Expression of histone deacetylases in cellular compartments of the mouse brain and the effects of ischemia

Abstract

Drugs that inhibit specific histone deacetylase (HDAC) activities have enormous potential in preventing the consequences of acute injury to the nervous system and in allaying neurodegeneration. However, very little is known about the expression pattern of the HDACs in the central nervous system (CNS). Identifying the cell types that express HDACs in the CNS is important for determining therapeutic targets for HDAC inhibitors and evaluating potential side effects. We characterized the cellular expression of HDACs 1-3, and HDACs 4 and 6, in the adult mouse brain in the cingulate cortex, parietal cortex, dentate gyrus, and CA1 regions of the hippocampus and subcortical white matter. Expression of class I HDACs showed a cell-and region-specific pattern. Transient focal ischemia induced by temporary middle cerebral artery occlusion, or global ischemia induced by in vitro oxygen-glucose deprivation, altered the extent of HDAC expression in a region- and cell-specific manner. The pan-HDAC inhibitor, SAHA, reduced ischemia-induced alterations in HDACs. The results suggest that in addition to promoting epigenetic changes in transcriptional activity in the nucleus of neurons and glia, HDACs may also have non-transcriptional actions in axons and the distant processes of glial cells and may significantly modulate the response to injury in a cell- and region-specific manner.

Fig. 1

Class 1 HDAC expression in the adult mouse brain. HDACs 1 (a), 2 (b), and 3 (c) are highly expressed in cortical and hippocampal neurons, with relative detection decreasing in other neuronal cells away from the cortex and low expression in areas of white matter. a In cingular (i) and parietal (iii) cortex, HDAC 1 expression was in neuronal cell bodies and in their axons. In hippocampal CA1–CA2 junction (ii), HDAC 1 expression was prominent in pyramidal cell bodies (stars) and was visible in some dendrites (arrowheads). In overlying subcortical white matter (SCWM), HDAC 1 expression was present in some nuclei and associated processes (arrows). b HDAC 2 expression was localized to neuronal nuclei in cingulate (i) and parietal cortex (iii) (arrows) and hippocampal pyramidal cells (ii) (stars). In some sections of the parietal cortex (iii), HDAC 2 labeling was faintly visible in axons (arrowheads). HDAC 2 expression was very prominent in SCWM and hippocampus (ii) in some astroglial nuclei and uniformly outlining their associated processes (arrows). c HDAC 3 was expressed in the nuclei of neurons and axons in cingulate and parietal cortex (i and iii) (arrows), but in the cytoplasm of pyramidal neurons of the hippocampus (stars), as well as in their axons and dendrites (ii) (arrowheads). Note HDAC 3 nuclear labeling associated with the synaptic domains of interneurons away from the pyramidal cell layer (dotted line)

Fig. 2

HDAC 1 expression in the cortex, corpus callosum, hippocampus, and subcortical white matter (SCWM). HDAC 1 displayed a clear cytoplasmic (arrows) and axonal (arrowheads) pattern in cortical neurons (cortex, upper panel) and co-localized with MAP2(+) neuronal cell bodies and axons (cortex, middle panel). Sytox(+) blue nuclei were visible in the merged images. Similarly, HDAC 1 (corpus callosum, upper panel) co-localized with NF-200(+) axons (arrowheads, left middle panel) in the corpus callosum. HDAC 1 was limited to the nuclei of GFAP(+) astrocytes in the hippocampus (gray matter, arrows). In SCWM, HDAC 1 labeling was detected in the nuclei (white matter, arrows) and to some extent in the proximal portions of the main processes and end-feet (arrowheads) of GFAP(+) astrocytes (green) outlining penetrating arterioles. Note the distinct co-localization of HDAC 1 and GFAP in the merged images, which confirmed that HDAC 1 exhibits a region (gray vs. white matter) specific labeling pattern in astrocytes

Fig. 3

HDAC 2 expression in neurons, astrocytes, and their end-feet. HDAC 2 (cortex, upper panel, arrows) demonstrated a distinct nuclear pattern in neurons, filling the center of MAP(+) neuronal cell bodies (cortex, middle panel). Consistent with this nuclear expression, HDAC 2 labeling was absent in NF-200(+) axons (corpus callosum, middle panel) in the corpus callosum. HDAC 2 is robustly expressed in GFAP(+) astrocyte nuclei and cell bodies (gray matter), filling astrocyte processes in the hippocampus (arrows) and SCWM (white matter). HDAC 2 is highly expressed in astrocyte end-feet (arrows), precisely outlining the interdigitating arterioles (arrows) in SCWM (merged, white matter lower panel). Note that HDAC 2 labeling identified astrocytes beyond their GFAP(+) immunoreactivity (white matter panel, stars)

