Levodopa-induced dyskinesia is associated with increased thyrotropin releasing hormone in the dorsal striatum of hemi-parkinsonian rats

Abstract

Background: Dyskinesias associated with involuntary movements and painful muscle contractions are a common and severe complication of standard levodopa (L-DOPA, L-3,4-dihydroxyphenylalanine) therapy for Parkinson's disease. Pathologic neuroplasticity leading to hyper-responsive dopamine receptor signaling in the sensorimotor striatum is thought to underlie this currently untreatable condition.

Methodology/principal findings: Quantitative real-time polymerase chain reaction (PCR) was employed to evaluate the molecular changes associated with L-DOPA-induced dyskinesias in Parkinson's disease. With this technique, we determined that thyrotropin releasing hormone (TRH) was greatly increased in the dopamine-depleted striatum of hemi-parkinsonian rats that developed abnormal movements in response to L-DOPA therapy, relative to the levels measured in the contralateral non-dopamine-depleted striatum, and in the striatum of non-dyskinetic control rats. ProTRH immunostaining suggested that TRH peptide levels were almost absent in the dopamine-depleted striatum of control rats that did not develop dyskinesias, but in the dyskinetic rats, proTRH immunostaining was dramatically up-regulated in the striatum, particularly in the sensorimotor striatum. This up-regulation of TRH peptide affected striatal medium spiny neurons of both the direct and indirect pathways, as well as neurons in striosomes.

Conclusions/significance: TRH is not known to be a key striatal neuromodulator, but intrastriatal injection of TRH in experimental animals can induce abnormal movements, apparently through increasing dopamine release. Our finding of a dramatic and selective up-regulation of TRH expression in the sensorimotor striatum of dyskinetic rat models suggests a TRH-mediated regulatory mechanism that may underlie the pathologic neuroplasticity driving dopamine hyper-responsivity in Parkinson's disease.

Figure 1. TRH increases with dyskinetic behavior.

(A) Behavioral scores assessed 20 min after L-DOPA administration. Red line: 6-OHDA/L-DOPA. The animals belonging to this group are the only ones developing dyskinesias. Black line: superimposed, identical values for 6-OHDA/saline; No lesion/L-DOPA; no lesion/saline. The animals in the three control groups never developed dyskinesias. (6-OHDA/L-DOPA vs. all other groups p<0.0001). (B) PreproTRH mRNA levels measured by qPCR. The mRNA levels for preproTRH in the right striatum (6-OHDA lesion side) of the animals chronically treated with L-DOPA were significantly increased (Bonferroni T **p<0.001) with respect to the striatum contralateral to the lesion of 6-OHDA/L-DOPA rats with LIDs and the non-dyskinetic control groups. (C) PreproTRH mRNA levels are associated with the presence of dyskinesias. PreproTRH mRNA levels in the striatum ipsilateral to the 6-OHDA lesion showed a positive correlation with the behavioral scores (Pearson's r = 0.725, p = 0.0002), but the relationship between the behavioral scores and the mRNA levels did not follow a linear relationship. No correlation was found between the mRNA levels for preproTRH and the behavioral scores of the control animals (Pearson's r = 0.313, p = 0.17). Shaded areas flanking the correlation curve represent the 95% confidence intervals. (D) Radioimmunoassay for TRH, pYE27, and pYE17. The TRH tripeptide is derived from the processing of the larger precursor proTRH, as are the peptides pYE27, and pYE17 (TRH structure shown in panel to right). Results of the RIA show that all three peptides derived from the larger preproTRH are greatly increased in the striatum ipsilateral to the lesion in the rats with LIDs, but not in the striatum contralateral to the lesion of the rats with LIDs, or in either striata of control treatment rats (Bonferroni T p<0.001).

Figure 2. Up-regulation of TRH is specific for the dopamine-depleted striatum of rats with L-DOPA-induced dyskinesias.

(A) Saline: proTRH immunoreactivity is very low absent in the dopamine-depleted striatum (right) of a control, saline-treated rat. Sections run from rostral (top) to caudal (bottom). L-DOPA: proTRH immunoreactivity is strong and variable in the dopamine-depleted striata of three exemplary L-DOPA-treated dyskinetic rats. Tyrosine hydroxylase (TH) immunostaining indicates loss of dopamine-containing cell bodies in the substantia nigra pars compacta (upper section) and uniform loss of dopamine-containing terminals in the striatum (lower section), ipsilateral to the lesion, of all four rats. (B) ProTRH immunostaining in fibers of the external globus pallidus (top panel), entopeduncular nucleus (middle panel) and substantia nigra pars reticularis (bottom panel) of a dopamine-depleted rat with LID. (C) ProTRH immunoreactivity in a putative medium spiny neuron in a striatal section from a rat with LID.

Figure 3. TRH shows a complex pattern of up-regulation that includes preferential expression in caudal striosomes.

(A) Non-uniform proTRH immunostaining in the dopamine-depleted striatum of three individual rats as compared to relatively uniform up-regulation of CalDAG-GEFII and down-regulation of MOR1 in the same rats. (B) The subcallosal streak (arrow) and putative striosome (asterisk) immunostained for proTRH (top panel) and the striosome marker CalDAG-GEFII (bottom panel) in neighboring striatal sections from a rat with LID.

Figure 4. TRH expression in pseudounipolar neurons of rats with LID-inducing treatments and control treatments.

(A) ProTRH⁺ periventricular neurons in the dorsal striatum contralateral to the 6-OHDA lesion in an animal that developed LIDs (magnification 10×). Full arrows show clusters of pseudounipolar neurons. (B) ProTRH ⁺ periventricular neurons and putative medium spiny neurons in the dorsal striatum ipsilateral to the 6-OHDA lesion in an animal that developed LIDs. Full arrows show clusters of pseudounipolar neurons; tail-less arrows show examples of putative medium spiny neurons. (C) ProTRH⁺ periventricular neurons in the dorsal striatum contralateral to the 6-OHDA lesion in an animal treated with saline (inset: higher magnification of the pseudounipolar neurons immunopositive for proTRH). Full arrows show clusters of pseudounipolar neurons. (D) ProTRH⁺ periventricular neurons in the dorsal striatum ipsilateral to the 6-OHDA lesion in an animal treated with saline. Full arrows show clusters of pseudounipolar neurons; Abbreviations: striatum: CP; lateral ventricle: V.