Parkinson-like wild-type superoxide dismutase 1 pathology induces nigral dopamine neuron degeneration in a novel murine model

Abstract

Atypical wild-type superoxide dismutase 1 (SOD1) protein misfolding and deposition occurs specifically within the degenerating substantia nigra pars compacta (SNc) in Parkinson disease. Mechanisms driving the formation of this pathology and relationship with SNc dopamine neuron health are yet to be fully understood. We applied proteomic mass spectrometry and synchrotron-based biometal quantification to post-mortem brain tissues from the SNc of Parkinson disease patients and age-matched controls to uncover key factors underlying the formation of wild-type SOD1 pathology in this disorder. We also engineered two of these factors - brain copper deficiency and upregulated SOD1 protein levels - into a novel mouse strain, termed the SOCK mouse, to verify their involvement in the development of Parkinson-like wild-type SOD1 pathology and their impact on dopamine neuron health. Soluble SOD1 protein in the degenerating Parkinson disease SNc exhibited altered post-translational modifications, which may underlie changes to the enzymatic activity and aggregation of the protein in this region. These include decreased copper binding, dysregulation of physiological glycosylation, and atypical oxidation and glycation of key SOD1 amino acid residues. We demonstrated that the biochemical profile introduced in SOCK mice promotes the same post-translational modifications and the development of Parkinson-like wild-type SOD1 pathology in the midbrain and cortex. This pathology accumulates progressively with age and is accompanied by nigrostriatal degeneration and dysfunction, which occur in the absence of α-synuclein deposition. These mice do not exhibit weight loss nor spinal cord motor neuron degeneration, distinguishing them from transgenic mutant SOD1 mouse models. This study provides the first in vivo evidence that mismetallation and altered post-translational modifications precipitates wild-type SOD1 misfolding, dysfunction, and deposition in the Parkinson disease brain, which may contribute to SNc dopamine neuron degeneration. Our data position this pathology as a novel drug target for this disorder, with a particular focus on therapies capable of correcting alterations to SOD1 post-translational modifications.

Keywords: Copper deficiency; Mouse model; Neurodegeneration; Oxidative stress; Parkinson disease; Post-translational modification; Protein misfolding; Substantia nigra pars compacta; Superoxide dismutase 1.

Fig. 1

Altered SOD1 post-translational modification in the post-mortem Parkinson disease SNc. a The ratio of Cu:Zn bound to SOD1 was decreased in the post-mortem SNc of Parkinson disease (PD) patients (n = 14) compared with controls (n = 14), but was unchanged in the PD OCx (n = 17) compared with controls (n = 18) (two-way ANOVA: p = 0.0039, F = 9.22; Sidak’s multiple comparisons post hoc tests: *p = 0.022 for PD vs Ct SNc, p = 0.24 for PD vs Ct OCx, #p = 0.0009 for PD SNc vs PD OCx). Data represent mean ± SEM. b Lower SOD1 Cu:Zn ratios were correlated with decreased SOD1 isoelectric point in the PD SNc. Spearman’s r coefficient, the p value, and the number of XY pairs analyzed (n) are stated within the panel. c Mature SOD1 is dimeric, with each monomer comprising an eight-stranded β-barrel (gray) that binds one Cu (orange) and Zn (cyan) ion. The electrostatic loop (blue) guides anionic superoxide toward Cu in the active site using a series of charged and polar residues. Zinc coordination is facilitated by three histidine residues and one aspartic acid residue (cyan) within the metal-binding loop (green), while copper coordination is mediated by four histidine residues (orange). The disulfide loop (yellow) is a substructure within the metal-binding loop, containing one of two cysteine residues that form an intramolecular disulfide bond within SOD1 protein (yellow). The Greek key loop (pink) forms a plug at one pole of the β-barrel and contributes to dimer interface stability. d Distribution of all residues identified as sites of PTMs in SOD1 protein isolated from the SNc of PD cases and controls (highlighted in red, residues listed in Supplementary Table 6). e Distribution of residues exhibiting significantly lower levels of physiological modifications (glycosylation, acetylglucosamination). f A significant decrease in glycosylation (glycosyl) of S98 and N131 and acetylglucosamination (GlcNAc) of S25, N26, and N131 was identified in the SNc of PD patients compared with controls, while atypical oxidation of H80 and W32, as well as carboxymethyllysine (CML; K36) and glycation of K9, K23, K91, R115, and K122, was increased in this region. g Distribution of residues exhibiting significantly higher levels of atypical modifications (oxidation, glycation). Residues in panels e and g are labeled using one letter amino acid codes with their side chains highlighted in red. Complete details of statistical analyses identifying PTM alterations are presented in Supplementary Table 6

