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A Single Day of 5-azacytidine Exposure During Development Induces Neurodegeneration in Neonatal Mice and Neurobehavioral Deficits in Adult Mice

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A Single Day of 5-azacytidine Exposure During Development Induces Neurodegeneration in Neonatal Mice and Neurobehavioral Deficits in Adult Mice

Shivakumar Subbanna et al. Physiol Behav.

Abstract

The present study was undertaken to evaluate the immediate and long-term effects of a single-day exposure to 5-Azacytidine (5-AzaC), a DNA methyltransferase inhibitor, on neurobehavioral abnormalities in mice. Our findings suggest that the 5-AzaC treatment significantly inhibited DNA methylation, impaired extracellular signal-regulated kinase (ERK1/2) activation and reduced expression of the activity-regulated cytoskeleton-associated protein (Arc). These events lead to the activation of caspase-3 (a marker for neurodegeneration) in several brain regions, including the hippocampus and cortex, two brain areas that are essential for memory formation and memory storage, respectively. 5-AzaC treatment of P7 mice induced significant deficits in spatial memory, social recognition, and object memory in adult mice and deficits in long-term potentiation (LTP) in adult hippocampal slices. Together, these data demonstrate that the inhibition of DNA methylation by 5-AzaC treatment in P7 mice causes neurodegeneration and impairs ERK1/2 activation and Arc protein expression in neonatal mice and induces behavioral abnormalities in adult mice. DNA methylation-mediated mechanisms appear to be necessary for the proper maturation of synaptic circuits during development, and disruption of this process by 5-AzaC could lead to abnormal cognitive function.

Keywords: DNA methylation; Developing brain; Epigenetics; Methyltransferase; Neuronal loss; Synaptic plasticity.