Fig. 4

HDAC 3 is expressed in neuronal cell bodies, axons, in the nuclei, and distal processes of astrocytes. HDAC 3 exhibited a cytoplasmic (arrows) and axonal (arrowhead) pattern in cortical neurons (cortex) and co-localized with MAP2(+) neuronal cell bodies and axons (cortex, middle panel). HDAC 3 (right, upper panel) also co-localized with NF-200(+) axons (left, middle panel) in the corpus callosum. HDAC 3 was displayed in the nuclei (arrows) and proximal main processes (arrowheads) of GFAP(+) astrocytes in hippocampus (arrows) and SCWM (merged panels). Note the distinct punctate nature of HDAC 3 expression in both gray matter regions, co-localizing with distal processes of GFAP(+) astrocytes (stars). In the lower right panel, HDAC 3 labeling was absent in astrocyte end-feet (blue arrowhead), but outlined the vasculature (blue arrow)

Fig. 5

HDAC 1–3 expression in the dentate gyrus and the CA1 regions of hippocampus. HDAC 1 (upper panels) was compared in the dentate gyrus (DG), a site of neurogenesis, and the CA1 region which is known to undergo apoptosis even after very transient ischemic injury. HDAC 1 labeling was mainly expressed in the cytoplasm of DG granule cells and in CA1 pyramidal neurons. This was confirmed with blue Sytox(+) nuclei filling the center of neurons (see insets). Note that HDAC 1 labeled dendrites of CA1 pyramidal neurons (upper right panel, arrows), but not neuronal processes in DG. HDAC 2 (middle panels) was expressed in the nuclei of DG granule cells and in the cytoplasm of CA1 pyramidal cells (see insets for blue nuclei labeled with Sytox). In addition, astrocytes diffusely expressed HDAC 2 in their nuclei, cell bodies, processes, and end-feet outlining the vasculature (left middle panel, arrows). HDAC 3 was mainly expressed in the nuclei of DG granule cells and in the nuclei and cytoplasm of CA1 pyramidal cells. Note that some cells in between the upper and lower blades of the DG express HDAC 3 in their cytoplasm (insets). The punctate nature of HDAC 3 expression was more prominent in the CA1 region, especially around interneurons, and outlined their synaptic domains (dotted circle). HDAC 3 expression was also present in CA1 pyramidal cell dendrites (arrows)

Fig. 6

Ischemia upregulates HDAC 1 and HDAC 2 expression. a Following MCAo (ipsilateral, 45 min), HDAC 1 was reduced in the ischemic core (dotted line separates the ischemic core from the penumbra), but upregulated in spared neurons of the surrounding penumbra in cortex, striatum (arrowheads), SVZ (star), as well as in glial cells in the SCWM (arrows, right panels) compared with the corresponding contralateral regions (left panels). Note how capillaries were encircled with bright HDAC 1 labeling in contralateral sections (arrows, lower left panel). b Following MCAo (ipsilateral, 45 min), HDAC 2 was reduced in the ischemic core, but upregulated in spared neurons of the surrounding penumbra in cortex, SVZ (star), as well as glial cells in the SCWM (arrows, right panels) compared with corresponding contralateral regions (left panels). Note that some striatal neurons (outlined with dotted lines) demonstrated loss of HDAC 2, while those adjacent to the SVZ upregulated HDAC 2. Capillaries were regularly encircled with bright HDAC 2 labeling (contralateral section; arrows, upper left panel)

Fig. 7

HDAC inhibition attenuates upregulation of class I HDAC isoforms. After 60 min of OGD, levels of HDAC 1–3 expression were upregulated in all glial cell nuclei (arrowheads) and processes of astrocytes (arrows) in MON. Note that OGD upregulated HDAC 3 most strongly (lower middle panel). HDAC inhibition with SAHA (1 µM), applied 30 min before, during, and 30 min after OGD, considerably attenuated HDAC 1–3 upregulation in MON

Fig. 8

HDAC 2 is robustly expressed in the neurovascular unit. HDAC 2 consistently labeled astrocyte end-feet in larger pial arteries in the cortex (left panels) and also penetrating small capillaries in the hippocampus (right panels). The HDAC 2 labeling was limited to astrocyte end-feet in the neurovascular unit. Endothelial cells (arrowheads, labeled blue with Sytox) did not express HDAC 2