Reversal of motor impairments in parkinsonian rats by continuous intrastriatal delivery of L-dopa using rAAV-mediated gene transfer

Abstract

Intrastriatal delivery of the tyrosine hydroxylase gene by viral vectors is being explored as a tool for local delivery of L-dopa in animals with lesions of the nigrostriatal pathway. The functional effects reported using this approach have been disappointing, probably because the striatal L-dopa levels attained have been too low. In the present study, we have defined a critical threshold level of L-dopa, 1.5 pmol/mg of tissue, that has to be reached to induce any significant functional effects. Using new generation high-titer recombinant adeno-associated virus vectors, we show that levels of striatal L-dopa production exceeding this threshold can be obtained provided that tyrosine hydroxylase is coexpressed with the cofactor synthetic enzyme, GTP-cyclohydrolase-1. After striatal transduction with this combination of vectors, substantial functional improvement in both drug-induced and spontaneous behavior was observed in rats with either complete or partial 6-hydroxydopamine lesions of the nigrostriatal pathway. However, complete reversal of motor deficits occurred only in animals in which part of the striatal dopamine innervation was left intact. Spared nigrostriatal fibers thus may convert L-dopa to dopamine and store and release dopamine in a more physiologically relevant manner in the denervated striatum to mediate better striatal output-dependent motor function. We conclude that intrastriatal L-dopa delivery may be a viable strategy for treatment and control of adverse side effects associated with oral L-dopa therapy such as on-off fluctuations and drug-induced dyskinesias in patients with Parkinson's disease.

Figure 1

TH immunohistochemistry from striatum (A, C, and E) and SN (B, D, and F). In partial lesions (C) striatal TH⁺ innervation is reduced largely in the lateral striatum, leaving some intact fibers medially, whereas TH⁺ fibers are abolished in the complete lesions (E) as compared with the intact side (A). Partial lesions destroyed dopamine neurons in the pars compacta (D), whereas the VTA cells were mostly spared (compare B and D). All TH⁺ cells were lost both in the SN and VTA in the complete lesions (F). ac, anterior commissure; cc, corpus callosum; lv, lateral ventricle; SNpc, SN pars compacta; SNpr, SN pars reticulata; STR, striatum. (The scale bar shown in A = 1 mm for A–C and 400 μm for D–F.)

Figure 2

Striatal l-dopa levels after i.p. injection. (A) Two groups of partially lesioned animals received injections of benz or l-dopa. In the benz group, low levels of l-dopa were recovered on the lesion or the intact sides. Injection of l-dopa caused accumulation of 1.30 pmol/mg of l-dopa on the lesion side above that of benz alone (arrow). (B) When central decarboxylation is blocked for 30 min before killing, 1.2 pmol/mg of l-dopa was recovered on the lesion side (NSD-1015 alone group). l-Dopa injection increased striatal l-dopa to 2.75 pmol/mg. Thus, we estimated that accumulation of 1.51 pmol/mg of l-dopa was induced by the injection (arrow).

Figure 3

Striatal levels of l-dopa and dopamine in completely lesioned animals injected with rAAV vectors. (A) Striatal TH enzyme activity was estimated 30 min after NSD-1015 by measuring the accumulation of l-dopa. Increased rAAV-mediated striatal l-dopa in the five-site 1:1 mix group was above the threshold value obtained with peripheral injection (dashed line). In contrast, animals receiving the TH vector alone or the 1:1 mixed vectors in only two sites did not produce this threshold l-dopa level. (B) Dopamine (DA) levels were reduced greatly on the lesioned side in all groups. Striatal dopamine was increased only in the 5 × 3-μl mixed-vector group.

Figure 4

TH immunohistochemistry in the striatum at 3 weeks after injection of the 1:1 rAAV-TH/rAAV-GCH1 vector mix. In large parts of the striatum (A–D) and globus pallidus (gp, C and D), cell bodies and fibers were TH⁺. Nearly all infected cells had neuron-like morphology both in striatum (E) and globus pallidus (F). ac, anterior commissure; cc, corpus callosum; lv, lateral ventricle; str, striatum. (The scale bar shown in A = 1 mm for A–D; the scale bars in E and F = 40 μm.)

Figure 5

Behavioral effects of l-dopa delivery. (A) Experimental time course. The 1:1 vector mix group showed clear improvements in the cylinder (B) and amphetamine-rotation (C) tests. Reduction in the apomorphine rotation (compared with the fourth pretest; Pre value in D) was observed only in the partially lesioned animals. *, significantly different from the control group and their baseline values before vector injection.