Lack of additive role of ageing in nigrostriatal neurodegeneration triggered by α-synuclein overexpression

Abstract

Introduction: Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons as well as the presence of proteinaceous inclusions named Lewy bodies. α-synuclein (α-syn) is a major constituent of Lewy bodies, and the first disease-causing protein characterized in PD. Several α-syn-based animal models of PD have been developed to investigate the pathophysiology of PD, but none of them recapitulate the full picture of the disease. Ageing is the most compelling and major risk factor for developing PD but its impact on α-syn toxicity remains however unexplored. In this study, we developed and exploited a recombinant adeno-associated viral (AAV) vector of serotype 9 overexpressing mutated α-syn to elucidate the influence of ageing on the dynamics of PD-related neurodegeneration associated with α-syn pathology in different mammalian species.

Results: Identical AAV pseudotype 2/9 vectors carrying the DNA for human mutant p.A53T α-syn were injected into the substantia nigra to induce neurodegeneration and synucleinopathy in mice, rats and monkeys. Rats were used first to validate the ability of this serotype to replicate α-syn pathology and second to investigate the relationship between the kinetics of α-syn-induced nigrostriatal degeneration and the progressive onset of motor dysfunctions, strikingly reminiscent of the impairments observed in PD patients. In mice, AAV2/9-hα-syn injection into the substantia nigra was associated with accumulation of α-syn and phosphorylated hα-syn, regardless of mouse strain. However, phenotypic mutants with either accelerated senescence or resistance to senescence did not display differential susceptibility to hα-syn overexpression. Of note, p-α-syn levels correlated with nigrostriatal degeneration in mice. In monkeys, hα-syn-induced degeneration of the nigrostriatal pathway was not affected by the age of the animals. Unlike mice, monkeys did not exhibit correlations between levels of phosphorylated α-syn and neurodegeneration.

Conclusions: In conclusion, AAV2/9-mediated hα-syn induces robust nigrostriatal neurodegeneration in mice, rats and monkeys, allowing translational comparisons among species. Ageing, however, neither exacerbated nigrostriatal neurodegeneration nor α-syn pathology per se. Our unprecedented multi-species investigation thus favours the multiple-hit hypothesis for PD wherein ageing would merely be an aggravating, additive, factor superimposed upon an independent disease process.

Fig. 1

rAAV2/9 vector-mediated overexpression of hα-syn in rat SNpc induces progressive dopaminergic neurodegeneration related to hα-syn expression dynamics. (a-e) Representative photomicrographs of dopaminergic markers and human α-syn immunostaining at striatal (a-c) and SNpc (d,e) levels at several time points after injection. (a,d): tyrosine hydroxylase (TH); (b): dopamine transporter (DAT); (c,e): human α-syn. (f) Quantification of the different markers over time. Briefly, stereological quantification of the number of TH-positive cells in the SNpc of rat are reported on the left axis, all staining intensity have been normalized between min (0 %) and max (100 %) and reported on the right axis to allow comparison of the evolution. Dot-lines represent actual values while plain curves are regression curves. Colors are the same as in the upper part of the panel: light blue: striatal TH, purple: striatal DAT, yellow: striatal α-syn, blue: SNpc TH, red: SNpc α-syn. Scales applies to all pictures: 1 mm

