Altered nucleolar morphology in substantia nigra dopamine neurons following 6-hydroxydopamine lesion in rats

Abstract

The nucleolus, the site of ribosomal ribonucleic acid (rRNA) transcription and assembly, is an important player in the cellular response to stress. Altered nucleolar function and morphology, including decreased nucleolar volume, has been observed in Parkinson’s disease; thus the nucleolus represents a potential indicator of neurodegeneration in the disease. This study determined the effects of a partial unilateral intrastriatal 6-hydroxydopamine (6-OHDA) lesion, which models the dopaminergic loss found in Parkinson’s disease, on the nucleoli of dopaminergic cells in the substantia nigra pars compacta (SNpc). Adult male Long-Evans rats underwent unilateral intrastriatal infusion of 6-OHDA (12.5 μg). Lesions were verified by amphetamine-stimulated rotation 7 days later, and rats were euthanized 14 days after infusion. Coronal sections (50μm) were stained for tyrosine hydroxylase-silver nucleolar (TH-AgNOR) stain using MultiBrain Technology (NeuroScience Associates), which resulted in clearly defined nucleoli and neuronal outlines. Stereological methods were used to compare dopaminergic morphology between lesioned and intact hemispheres in each rat. In cells exhibiting a definable nucleolus, nucleolar volume was decreased by 16% on the ipsilateral side. The ipsilateral SNpc also exhibited an 18% decrease in SNpc planimetric volume, a 46% decrease in total TH-positive neuron number, and an 11% decrease in neuronal body volume (all P<0.05 by paired t-test). These findings suggest that the 6-OHDA lesion alters nucleolar morphology and that these changes are similar to those occurring in Parkinson’s disease.

Differential effects of intrastriatal 6-hydroxydopamine on cell number and morphology in midbrain dopaminergic subregions of the rat

“…This procedure stains the nucleolus of tyrosine-hydroxylase-positive neurons, thus facilitating both stereological cell counting and assessment of nucleolar morphology (Healy-Stoffel et al, 2013). The antibody was rabbit polyclonal anti-tyrosine hydroxylase (Pel-Freez Biologicals, Rogers, AR; P40101-0, Lot# 06432; working dilution 1:1000), which was generated using SDS-denatured tyrosine hydroxylase purified from rat pheochromocytoma.…”

“…The double nucleator was then employed by automatic software placement of four randomly oriented crossed rays centered at the counting point, and four discriminately placed markers on the outside borders of each of the nucleolus and the outline of the cell body, respectively as previously described in detail (Healy-Stoffel et al, 2013). In some neurons, the nucleolus was visible within the nucleus, but dark staining of the nucleus made it impossible to accurately distinguish the borders of the nucleolus for placement of the nucleator probe (SNpc: 9.8 ± 1.1% on the ipsilateral side and 9.9 ± 0.9% on the contralateral side; VTA: 7.1 ± 1.5% on the ipsilateral side and 10.2 ± 1.4% on the contralateral side; RRF: 9.6 ± 2.1% on the ipsilateral side and 11.2 ± 1.2% on the contralateral side; all not significant by paired t-test).…”

“…The partial unilateral lesion results in a moderate loss of midbrain dopamine neurons compared to more extensive lesions (Kirik et al, 1998), and is thus relevant to early-stage PD. Using this model, we have demonstrated decreased SNpc neuronal number and neuronal volume (Healy-Stoffel et al, 2012), as well as decreased volume of the nucleolus in SNpc dopaminergic cells (Healy-Stoffel et al, 2013). The nucleolus, the site of rRNA synthesis, is of particular interest as it is involved in directing the cellular response to stress (Boulon et al, 2010), and is thus a likely participant in the neurodegenerative processes in PD.…”

The nucleolar protein nucleophosmin is essential for autophagy induced by inhibiting Pol I transcription

“…Furthermore, nucleolar disruption and autophagy are enhanced in mouse medium spiny neurons by conditional knockout of the RNA Pol I-specific transcription initiation factor-IA (TIF-IA) 22 . A decrease in rRNA synthesis and nucleolar disruption have been reportedly observed in animal models for a variety of neurodegenerative diseases, including Huntington's disease and Parkinson's disease 22 23 24 25 , against which autophagy has protective roles 3 26 27 . In contrast, increased rRNA synthesis and an enlarged nucleolus are observed in tumour cells 28 29 30 with high levels of autophagy 31 32 33 .…”

The role of the nucleolus in neurodegeneration.

“…Decreased nucleolar volume has been documented in human PD tissue and two pharmacological PD models, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA) [88,156,214]. In addition, increased frequency of dopaminergic neurons with dispersion of nucleolar nucleophosmin, (NPM1) was observed in substantia nigra from PD brains as compared to controls [214].…”