The transfection of BDNF to dopamine neurons potentiates the effect of dopamine D3 receptor agonist recovering the striatal innervation, dendritic spines and motor behavior in an aged rat model of Parkinson's disease.

The progressive degeneration of the dopamine neurons of the pars compacta of substantia nigra and the consequent loss of the dopamine innervation of the striatum leads to the impairment of motor behavior in Parkinson's disease. Accordingly, an efficient therapy of the disease should protect and regenerate the dopamine neurons of the substantia nigra and the dopamine innervation of the striatum. Nigral neurons express Brain Derived Neurotropic Factor (BDNF) and dopamine D3 receptors, both of which protect the dopamine neurons. The chronic activation of dopamine D3 receptors by their agonists, in addition, restores, in part, the dopamine innervation of the striatum. Here we explored whether the over-expression of BDNF by dopamine neurons potentiates the effect of the activation of D3 receptors restoring nigrostriatal innervation. Twelve-month old Wistar rats were unilaterally injected with 6-hydroxydopamine into the striatum. Five months later, rats were treated with the D3 agonist 7-hydroxy-N,N-di-n-propy1-2-aminotetralin (7-OH-DPAT) administered i.p. during 4½ months via osmotic pumps and the BDNF gene transfection into nigral cells using the neurotensin-polyplex nanovector (a non-viral transfection) that selectively transfect the dopamine neurons via the high-affinity neurotensin receptor expressed by these neurons. Two months after the withdrawal of 7-OH-DPAT when rats were aged (24 months old), immunohistochemistry assays were made. The over-expression of BDNF in rats receiving the D3 agonist normalized gait and motor coordination; in addition, it eliminated the muscle rigidity produced by the loss of dopamine. The recovery of motor behavior was associated with the recovery of the nigral neurons, the dopamine innervation of the striatum and of the number of dendritic spines of the striatal neurons. Thus, the over-expression of BDNF in dopamine neurons associated with the chronic activation of the D3 receptors appears to be a promising strategy for restoring dopamine neurons in Parkinson's disease

Transfection of the BDNF Gene in the Surviving Dopamine Neurons in Conjunction with Continuous Administration of Pramipexole Restores Normal Motor Behavior in a Bilateral Rat Model of Parkinson’s Disease

In Parkinson’s disease (PD), progressive degeneration of nigrostriatal innervation leads to atrophy and loss of dendritic spines of striatal medium spiny neurons (MSNs). The loss disrupts corticostriatal transmission, impairs motor behavior, and produces nonmotor symptoms. Nigral neurons express brain-derived neurotropic factor (BDNF) and dopamine D3 receptors, both protecting the dopamine neurons and the spines of MSNs. To restore motor and nonmotor symptoms to normality, we assessed a combined therapy in a bilateral rat Parkinson’s model, with only 30% of surviving neurons. The preferential D3 agonist pramipexole (PPX) was infused for four ½ months via mini-osmotic pumps and one month after PPX initiation; the BDNF-gene was transfected into the surviving nigral cells using the nonviral transfection NTS-polyplex vector. Overexpression of the BDNF-gene associated with continuous PPX infusion restored motor coordination, balance, normal gait, and working memory. Recovery was also related to the restoration of the average number of dendritic spines of the striatal projection neurons and the number of TH-positive neurons of the substantia nigra and ventral tegmental area. These positive results could pave the way for further clinical research into this promising therapy

BDNF gene transfection by NTS-polyplex to dopamine neurons of substantia nigra pars compacta.

<p>(A) Panoramic view of the labeled TH+ neurons. (B) Amplification of the zone indicated by the frame in A. The arrowheads show the co-localization of the BDNF-flag in the TH+ neurons. This is a representative illustration of the BDNF-flag expression.</p

The combined treatment did not recover the ambulatory activity and rearing in the open field.

<p>The lesion reduced the ambulatory activity evaluated by the distance traveled (A) and frequency of rearing (B) during the first ten min in the open field. However, it did not produce bradykinesia assessed by the speed of walking (C). None of the treatments recovered the ambulatory activity or rearing (A and B). Intact rats did not showed changes either in ambulation or in rearing, throughout the experiment. The 6-OHDA lesion reduced significantly the traveled distance and frequency of rearing (A: F_{3, 6} = 73.75, <i>P</i> < 0.0001 and B: F_{3, 6} = 57.30, <i>P</i> < 0.0001, One-way repeated-measures ANOVA) but it did not produce a significant effect on bradykinesia (C: F_{3, 6} = 3.563, <i>P</i> < 0.0868, One-way repeated-measures ANOVA). *** <i>P</i> < 0.001 compared with saline-treated rats. ^# <i>P</i> = No significant difference compared with saline-treated rats (Bonferroni’s Multiple Comparison Post tests). The data are given as the mean ± SEM (<i>n</i> = 5–6 rats per group).</p

Illustration of the experimental design.

<p>Arrows with asterisk indicate an evaluation of the motor behavior, which included gait analysis, rotarod performance and ambulatory activity in the open field. Double asterisks indicate EMG recordings. Rats were 12 months old at the beginning of the experiment.</p

The combined treatment recovered normal muscle tone.

