Abstract
PML is a tumor suppressor protein involved in the pathogenesis of promyelocytic leukemia. In non-neuronal cells, PML is a principal component of characteristic nuclear bodies. In the brain, PML has been implicated in the control of embryonic neurogenesis, and in certain physiological and pathological phenomena in the adult brain. Yet, the cellular and subcellular localization of the PML protein in the brain, including its presence in the nuclear bodies, has not been investigated comprehensively. Because the formation of PML bodies appears to be a key aspect in the function of the PML protein, we investigated the presence of these structures and their anatomical distribution, throughout the adult mouse brain. We found that PML is broadly expressed across the gray matter, with the highest levels in the cerebral and cerebellar cortices. In the cerebral cortex PML is present exclusively in neurons, in which it forms well-defined nuclear inclusions containing SUMO-1, SUMO 2/3, but not Daxx. At the ultrastructural level, the appearance of neuronal PML bodies differs from the classic one, i.e., the solitary structure with more or less distinctive capsule. Rather, neuronal PML bodies have the form of small PML protein aggregates located in the close vicinity of chromatin threads. The number, size, and signal intensity of neuronal PML bodies are dynamically influenced by immobilization stress and seizures. Our study indicates that PML bodies are broadly involved in activity-dependent nuclear phenomena in adult neurons.
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Acknowledgments
This work is supported by the grant No. 2012/05/E/NZ4/02997 from National Science Center, and by the European Regional Development Fund POIG 01.01.02-00-008/08. The authors are grateful to Dr. Tomasz Prószyński for his generous gift of immortalized fibroblasts.
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429_2015_1053_MOESM1_ESM.tif
Supplemental Fig. 1. Control reaction for DAB immunostaining. (a) DAB immunostaining with a non-immune mouse IgG1. The strong signal in the fimbria hippocampi is unspecific. (b) DAB immunostaining with the mouse anti-PML antibody. (TIFF 4837 kb)
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Supplemental Fig. 2. Immunofluorescence staining for the PML protein (green) in conditions of signal oversaturation. Note the presence of specific signal in the nucleoplasm between the PML bodies (arrowheads), and in the cytoplasm (arrows) in the cortex (a–c), hippocampus (g–i) and striatum (m–o), as compared to non-immune IgG1 (d-f, j-l, p-s, respectively). DNA was counterstained with Hoechst 33342 (blue). The neuronal marker NeuN, which is present in both the nucleus and the cytoplasm, is presented in red color (TIFF 11558 kb)
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Supplemental Fig. 3. Colocalization of PML and SUMO-2/3. Confocal images demonstrating the colocalization of PML (green) with SUMO-2/3 (red in b,c) in a cortical neuron. Note that only few bodies contain SUMO-2/3. The cells were visualized using neuronal marker NeuN (blue). The white dashed outline indicates the circumference of the nucleus. The scale bar—5 μm (TIFF 6301 kb)
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Supplemental Fig. 4. Quantitative analysis of colocalization between PML and SUMO-1 (a) or SUMO-2/3 (b); there is lack of significant changes in PML-SUMO-1 or PML-SUMO-2/3 overlap. Error bars represent the SEM; Mann–Whitney test showed no statistical significant difference (P > 0.05). For (a) and (b) at least 150 nuclei from 3 different individuals per each group was analyzed (TIFF 11306 kb)
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Supplemental Fig. 5. RT-PCR and Western blotting for PML. (a) Fold change in the amounts of PML mRNA in PTZ seizures and stress; the limits of each box plot represents the rank of the data; the lower and upper edges of the box are the first and third quartiles, therefore 50 % of the data occurs in this range; the thick-dark horizontal segment represents the median; p value for PTZ seizures < 0.2005, p value for stress < 0.183, Bootstrap method (Cleries et al. 2012); note that there is no significant change in either condition. (b) Western blotting for PML (upper lane) and nuclear marker matrin-3 (lower lane) in control brain, pentylenetetrazole group (PTZ, 2 h), and stress group. Note the apparent lack of consistent changes of PML compared to matrin-3; each column represents the distinct animal (TIFF 11827 kb)
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Hall, M.H., Magalska, A., Malinowska, M. et al. Localization and regulation of PML bodies in the adult mouse brain. Brain Struct Funct 221, 2511–2525 (2016). https://doi.org/10.1007/s00429-015-1053-4
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DOI: https://doi.org/10.1007/s00429-015-1053-4