Fig. 2

Novel SOCK mice recapitulate elevated SOD1 protein levels and brain copper deficiency observed in the post-mortem Parkinson disease SNc. a Novel SOCK mice were developed by crossbreeding hSOD1^WT mice, which overexpress wild-type human SOD1, with Ctr1^+/- mice exhibiting decreased cellular copper within the central nervous system due to a knockdown of the neuronal copper import protein Ctr1. b Midbrain copper levels quantified using inductively coupled plasma-mass spectrometry varied significantly between wild-type, hSOD1^WT, Ctr1^+/- and SOCK mice across all ages, with decreases observed in Ctr1^+/- and SOCK mice compared with wild-type mice. c Levels of SOD1 protein in the midbrain quantified using immunoblotting varied significantly between all mouse strains, with increases observed in hSOD1^WT and SOCK mice compared with wild-type mice across all ages. No differences were observed between hSOD1^WT and SOCK mice at any age. Similar trends were observed for copper levels (d) and SOD1 protein levels (e) in the cortex. f Copper levels in the liver varied significantly between mouse strains, with increases observed in hSOD1^WT and SOCK mice compared with wild-type mice across all ages. g SOD1 protein levels in the liver varied significantly between mouse strains, with increases observed in hSOD1^WT and SOCK mice compared with wild-type mice across all ages. Data in panels b-g represent mean ± SEM. Comparisons marked with an asterisk (*) denote comparisons made to wild-type mice, while those marked with a bullet point (•) demarcate those made between SOCK and Ctr1^+/- mice. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, • p < 0.0001, # p < 0.0001. Statistical main effects, post hoc comparisons, and sample sizes are listed in Supplementary Tables 8 and 9

Fig. 3

Post-translational modification of SOD1 is altered in the midbrain of SOCK mice. Atypical SOD1 PTMs were increased in the SOCK mouse midbrain (a), some of which were also shared with hSOD1^WT mice, while others were unique to SOCK mice (b). These include oxidation of metal-binding histidine residues (c; H46, H48, H71) and glycation of a solvent-accessible lysine residue (d; K23). Side chains of labeled residues are highlighted in black. Similar to atypical SOD1 PTMs, a number of physiological PTMs were significantly dysregulated in the SOCK mouse midbrain (e), some of which were shared with hSOD1^WT mice, while others were unique to SOCK mice (f). GlyGly modifications result from tryptic digestion of ubiquitin-conjugated proteins, which serve as indicators of protein ubiquitination. g Distribution of physiological SOD1 PTM alterations. h SOD1 PTM alterations in the SOCK mouse midbrain largely overlap with those observed in the Parkinson disease SNc. Residues in panels c, d and g are labeled using one letter amino acid codes. Copper and zinc ions are highlighted in orange and cyan respectively. Complete details of statistical analyses identifying PTM alterations in SOCK and hSOD1^WT mice are presented in Supplementary Tables 10 and 11. CML carboxymethyllysine, Glycosyl glycosylation, Acetyl acetylation, GlcNAc acetylglucosamination, Succinyl succinylation, Phosphoryl phosphorylation, Deamid deamidation

