Alpha-synuclein increases in rodent and human spinal cord injury and promotes inflammation and tissue loss

Abstract

Synucleinopathies are neurodegenerative diseases in which α-synuclein protein accumulates in neurons and glia. In these diseases, α-synuclein forms dense intracellular aggregates that are disease hallmarks and actively contribute to tissue pathology. Interestingly, many pathological mechanisms, including iron accumulation and lipid peroxidation, are shared between classical synucleinopathies such as Alzheimer's disease, Parkinson's disease and traumatic spinal cord injury (SCI). However, to date, no studies have determined if α-synuclein accumulation occurs after human SCI. To examine this, cross-sections from injured and non-injured human spinal cords were immunolabeled for α-synuclein. This showed robust α-synuclein accumulation in profiles resembling axons and astrocytes in tissue surrounding the injury, revealing that α-synuclein markedly aggregates in traumatically injured human spinal cords. We also detected significant iron deposition in the injury site, a known catalyst for α-synuclein aggregation. Next a rodent SCI model mimicking the histological features of human SCI revealed aggregates and structurally altered monomers of α-synuclein are present after SCI. To determine if α-synuclein exacerbates SCI pathology, α-synuclein knockout mice were tested. Compared to wild type mice, α-synuclein knockout mice had significantly more spared axons and neurons and lower pro-inflammatory mediators, macrophage accumulation, and iron deposition in the injured spinal cord. Interestingly, locomotor analysis revealed that α-synuclein may be essential for dopamine-mediated hindlimb function after SCI. Collectively, the marked upregulation and long-lasting accumulation of α-synuclein and iron suggests that SCI may fit within the family of synucleinopathies and offer new therapeutic targets for promoting neuron preservation and improving function after spinal trauma.

Figure 1

α-synuclein and iron increase in the human spinal cord after SCI. (A) LB509 + α-synuclein was present in injured white matter at 12d post-injury, and localized to large aggregates resembling axons within the injured white matter (red arrowheads). (B) Pathological α-synuclein was not present in white matter from non-injured human spinal cords. (C) α-synuclein was also present after human SCI in cells resembling astrocytes around the lesion site at 12d post-injury. (D,E) High magnification of cells boxed in panel (C). (F) Human SCI tissue stained for ferric iron using Perls stain shows dense iron accumulation within the injured region; adjacent spared tissue (*) had no visible iron stain. (G) Section of human SCI showing spared neurons close to lesioned tissue appear to have iron + inclusions in their cytoplasm (arrowhead). Scale bars: 50 µm in (A) and (B); 20 µm in (C,E,G); 400 µm in (F).

Figure 2

Aberrant forms of native α-synuclein are present after spinal cord injury in rats. (A) Antibody labeling for native monomeric α-synuclein (D37A6) revealed the expected immunoreactivity in the grey matter in uninjured rat spinal cords. (B) Following SCI, native α-synuclein was lost in areas of damaged grey matter. Image taken from Sect. 21d post-injury (dpi). (C) Representative western blot of monomeric α-synuclein stained with the D37A6 antibody illustrating protein modifications after SCI. Protein was isolated from naïve, 7d, 21d and 42d post-injury. White dividing lines indicate borders of cropped representative fields from the same western blot. (D) Quantification of normal molecular weight isoform of α-synuclein protein showed a non-significant reduction after SCI. (E,F) Truncated and larger molecular weight isoforms of α-synuclein were significantly increased 7d–42d after spinal cord injury compared to uninjured controls. n = 3–4/group. Two-way repeated measures ANOVA with Bonferroni post-hoc test. *p < 0.05, **p < 0.01.

Figure 3

Neuron loss and iron deposition are reduced after SCI in male α-synuclein knockout mice. (A) Representative images of NeuN stained tissue at 600 µm rostral to the injury epicenter. (B) Quantification of NeuN positive neurons ± 600 µm of the lesion epicenter. Male α-synuclein knockout mice had significantly more spared neurons in tissue ± 600 µm of the lesion epicenter compared to controls. Neuron sparing was not different in female mice. n = 4–9/group. Two-tailed t-test *p < 0.05. (C) Quantification of intraspinal iron accumulation 600 µm rostral or caudal to the injury epicenter in male mice. α-synuclein knockout mice had significantly lower intraspinal iron caudal to the injury epicenter compared to control mice. n = 4–7/group. Two-way repeated measures ANOVA with Bonferroni post-hoc test. *p < 0.05. Scale bar: 500 µm.

Figure 4

Axon sparing is increased in α-synuclein knockout mice after SCI. (A) Quantification of neurofilament-heavy positive axons 600 µm rostral of the lesion epicenter. Both male and female α-synuclein mice had significantly increased axon sparing after SCI. (B) Schematic representation of regions of axon quantification. White boxes represent quantified areas. Red area represents region of frankly damaged tissue 600 µm rostral of the lesion epicenter. (C) Representative images of neurofilament positive axons from sampled regions. n = 3–4/group. Two-way repeated measures ANOVA with Bonferroni post-hoc test. *p < 0.05, **p < 0.01. Scale bar equals 12.5 µm.

Figure 5

α-synuclein knockout reduces intraspinal macrophage accumulation after SCI. (A) Representative images of Cd11b-positive macrophages 600 µm rostral to the injury epicenter. (B) Male KO mice had significantly reduced macrophage accumulation (Mac-1 immunoreactivity) compared to controls across the lesion extent (± 600 µm from epicenter). Macrophage accumulation was not different in female KO and control mice; however female control mice had significantly reduced overall Mac-1 immunoreactivity compared to male controls. n = 4–5/group. Two-way repeated measures ANOVA with Bonferroni post-hoc test. ***p < 0.001. Scale bar equals 500 µm.

Figure 6

α-synuclein knockout mice have altered inflammatory markers after SCI. (A–C) Real-time PCR of RNA collected from the injury site at 21d post-injury showed a significant reduction in CD68, IL-1β and Iκβα RNA in KO mice compared to controls. (D–E) There was a trend towards a decrease in RNA levels of CXCL1 and TNFα. n = 4–6/group. Two-tailed t-test. *p < 0.05.

Figure 7

Mice lacking α-synuclein had worse locomotor recovery after spinal cord injury, likely due to dopamine deficiency. (A) Hindlimb function assessed with the BMS rating scale revealed KO mice had significantly worse locomotor recovery, including a marked reduction in stepping ability, compared to controls 7–20 days post-injury (dpi). Horizontal line indicates a BMS score of 4, which is the threshold for achieving plantar stepping. (B) Movement time was not different between genotypes at any time post-injury. (C,D) Treatment with the dopamine agonist SKF-81297 at 7dpi transiently reversed the stepping impairment in the KO mice and improved BMS scores to control levels (C). The number of KO mice stepping increased from zero before treatment to three 15 min after treatment (D). (E,F) Treatment with the dopamine agonist at 14dpi slightly increased locomotor function in KO mice (E) and significantly increased the number of KO mice stepping 15 min after treatment (F). n = 5–6/group. (A–C,E): Two-way repeated measures ANOVA with Bonferroni post-hoc test. D, F: Chi-squared test. *p < 0.05, ***p < 0.001.