Fig. 2

Behavioral impairments associated with hα-syn overexpression in rats. a Spontaneous locomotor activity at baseline (Bsl) and at 4, 8, 12 and 16 weeks after surgery. b Stepping test at baseline and at 4,8,12 and 16 weeks after surgery. c Color-coded stick diagram decomposition of hindlimb movement for a representative healthy and AAV-hα-syn rat during crossing of an elevated horizontal ladder with irregularly spaced rungs (spacing 5.2 +/− 0.3 cm). The corresponding endpoint trajectory is shown below. The colour code indicated the instantaneous velocity of the endpoint, while the arrows report the orientation and intensity of the foot push-off at swing onset. Light grey: stance; dark grey: hit; yellow: slip; dark red: miss. d Pie charts summarize total percentage of hits, slips, and misses (total of 182 steps evaluated, n are indicated in the legend boxes). Below percentage of hits, slips and misses are quantified as histogram plots. e The same representation is shown for a representative healthy and Parkinsonian rat during overground locomotion. f Principal component (PC) analysis was applied on 89 gait parameters measured during overground locomotion (10–15 gait cycles/hindlimb/rat, n = 4 healthy and n = 6 AAV-hα-syn rats). Each gait cycle is represented as a dots in the new space created by PC1–3. Histogram plot depicts mean values of PC1 scores for each experimental group. g Histogram plots represent mean values of parameters with high factor loading (|factor loading| > 0.5) on PC1, illustrating the most salient differences in motor control between the two groups. Numbers refer to Additional file 1: Table S1. *p < 0.05; **p < 0.01; ***p < 0.001; $ p < 0.05 vs 4 weeks time point; error bars, SEM; PC, Principal Component; a.u., arbitrary unit

Fig. 3

Human α-syn overexpression induces dopaminergic neurodegeneration associated with α-syn pathology in mice. a Stereological cell counts of SNpc TH-positive neurons (left two panels, n = 4 animals for each genotype) and mean grey values of striatal TH immunoreactivity (right two panels) at 20 weeks after surgery compared to non-injected side (white bars). Top panels display representative TH immunostaining at SNpc (left) and striatum (right) levels in the three mouse strains. b Volume quantification of the hα-syn immunostaining at the SNpc level (left two panels) and surface quantification of the hα-syn immunostaining at the striatal level (right two panels) at 20 weeks after surgery. Top panels display representative hα-syn immunostaining at SNpc (left) and striatum (right) levels in the mouse strains used. c Volume quantification of the p-α-syn immunostaining at the SNpc level at 20 weeks after surgery. Left panels display representative p-α-syn immunostaining at SNpc at macroscopic (left) and microscopic (right) levels in all mouse strains. d Covariance analysis of each parameter measured in all mouse strains. Data regarding TH pathway are expressed as a percentage of loss of TH fibers or cell bodies. Syn data are expressed as a percentage of immunopositive surface of the structure of interest. STR: striatum; * p < 0.05 vs non injected side of C57Bl/6 J mice; # p < 0.05 vs non injected side of the AKR/J background mice concerned. Scales: 1.5 mm for STR, 0.5 mm for SNpc and 20 μm for high magnification pictures of the C panel. hα-syn: human α-syn; p-α-syn: S129 phosphorylated α-syn

Fig. 4

Human α-syn overexpression induces dopaminergic neurodegeneration associated with α-syn pathology in young and old marmoset monkeys. a Stereological cell counts of SNpc TH-positive neurons (left panel, n = 8 for young animals and n = 5 for old animals) and mean grey values of striatal TH-positive fibers (right panel) at 11 weeks after surgery. Colors: blue: injected side of young animals; red: injected side of old animals; white and light grey: non-injected side of young and old animals, respectively. Top panels display representative TH immunostaining at SNpc (left) and striatum (right) levels. b Volume quantification of the hα-syn immunostaining at the SNpc level (left panel) and surface quantification of the hα-syn immunostaining at the striatal level (right panel) at 11 weeks after surgery. Top panels display representative hα-syn immunostaining at SNpc (left) and striatum (right) levels. c Volume quantification of the p-α-syn immunostaining at the SNpc level at 11 weeks after surgery. Left panels display representative human p-α-syn immunostaining at SNpc at macroscopic (left) and microscopic (right) levels. d Covariance analysis of each parameter measured in young and old marmosets. Data regarding TH pathway represent a loss of TH fibers or cell bodies. STR: striatum; * p < 0.05 vs non injected side of young animals; # p < 0.05 vs non injected side of old animals; & p < 0.05 vs injected side of young animals. Scales: 1.5 mm for STR, 0.5 mm for SNpc, 250 μm and 20 μm for high magnification pictures of the (c) panel. hα-syn: human α-syn; p-α-syn: S129 phosphorylated α-syn