) and harvested at 12 months p.i. (n ≥ 6 animals per cohort). Representative images show the lack of αS pathology in both the ipsilateral and contralateral DRG and several CNS regions, depicted by cartoons with black dots representing the specific locations the images were taken. Tissue sections were stained with an antibody to αS phosphorylated at Ser129 (2G12) and counterstained with hematoxylin. Scale bars = 50 μm.