Pathologic modifications of alpha-synuclein in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated squirrel monkeys

Abstract

alpha-Synuclein expression is increased in dopaminergic neurons challenged by toxic insults. Here, we assessed whether this upregulation is accompanied by pathologic accumulation of alpha-synuclein and protein modifications (i.e. nitration, phosphorylation, and aggregation) that are typically observed in Parkinson disease and in other synucleinopathies. A single injection of the neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to squirrel monkeys caused a buildup of alpha-synuclein but not of beta-synuclein or synaptophysin within nigral dopaminergic cell bodies. Immunohistochemistry and immunoelectron microscopy also revealed large numbers of dystrophic axons labeled with alpha-synuclein. Antibodies that recognize nitrated and phosphorylated (at serine 129) alpha-synuclein stained neuronal cell bodies and dystrophic axons in the midbrain of MPTP-treated animals. After toxicant exposure, alpha-synuclein deposition occurred at the level of neuronal axons in which amorphous protein aggregates were observed by immunoelectron microscopy. In a subset of these axons, immunoreactivity for alpha-synuclein was still evident after tissue digestion with proteinase K, further indicating the accumulation of insoluble protein. These data indicate that toxic injury can induce alpha-synuclein modifications that have been implicated in the pathogenesis of human synucleinopathies. The findings are also consistent with a pattern of evolution of alpha-synuclein pathology that may begin with the accumulation and aggregation of the protein within damaged axons.

FIGURE 1

α-Synuclein but not other synaptic proteins is accumulated within nigral dopaminergic cell bodies of MPTP-treated monkeys. Squirrel monkeys were given a single subcutaneous injection of either saline or MPTP and killed four weeks later. Representative midbrain sections from control (A, C, E) and MPTP-treated (B, D, F) animals were immunostained for α-synuclein (A, B), β-synuclein (C, D) or synaptophysin (E, F), and counterstained with Cresyl violet (purple). Increased immunoreactivity (brown) for α-synuclein, but not β-synuclein or synaptophysin, is evident within neuromelanin-loaded (black granules) nigral dopaminergic cell bodies in the MPTP-treated monkey. Scale bar in (F) corresponds to 10 μm (A-F).

FIGURE 2

Neuritic accumulation of α-synuclein in the midbrain of MPTP-treated monkeys resembles pathologic changes in PD patients. (A-C) Representative sections of the ventral midbrain from monkeys injected once with either saline (A) or MPTP (B, C) and killed four weeks later. Tissues were immunostained with an antibody against α-synuclein and counterstained with Cresyl violet. Numerous α-synuclein-immunoreactive (brown) neurites are shown at low (B) and high (C) magnification in the ventral midbrain of the MPTP-treated animal. (D, panel and inset) Pathological neurites in the ventral midbrain of a PD patient. A representative midbrain section was immunostained with an antibody against α-synuclein and counterstained with Cresyl violet. Scale bar in (B) corresponds to 100 μm (A, B). Scale bars in (C, D and inset) correspond to 20 μm.

FIGURE 3

Immunoelectron microscopy (IEM) images of α-synuclein accumulation within dystrophic neurites. Tissue punches of the ventral midbrain of monkeys treated with saline or MPTP were processed for IEM with an anti-α-synuclein antibody. (A, B) Representative electron micrographs show increased α-synuclein immunolabeling (black particles) within myelinated (B) and unmyelinated (inset of panel B) neurites in the MPTP-treated monkey compared to the control (A). (C, D) Representative electron micrographs show normal tissue architecture in a control (C) and an enlarged dystrophic axon in an MPTP-injected animal (arrow in D). (E) At higher magnification, accumulation of α-synuclein (black particles) is more evident within a dystrophic axon in an MPTP-treated monkey. Scale bar in (B) corresponds to 1 μm (A, B). Scale bar in (inset) corresponds to 1 μm. Scale bar in (D) corresponds to 4 μm (C, D). Scale bar in (E) corresponds to 1 μm.

