CCCTC-binding factor (CTCF) is a multifunctional nuclear protein with 11 zinc finger DNA-binding motifs in mice (1). Neural CTCF is linked to neurodevelopment, including neural progenitor cell proliferation, differentiation, and survival (2, 3).
Furthermore, CTCF is involved in neuroinflammation and neuronal death, which is highly associated with various neurodegenerative and neurological disorders (8). Sams
Based on these findings, although it has been reported that CTCF regulates age-dependent axonal regeneration and neurodegeneration via the physiological downregulation of
Loss of CTCF expression in the hippocampi of adult
To confirm whether the decrease in the width and neuronal number of the SP in CA1 of cKO mice was due to neuronal cell death, we investigated the immunoreactivity of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining, which is most widely used to detect cells undergoing apoptotic cell death (11, 12), in NeuN+ neurons of CA1 (Fig. 1G). As the number of NeuN+ neurons was significantly lower in the SP of CA1 in cKO mice than in the SP of CA1 in CTL mice (Fig. 1D), we compared the number of NeuN+ neurons showing TUNEL immunoreactivity (TUNEL+NeuN+ neurons) per area in the SP of CA1 between control and cKO mice at 7.5-10 months of age. We found a remarkably increased number of TUNEL+NeuN+ neurons in the SP layer (Fig. 1H, white arrow and Fig. 1I; CTL, 1.39 ± 0.24/67 μm2; cKO, 7.0 ± 1.10/67 μm2, t(9) = −4.933, **P = 0.0011), suggesting that decreases in the neuronal number and width of the SP were due to apoptotic neuronal death in 7.5-10-month-old
Based on a previous report demonstrating an increase in inflammation-related gene expression without hippocampal neuronal death in cKO mice at 3-4 months of age (9), we hypothesized that neuroinflammatory responses induced by a long-term deficiency in CTCF expression of glutamatergic neurons would result in neuronal death in the hippocampus of cKO mice over 7 months of age. To address this, we investigated glial cells associated with neuroinflammation in the hippocampus of 7.5-10-month-old cKO mice. As expected, reactive microgliosis was clearly observed in all layers of CA1 in
Next, we examined the changes in glial cells in the hippocampal CA3 and DG of cKO mice, which did not show any neuronal loss compared to the CTL (Fig. 2). Surprisingly, cKO mice exhibited abnormal gliosis in both regions of the hippocampus. In CA3, only reactive microgliosis was observed in the SO layer of cKO mice (Fig. 4A, B; t(8) = −2.503, *P = 0.0367) and we found neither abnormal microgliosis in the SP, stratum lucidum (SL), and SR layers (Fig. 4A, C, D) nor astrogliosis in SO, SP, SL, and SR layers (Fig. 4E-H). Unlike CA3, dramatic reactive microgliosis (Fig. 4I) and astrogliosis (Fig. 4M) were visible in the molecular layer (ML) and hilus of DG and quantitative analysis of cell number per area revealed significant increases in Iba1+ microglia (Fig. 4J for ML: t(8) = −6.634, ***P = 0.00016; Fig. 4L for hilus: t(4.175) = −3.068, *P = 0.0353) and GFAP+ astrocytes (Fig. 4N for ML: t(8) = −9.717, ***P = 0.0000105 ; Fig. 4P for hilus: t(8) = −2.997, *P = 0.0171) in ML and hilus, but not the granule cell layer (GCL) (Fig. 4K, O) of DG in cKO mice. However, in contrast with CA1, bipolar rod-shaped microglia was not prominently observed in CA3 and DG of cKO mice at the same age (Supplementary Fig. 2). As a result, these findings indicate that neuroinflammatory responses to potent neuronal damage induced by
In a previous report, 3-4-month-old
Interestingly, we observed abnormal microgliosis and astrogliosis in the DG and only microgliosis in CA3 without neuronal loss in 7.5-10-month-old cKO mice. Based on these findings, we speculate that neuronal damage induced by the depletion of CTCF causes abnormal gliosis of microglia, followed by reactive astrogliosis and neuronal death in hippocampal CA1 at 3-10 months of age. Furthermore, neuronal damage and neuroinflammation occur first in the hippocampal CA1 and next in the DG, and finally in the CA3 of
Neuronal death following sequential microgliosis and astro-gliosis was also observed in the anterior cingulate cortex (ACC) of cKO mice. Indeed, our previous study on the ACC demonstrated apoptotic cell death of cortical neurons in cKO mice over 5 months of age, subsequent to age-dependent aggravation in reactive gliosis at 4 months of age (15). However, in the present study, we found that hippocampal neuronal damage and death induced by CTCF deficiency occur later than cortical neuronal death of the ACC and we assume that it might attribute to enrichment of neurogenesis-related genes in the hippocampus.
We also found microglia with a bipolar rod shape, which is considered a transitional stage between the ramified microglia for immune surveillance (16) and amoeboid microglia for phagocytosis (17), specifically in the SR layer of the CA1, but neither CA3 nor DG, in 7.5-10-month-old cKO mice. In humans, the presence of rod-shaped microglia in the hippocampus is directly associated with age-dependent increases in senescence and neurodegeneration-related pathologies, such as dementia and senile plaque formation in the brain (18). In addition, CTCF itself as an epigenetic factor has known to play a pivotal role in transcriptional regulation of
Brain sample preparation and immunohistochemistry were performed as previously described (24). After tissue preparation (coronal slices, 40 μm thickness), the brain sections were incubated with the following primary antibodies at 1:1,000 dilution: mouse anti-NeuN (MAB377; Merck Millipore, Burlington, MA, USA), rabbit anti-GFAP (Z0334; Dako, Glostrup, Denmark) overnight at 4°C, and goat anti-ionized calcium-binding adapter protein-1 (Iba1) (NB100-1028; Novus Biologicals, Littleton, CO, USA) for 2 days at 4°C. The sections were then incubated with the following secondary antibodies at 1:200 dilutions for 3 h at room temperature: Cy5-conjugated anti-mouse (for anti-NeuN), Alexa Fluor 546 donkey anti-rabbit IgG (for anti-GFAP), and Alexa Fluor 488 donkey anti-goat IgG (for anti-Iba1). For TUNEL assay, sections were labeled with an
All experiments were performed in a blinded fashion, with the minimum number of mice required to achieve statistical validity. Data are shown as the mean ± standard error of the mean. For each variable, two groups were compared using a two-tailed
This study was supported by the National Research Foundation (NRF) of Korea grants funded by the Korean government (MSIP) [NRF-2019R1F1A1063932] to K.L.
The authors have no conflicting interests.