<p>The lesion increased the EMG activity of the contralateral gastrocnemius but did not affect the EMG of the ipsilateral one (A). The combined treatment normalized the EMG activity (A and B) and the effect persisted two months after the treatment (B). The D3 agonist also reduced the EMG, but the reduction was partial and occurred two months after the treatment (B). The recovery produced by the combined treatment was significant (F_{2, 6} = 5.674, <i>P</i> < 0.0414, One-way repeated-measures ANOVA). * <i>P</i> < 0.05; ** <i>P</i> < 0.01 compared with saline-treated rats (Bonferroni’s Multiple Comparison Post tests). The data are given as the mean ± SEM (<i>n</i> = 5 rats per group). A, shows a representative case.</p

Administration of the D3 agonist associated with the BDNF transfection restored motor coordination.

<p>The lesion reduced the time on the rod (A-I) and the rotarod performance (B). The combined treatment normalized the time on the rod (A-II) and the rotarod performance (B). The effect persisted two months after the treatment (A-III and B). The D3 agonist alone also recovered the motor coordination (A, B) but the rats dragged the contralateral hind limb during rod rotation (arrows in C and supplementary videos <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117391#pone.0117391.s003" target="_blank">S3 Video</a>—ROTAROD PERFORMANCE IN NORMAL SPEED and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117391#pone.0117391.s004" target="_blank">S4 Video</a>- ROTAROD PERFORMANCE IN SLOW MOTION). Saline-treated rats did not recover the rotarod performance (A, B). The recovery produced by the treatments were significant (AI: F_{3, 12} = 9.881, <i>P</i> < 0.0015; AII: F_{3, 12} = 8.879, <i>P</i> < 0.0023; AIII: F_{3, 12} = 12.49, <i>P</i> < 0.0005 and B: F_{3, 15} = 21.84, <i>P</i> < 0.0001, One-way repeated-measures ANOVA). * <i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> < 0.001 compared with saline-treated rats. ^# <i>P</i> = No significant difference compared with intact rats (Bonferroni’s Multiple Comparison Post tests). The data are given as the mean ± SEM (<i>n</i> = 5–6 rats per group). C illustrates representative cases.</p

The combined treatment recovered the nigro-striatal innervation.

<p>(A) Representative micrographs of the substantia nigra pars compacta showing TH+ neurons with the presence of rhodamine-labeled dextran amine (arrowheads), previously injected into the striatum. (B) Graph showing the percentage of rhodamine-labeled TH+ neurons with respect to the total of TH+ neurons of the same substantia nigra pars compacta, in each experimental condition. The recovery produced by the treatments was significant (F_{2, 8} = 18.34, <i>P</i> < 0.0010, One-way ANOVA). * <i>P</i> < 0.05; ** <i>P</i> < 0.01 compared with saline-treated rats (Bonferroni’s Multiple Comparison Post tests). The data are given as the mean ± SEM (<i>n</i> = 4 rats per group).</p

The combined treatment restored the TH immunoreactivity of the striatum.

<p>(A) Representative micrographs of TH immunoreactivity showing that the lesion extends along the rostro-caudal extention of the striatum. (B). Representative micrographs showing the effect of the treatments on TH immunoreactivity of the striatum. The black arrows show lesion produced by 6-OHDA. (C) Optical densitometry analysis of TH-immunoreactivity in dorso-medial (DMS), dorso-lateral (DLS) and ventral (VS) regions of the striatum. The recovery produced by the treatments was significant (DMS: F_{2, 9} = 9.694, <i>P</i> < 0.0057; DLS: F_{2, 9} = 40.46, <i>P</i> < 0.0001 and VS; F_{2, 9} = 19.93, <i>P</i> < 0.0005, One-way ANOVA) * <i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> < 0.001 compared with saline-treated rats; ^& <i>P</i> < 0.01 compared with the 7-OH-DPAT alone (Bonferroni’s Multiple Comparison Post tests). The data are given as the mean ± SEM (<i>n</i> = 5 rats per group). The insert shows the three different sectors where the OD was determined.</p

The combined treatment restored gait.

<p>The lesion increased the angle of the contralateral ankle during the dorsiflexion of the swing phase of gait (arrows) and the undulatory displacement of the hip (limping), red line in A. The combined treatment normalized both conditions (A, B) and supplementary videos (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117391#pone.0117391.s001" target="_blank">S1 Video</a>—GAIT ANALYSIS IN NORMAL SPEED and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117391#pone.0117391.s002" target="_blank">S2 Video</a>—GAIT ANALYSIS IN SLOW MOTION). The D3 agonist alone did not restore neither the hip displacement (not shown) nor the angle of the ankle during gait. The increase in the angle produced by the lesion persisted all along the experiment (saline-treated rats) in B. The recovery produced by the combined treatment was significant (F_{3, 15} = 26.09, <i>P</i> < 0.0001, One-way repeated-measures ANOVA). *<i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> < 0.001compared with saline-treated rats; ^# <i>P</i> = No significant difference compared with intact rats (Bonferroni’s Multiple Comparison Post tests). A, illustrate a representative case. The data are given as the mean ± SEM (<i>n</i> = 5–6 rats per group).</p