Knockdown of α-syn in the non-human primate results in dose-dependent nigrostriatal degeneration: A case study
To begin to further characterize the consequences of knockdown of endogenous α-syn and determine whether effects seen in rats were reproducible in a species more closely related to humans, we generated an α-syn shRNA specific for St.Kitts green monkeys (Chlorocebus sabaeus). We injected α-syn shRNA or scrambled shRNA, of two different titers, into the SN of individual monkeys (N = 4 total), waited 3 months, and examined DA neuron numbers, morphology, striatal innervation, and striatal DA content. α-Syn shRNA produced region-specific, titer-related, degeneration of SN tyrosine hydroxylase-positive (TH+) neurons and innervation of the striatum, reproducing the pattern of nigrostriatal degeneration observed in PD: SN degeneration was exaggerated in ventral tier neurons (vtSN; Gibb and Lees, 1991) with relative sparing of adjacent ventral tegmental area (VTA) DA neurons, and loss of TH+ fibers in the putamen (Pt) exceeded denervation of the caudate nucleus (Cd) (Kish et al., 1988; Figure 1). Stereologic quantification of TH+ neurons confirmed the qualitative observations: loss of TH+ neurons was greatest in vtSN with the VTA exhibiting relatively little neuron loss. However, the general pattern of TH+ neuron loss was not different in the High and Low titer α-syn shRNA conditions. These neurons also contain neuromelanin, and when counts of neuromelanin-only positive cells were added to the analysis, titer-related differences emerged. The Low titer shRNA condition was associated with the preservation of significantly more neuromelanin-only neurons, suggesting that while loss of TH+ phenotype was equivalent across titers, overt loss of neurons was greater in the High titer condition. The presence of TH-negative, neuromelanin-positive neurons is suggestive of ongoing pathology, an observation that also is seen in early PD. Importantly, co-localization of green fluorescent protein (GFP) as a marker of viral transduction within surviving midbrain DA neurons confirmed that dorsal SN and VTA neurons were transduced, but showed less degeneration (Figure 1). Titer-related differences in striatal DA depletion also were detected. Deficits in DA in the caudate nucleus and putamen were exaggerated in the α-syn shRNA High titer condition and a significant increase in the homovanillic acid (HVA)/DA ratio was only detected in the High titer subject. Increase in the HVA/DA ratio is known to be associated with a compensatory response to ongoing significant degeneration of DA neurons (Zigmond et al., 1990).
Figure 1  Knock-down of endogenous α-syn in midbrain dopamine neurons reproduces a PD-like pattern of nigrostriatal degeneration. rAAV- α-syn-shRNA treatment produces exaggerated degeneration of ventral tier substantia nigra (vtSN) dopamine neurons and denervation of the putamen (Pt) that exceeds that in the caudate (Cd), reproducing the pattern of degeneration observed in Parkinson's disease [A,B,E,G,H, transduced hemisphere on right side of micrographs, staining for tyrosine hydroxylase (TH)]. This pattern is not observed with rAAV-scr-shRNA treatment (C,D,F,I,J, arrows in (J) indicate injection site). Dorsal tier substantia nigra (dtSN) neurons surviving α-syn-shRNA treatment (white asterisk in H) were transduced by the vector, but resistant to degeneration (K, brown stain for GFP vector tag). While rAAV- α-syn-shRNA of both High and Low titers produced equivalent loss of TH+ vtSN neurons, greater numbers of neuromelanin-positive neurons surviving in this region following Low titer treatment (M) indicates phenotype suppression with cell survival, and the low number of these cells in the High titer treatment (L) suggests greater overt cell loss [black asterisk in (H) indicates vtSN region shown in (L) and equivalent region in (M) with greater (Low shRNA titer M) and lesser (High titer in L) numbers of surviving neuromelanin neurons (brown pigment)]. Abbreviations: Cd, caudate nucleus; Pt, putamen; ic, internal capsule; dtSN, dorsal tier substantia nigra; vtSN, ventral tier substantia nigra; Calibration bar in (A) = 100 μm and applies to (A–F); bar in (G) = 500 μm and applies to (G–J); bar in (M) = 100 μm and applies to (K–L).