Fig. 4

Immature, catalytically dysfunctional SOD1 accumulates and aggregates in the SOCK mouse SNc. a Total SOD activity varied significantly in the midbrain between wild-type, hSOD1^WT, Ctr1^+/-, and SOCK mice aged 1.5–12 months old. Activity was increased in the midbrains of both hSOD1^WT and SOCK mice compared with wild-type mice at all ages except 1.5-month-old SOCK mice, yet was decreased in SOCK mice compared with hSOD1^WT mice at all ages. b SOD activity per unit of SOD1 protein also varied significantly in the midbrain between all four mouse strains and was decreased in the midbrains of both hSOD1^WT and SOCK mice compared with wild-type mice at all ages as well as in SOCK mice compared with hSOD1^WT mice at all ages. Immunofluorescent staining of fixed midbrain tissues from hSOD1^WT (c) and SOCK (d) mice with the unfolded beta barrel (UβB) conformation-specific SOD1 antibody revealed the presence of disSOD1 aggregates (double white arrowheads) within and outside of dopamine neuron [tyrosine hydroxylase (TH)-positive] soma at all ages examined in SOCK mice, which was present at much lower levels in hSOD1^WT mice. Corresponding images for Ctr1^+/- and wild-type mice are presented in Supplementary Fig. 8. DisSOD1 staining was occasionally colocalized with the astrocyte marker, GFAP (e), but rarely with the microglial marker Iba1 (f). Images in panels c–f were acquired from 12-month mice. Antibody details are presented in Supplementary Table 4. g Three-dimensional reconstruction of SOD1 aggregates in the SNc of 12-month-old hSOD1^WT and SOCK mice (gray, TH-positive neuron soma; magenta, disSOD1 within TH-positive soma; yellow, extrasomal disSOD1). Scale bars represent 10 µm in panels c–g. h The volume of disSOD1, expressed as a % of the volume of tissue within which it was quantified, varied significantly between genotypes across all ages and was elevated in SOCK mice compared with hSOD1^WT mice across all ages examined. i Mean volume of disSOD1 increased with age in all four mouse strains (linear regression, significantly non-zero; WT (F_{(1, 9341)} = 9991, p < 0.0001), Ctr1^+/- (F_{(1, 2787)} = 3682, p < 0.0001), hSOD1^WT (F_{(1, 24065)} = 53,521, p < 0.0001), SOCK (F_{(1, 68826)} = 636,972, p < 0.0001), with a steeper increase observed in SOCK mice compared with hSOD1^WT mice (statistics displayed in the panel). j Total disSOD1 volume varied within and outside of SNc dopamine (DA) neuron soma with age in SOCK mice and was increased within DA neuron soma at 12 months-of-age compared with 1.5 months-of-age in these mice. Data in panels a, b, h–j represent mean ± SEM. Comparisons marked with an asterisk (*) denote comparisons made to wild-type mice (or 1.5-month-old SOCK mice in panel j), while those marked with a hashtag (#) demarcate those made to hSOD1^WT mice. # p < 0.05, ### p < 0.01, #### p < 0.0001, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Complete statistical details for panels a, b, h, j are presented in Supplementary Tables 12 and 13

Fig. 5

Dopamine neuron degeneration and perturbed dopamine metabolism occur in the absence of substantial α-synuclein aggregation in the SOCK mouse SNc. a Quantitative stereology revealed significant variation in the density of dopamine neurons in the SNc of 1.5-to-12-month-old wild-type (WT), Ctr1^+/, hSOD1^WT and SOCK mice, which was decreased in 6- and 12-month-old SOCK mice compared with WT mice. The density of nigral dopamine neurons was also significantly lower in SOCK mice compared with hSOD1^WT mice at 12 months-of-age but not 6 months-of-age. Data represent the density of TH neurons as a proportion of the average number of TH neurons quantified for age-matched wild-type mice, with the red dotted line representing 100% of this neuronal density. Raw neuronal densities are reported in Supplementary Fig. 12. b Nigral TH⁺ neuron density significantly decreased with age in all four mouse strains [linear regression, significantly non-zero; WT (F_(1,41) = 10.35, p = 0.0025), Ctr1^+/- (F_(1,41) = 32.61, p < 0.0001), hSOD1^WT (F_(1,22) = 8.86, p = 0.007) and SOCK (F_(1,36) = 34.45, p < 0.0001)], with a more rapid decrease observed in SOCK mice compared with hSOD1^WT mice (statistics displayed in the panel). Data represent the density of TH neurons as a proportion of the average number of TH neurons quantified at 1.5 months-of-age for that same mouse genotype. c. Decreases in dopamine neuron density were correlated with higher disSOD1 pathological burden in SOCK mice (statistics displayed in panel). Striatal dopamine levels (d) were unchanged between genotypes across all ages, as were striatal levels of homovanillic acid. Striatal dopamine turnover (f; calculated by normalizing the amount of HVA to dopamine levels) varied significantly and was increased in 6- and 12-month-old SOCK mice compared with WT mice and hSOD1^WT mice. g Immunofluorescent staining of fixed midbrain tissues from 12-month-old SOCK mice with antibodies recognizing pS129 α-synuclein and SOD1 in an unfolded beta barrel (UβB) conformation revealed a small population of aggregates containing both proteins in the SOCK mouse SNc (double white arrowheads) that was absent in the WT SNc. Scale bars represent 10 µm. Antibody details are presented in Supplementary Table 4. h The proportion of α-synuclein phosphorylated at Ser129 was significantly increased in the SOCK mouse midbrain compared with WT mice across all ages (two-way ANOVA: age—F_{(3, 46)} = 4.08, p = 0.012; genotype—F_{(1, 46)} = 37.11, p < 0.0001; Sidak’s multiple comparisons post hoc test: p values for 1.5–12 months = 0.0045, 0.028, 0.038, 0.0098, respectively). Comparisons marked with an asterisk (*) denote comparisons made to wild-type mice, while those marked with a hashtag (#) demarcate those made to hSOD1^WT mice. Data in a, d, e, f, h represent mean ± SEM. # p < 0.05, *p < 0.05, **p < 0.01. Complete statistical details for panels a, d, e, f are presented in Supplementary Tables 14 and 15

