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. 2013 Feb 12;231:282-95.
doi: 10.1016/j.neuroscience.2012.11.049. Epub 2012 Dec 5.

Neural Expression of the Transcription Factor THAP1 During Development in Rat

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Free PMC article

Neural Expression of the Transcription Factor THAP1 During Development in Rat

Y Zhao et al. Neuroscience. .
Free PMC article

Abstract

Loss of function mutations in THAP1 has been associated with primary generalized and focal dystonia in children and adults. THAP1 encodes a transcription factor (THAP1) that harbors an atypical zinc finger domain and plays a critical role in G(1)-S cell cycle control. Current thinking suggests that dystonia may be a neurodevelopmental circuit disorder. Hence, THAP1 may participate in the development of the nervous system. Herein, we report the neurodevelopmental expression patterns of Thap1 transcript and THAP1 protein from the early postnatal period through adulthood in the rat brain, spinal cord and dorsal root ganglia (DRG). We detected Thap1 transcript and THAP1-immunoreactivity (IR) in the cerebral cortex, cerebellum, striatum, substantia nigra, thalamus, spinal cord and DRG. Thap1 transcript expression was higher in the brain than in spinal cord and DRG at P1 and P7 and declined to similar levels at P14 and later time points in all regions except the cerebellum, where it remained high through adulthood. In the brain, THAP1 expression was highest in early development, particularly in the cerebellum at P7. In addition to Purkinje cells in the cerebellum, THAP1-IR was also localized to pyramidal neurons in the cerebral cortex, relay neurons in the thalamus, medium spiny and cholinergic neurons in the striatum, dopaminergic neurons in the substantia nigra, and pyramidal and interneurons in the hippocampus. In the cerebellar cortex, THAP1-IR was prominently distributed in the perikarya and proximal dendrites of Purkinje cells at early time-points. In contrast, it was more diffusely distributed throughout the dendritic arbor of adult Purkinje cells producing a moderate diffuse staining pattern in the molecular layer. At all time points, nuclear IR was weaker than cytoplasmic IR. The prominent cytoplasmic and developmentally regulated expression of THAP1 suggests that THAP1 may function as part of a cell surface-nucleus signaling cascade involved in terminal neural differentiation.

Figures

Fig. 1
Fig. 1
Relative quantitative real-time RT-PCR for Thap1. (A) Relative expression of Thap1 isoform 1 normalized to P21 cerebral cortex. (B) Relative percent contribution of Thap1 isoform 2 to total Thap1 transcripts.
Fig. 2
Fig. 2
In situ hybridization for Thap1 at E15, P1, P7, P14, P21, P28, 2M, and 6M developmental time points. Roughly equivalent autoradiographic images of parasagittal sections hybridized with radiolabeled (35S-UTP) anti-sense cRNA probes are shown along with a parasagittal P14 brain section hybridized with a Thap1 sense probe. Scale bar, 5 mm.
Fig. 3
Fig. 3
Western blot analysis with ProteinTech affinity-purified rabbit anti-THAP1 polyclonal antibody. (A) Signal specificity was evaluated in HEK293 protein extracts without and with antibody pre-absorption. THAP1 was also detected with mouse anti-V5 and Novus affinity-purified polyclonal rabbit anti-THAP1 antibodies. β-tubulin served as the endogenous control. (B) A single band was seen with protein extracts from striatum, thalamus, hippocampus, cerebellum and cerebral cortex. β-tubulin was the endogenous control. (C) Immunohistochemical staining patterns in P14 and 2M rat cerebellum were similar with ProteinTech and Novus anti-THAP1 antibodies. Scale bar, 100 μm. (D) Relative THAP1 expression in comparison to 2M cerebral cortex with β-tubulin as the endogenous control.
Fig. 4
Fig. 4
Immunofluorescence localization of THAP1 in hTHAP1 transfected HEK293 cells. THAP1 was detected with affinity-purified polyclonal rabbit anti-THAP1 antibody. Nuclei were counterstained with DAPI, and cells were visualized by differential interference contrast (DIC) microscopy. MERGE is an overlay of the THAP1, DAPI, and DIC images. Scale bar, 10 μm.
Fig. 5
Fig. 5
Nickel-intensified DAB THAP1-IR in rat brain at multiple developmental time points. m, molecular layer; p, Purkinje cell layer; g, granular cell layer. Scale bar, 200 μm.
Fig. 6
Fig. 6
THAP1-IR in hippocampus. THAP1-IR was striking in hippocampal pyramidal and interneuon cell bodies and proximal dendrites, and weak in granule cell layer in dentate gyrus in early development. THAP1-IR was decreased with increase of age especially in pyramidal cells. In adult hippocampus, THAP1-IR was mainly mildly and diffusely distributed in the layers containing neuronal processes with sporadic intermediately stained neuronal bodies. Scale bar, 200 μm.
Fig. 7
Fig. 7
THAP1-IR in spinal cord and DRG. THAP1-IR was also present in motor neurons in spinal cord (left panel) and sensory ganglion cells in DRG (right panel) with high level in early development and weaker expression in adulthood. Scale bars, 100 μm in spinal cord; 50 μm in DRG.
Fig. 8
Fig. 8
Double-label fluorescent immunocytochemistry for simultaneous detection of Purkinje cell marker calbindin (red) and THAP1 (green) in rat cerebellum. THAP1-IR was prominent in the perikarya and proximal dendrites of Purkinje cells at early postnatal time points. In contrast, THAP1-IR was more diffusely distributed throughout the dendritic arbors of adult Purkinje cells producing a moderately strong diffuse staining pattern in the molecular layer. m, molecular layer; p, Purkinje cell layer; g, granular cell layer. Scale bar, 50 μm.
Fig. 9
Fig. 9
Subcellular localization of THAP1-IR. Double-label fluorescent immunocytochemistry for simultaneous detection of THAP1 (green) with histone 1 (red) or lamin A&C (red). Some THAP1-IR appeared concentrated near the nuclear envelope within the nucleus and showed modest co-localization with histone 1, and minimal co-localization with lamin A&C. Scale bar, 25 μm.
Fig. 10
Fig. 10
Double-label fluorescent immunocytochemistry for simultaneous detection of THAP1 (green) and parvalbumin (red) in rat cerebellum. THAP1-IR was not localized to interneurons within molecular layer. Arrows are used to mark a subset of neurons within the molecular layer. m, molecular layer; p, Purkinje cell layer; g, granular cell layer. Scale bar, 50 μm.
Fig. 11
Fig. 11
Double-label fluorescent immunocytochemistry for simultaneous detection of THAP1 (green) with TH or ChAT (red) in P14 rat brain. THAP1-IR was present in TH-positive dopaminergic neurons in substantial nigra and ChAT-positive cholinergic neurons in striatum. Scale bar, 50 μm.

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