FIGURE 4

α-Synuclein does not colocalize with astrocytic or microglial markers. (A-C) A representative section of the ventral midbrain from a monkey injected once with MPTP and killed four weeks later was co-labeled with anti-α-synuclein (A) and anti-GFAP (an astrocytic marker, B). The arrow in panel A indicates an enlarged dystrophic axon positively stained for α-synuclein. The merged image (C) shows lack of α-synuclein immunoreactivity within astrocytes. (D-F) A representative section of the ventral midbrain from an MPTP-treated monkey was co-labeled with anti-α-synuclein (D) and anti-Iba1 (a microglial marker, E). The arrow in panel D indicates an enlarged α-synuclein-immunoreactive axon. The merged image (F) shows lack of α-synuclein and Iba1 colocalization. Scale bar in (F) corresponds to 10 μm (A-F).

FIGURE 5

Nitrated α-synuclein within nigral dopaminergic cell bodies and dystrophic axons of MPTP-treated monkeys. Squirrel monkeys received a single subcutaneous injection of either saline or MPTP and were killed four weeks later. (A-C) Representative midbrain sections from a control (A) and an MPTP-treated (B, C) animal were immunostained with anti-nitrated α-synuclein, and counterstained with Cresyl violet (purple). Immunoreactivity for nitrated α-synuclein (brown) is shown within a neuromelanin-containing nigral neuron (B) and dystrophic neurites (C) after MPTP treatment. (D-F) A representative midbrain section from an MPTP-treated animal was processed for confocal microscopy. Tissue was dual-labeled with anti-α-synuclein (D) and anti-nitrated α-synuclein (E) antibodies. The merged image (F) shows that α-synuclein is nitrated in some but not all dystrophic neurites. Scale bar in (B) corresponds to 10 μm (A, B). Scale bar in (C) corresponds to 10 μm. Scale bar in (F) correspond to 10 μm (D-F).

FIGURE 6

Phosphorylated α-synuclein within nigral dopaminergic cell bodies and axonal profiles of MPTP-exposed monkeys. Squirrel monkeys received a single subcutaneous injection of either saline or MPTP and were killed four weeks later. (A-C) Representative midbrain sections from a control (A) and an MPTP-treated (B, C) animal were immunostained with an anti-phospho-Ser 129 α-synuclein antibody and counterstained with Cresyl violet (purple). Immunoreactivity for phospho-Ser 129 α-synuclein (brown) is shown within a nigral neuron (B) and dystrophic neurites (C) after MPTP treatment. (D-F) A representative midbrain section from an MPTP-treated animal was processed for confocal microscopy. Tissue was dual-labeled with anti-α-synuclein (D) and anti-phospho-Ser 129 α-synuclein (E) antibodies. The merged image (F) shows that α-synuclein is phosphorylated at Ser129 in some but not all dystrophic neurites. Scale ba rin (B) corresponds to 10 μm (A, B). Scale bar in (C) corresponds to 10 μm. Scale bar in (F) corresponds to 10 μm (D-F).

FIGURE 7

Proteinase K-resistant axonal pathology in MPTP-exposed monkeys. Squirrel monkeys received a single subcutaneous injection of either saline or MPTP and were killed four weeks later. Representative midbrain sections from a saline- (A, B) and an MPTP-treated (C-F) animal were incubated in either buffer (A, C, E) or proteinase K (B, D, F). Sections were subsequently immunostained with an antibody against α-synuclein (brown) and counterstained with Cresyl violet (purple). The robust immunoreactivity observed within nigral cell bodies of the MPTP-exposed animal (C) was removed by proteinase K digestion leaving only neuromelanin granules (black) (D). A subset of dystrophic axons in the MPTP-treated monkey retained α-synuclein immunoreactivity even after proteinase K-treatment (F). Scale bar in (D) corresponds to 10 μm (A-D). Scale bar in (F) corresponds to 10 μm (E, F).