Fig. 6

Neither body weight, nor spinal cord motor neuron density, are altered in SOCK mice despite poorer motor performance on the rotarod apparatus. Assessment of motor performance was conducted for wild-type (WT), Ctr1^+/-, hSOD1^WT, and SOCK mice on an accelerating rotarod apparatus for a maximum of 180 s. a All mouse strains exhibited progressively poorer motor performance as they aged, with SOCK and hSOD1^WT mice exhibiting a greater decline in performance compared with WT and Ctr1^+/- mice. There was no difference in the rate of decline in motor performance between SOCK and hSOD1^WT mice (statistics displayed in panel), resulting in significant differences between these strains and wild-type mice across most ages examined. b SOCK mice also exhibited significantly poorer motor performance compared with hSOD1^WT mice at 1.5 and 12 months-of-age. c Body weight was measured as an index of general animal health throughout their lifespan. No significant changes in body weight were observed between genotypes at any experimental age, with all mouse strains gaining weight at a similar rate (d; statistical comparison of slopes presented in panel). e The number of spinal motor neurons co-expressing choline acetyltransferase (ChAT) and islet 1 (ISL-1) proteins did not differ between wild-type and SOCK mice at 6 months-of-age (unpaired t test: t = 1.003, df = 12, n = 7/group, p = 0.33). f Representative immunostaining of 6-month-old wild-type and SOCK spinal cord tissues for ChAT, ISL-1, and the unfolded β-barrel (UβB) conformation-specific disSOD1 antibody, counterstained with Hoechst. Scale bars represent 30 µm. Comparisons marked with an asterisk (*) denote comparisons made to wild-type mice, while those marked with a hashtag (#) demarcate those made to hSOD1^WT mice. Data in panels b, c, and e represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, # # # p < 0.001. Complete statistical details for panels b, c are presented in Supplementary Tables 16 and 17

Fig. 7

Proposed role of disSOD1 in nigral dopamine neuron death in Parkinson disease. An overall reduction in brain copper levels, as well as decreased expression of CTR1 in Parkinson disease SNc, together reducing brain copper bioavailability and copper binding to SOD1. This creates mismetallated disSOD1, which exhibits lower antioxidant activity per unit of protein and is prone to aggregation. Oxidative stress resulting from other etiological factors stimulates increased SOD1 expression and promotes oxidation and glycation of solvent-accessible residues within a now growing pool of disSOD1. Other pathologies may contribute to disSOD1 aggregation within the complex Parkinson disease (PD) degenerative cascade, which may themselves be exacerbated by brain copper deficiency. Combined, these changes may contribute to damage and death of dopamine neurons within the SNc. Changes indicated in bold text represent the hypothesized key driving mechanisms leading to disSOD1 formation