Figures

Fig. 1
Fig. 1
5-AzaC treatment of P7 mice inhibits DNA methylation in the mouse brain. (A) The global DNA methylation levels were quantified using DNA from the hippocampus (HP), neocortex (NC) and cerebellum (CB) tissues. HP, NC and CB of P7 mice after treatment with saline or various doses of 5-AzaC (0–10 mg/kg body weight, s. c. 8 h). *p < 0.01 vs. 0 mg (n= 6 pups/group). (B) Global 5-mC-specific dot-blot intensities of genomic DNA prepared from the HP, NC and CB (150 ng, 100 ng and 5 ng of genomic DNA) of P7 mice after treatment with saline or 5-AzaC (5 mg/kg body weight, s. c. 8 h). *p < 0.01 vs. saline. The amounts of 5-mC are shown as a percentage of the 5-mC-specific dot-blot intensities compared to the saline controls (n = 8 pups/group). *p < 0.01 vs. Saline. (C) Free-floating coronal brain sections (hippocampus, retrosplenial cortex and cerebellum) from both the groups (saline and 5-AzaC) were subjected to immunohistochemistry with anti-mouse-5mC and anti-rabbit-NeuN to label methylated cytosine in neurons. Manders coefficient analysis was used to evaluate 5-mC positive NeuN neurons in the hippocampus (CA1 region), retrosplenial cortex (RSC) and cerebellum (CB) (cerebellar folia region). Error bars, SEM (n = 6 pups/group). *p < 0.01 vs. Saline. Scale bars = 10 μm.
Fig. 2
Fig. 2
The 5-AzaC treatment of P7 mice induces neurodegeneration. (A) Western blot analysis of CC3 HP, NC and CB in cytosolic extracts from the saline- and 5-AzaC-treated (0–10 mg/kg, s. c. 8 h) groups. (B) Western blot analysis of CC3 using cytosolic extracts of the HP and NC from the saline- and 5-AzaC-treated (5 mg/kg, s. c.) groups at various time points. Ponceau S staining confirmed equal loading of the protein, and β-actin was used as a loading controls Error bars, SEM (n = 10 pups/group). (C) Free-floating coronal brain sections (hippocampus and retrosplenial cortex) from the saline- and 5-AzaC-treated (5 mg/kg, s. c. 8 h) animals were immunostained with an anti-rabbit CC3 antibody. The white arrows indicate the CC3-positive neurons in the hippocampus and retrosplenial cortex. Scale bars = 200 μm. The hippocampal region was enlarged to show the CC3-positive cells (*). The scale bars represent 50 μm. The CC3-positive cells in the hippocampus and retrosplenial cortex were counted. Error bars, SEM (n = 5 pups/group). *p < 0.01 vs. 0 h (saline).
Fig. 3
Fig. 3
5-AzaC inhibits ERK1/2 phosphorylation and Arc protein expression in the neonatal brain. Western blot analysis of pERK1/2, ERK1/2 and Arc in cytosolic extracts from the hippocampus (HP) and neocortex (NC) of the saline- or 5-AzaC-treated groups; β-actin was used as a loading control. Error bars, SEM (n = 10 pups/group) (*p < 0.01 vs. 0 h (saline)).
Fig. 4
Fig. 4
Pharmacological inhibition of G9a/CB1R or genetic ablation of CB1R fails to rescue 5-AzaC-induced caspase-3 activation in P7 mice. (A) The mice were pre-treated with Bix (1 mg/kg) or vehicle for 30 min and then exposed to 5-AzaC (5 mg/kg). The CC3 levels in cytosolic extracts of the hippocampus (HP) and neocortex (NC) were determined by Western blot analysis. (B) P7 CB1RWT mice were pre-treated with SR (1 mg/kg) or vehicle for 30 min, and P7 CB1RKO mice were exposed to 5-AzaC (5 mg/kg). The CC3 levels in cytosolic extracts of the hippocampus (HP) were determined by Western blot analysis. β-actin was used as a loading control. Error bars, SEM (n = 10 pups/group) (*p < 0.01 vs. vehicle).
Fig. 5
Fig. 5
5-AzaC-induced deficits in Arc protein expression are long-lasting and persist into adulthood but not ERK1/2 phosphorylation deficits. Western blot analysis of pERK1/2, ERK1/2 and Arc in cytosolic extracts of the hippocampus (HP) from the P7 saline- or 5-AzaC-treated adult mice; β-actin was used as a loading control. Error bars, SEM (n = 10 pups/group) (*p < 0.01 vs. 0 h (saline)).
Fig. 6
Fig. 6
5-AzaC treatment of P7 mice induces long-lasting impairments in spatial memory and social recognition memory in adulthood. Spatial memory performance was measured using the Y-maze. (A) Discrimination index [preference for the novel arm over the familiar other arm (Novel/Novel + Other)] for arm entries and (B) dwell time of the saline- and 5-AzaC-treated male and female mice at 24 h after the first encounter with the partially opened maze. The dashed line denotes chance performance (0.5). (C) The percentage of saline- and 5-AzaC-treated animals (male and female combined) that selected the novel arm as the first choice 24 h after the first encounter with the partially open maze is shown. (D) Exposure to 5-AzaC at P7 induces a loss of social recognition memory in adult male mice. The percent of time spent in social investigation at 24 h after the first encounter with the same juvenile mouse is shown for the saline- and 5-AzaC-treated mice. Error bars, SEM (n = 8 mice/group). *p < 0.01 vs. saline.
Fig. 7
Fig. 7
Treatment of P7 mice with 5-AzaC causes objects recognition memory deficits in adulthood. (A) The level of exploration was measured at el and e2 (24 h), namely the time the saline- and 5-AzaC-treated male mice spent exploring the two objects in T1 and T2 (24 h). (B) Discrimination indices (d2) obtained from the saline- and 5-AzaC-treated mice after 24 h retention intervals. e1 and e2 are measures of the total time spent exploring both objects during T1 and T2, respectively. d2 is an index measuring discrimination between the new and the familiar objects. Error bars, SEM (n = 8 mice/group). *p < 0.01 vs. saline.
Fig. 8
Fig. 8
Exposure of P7 mice to 5-AzaC induces LTP deficits in adult mice. (A) A schematic drawing showing the positions of the stimulating and recording electrodes in the CA1 region of the hippocampus. (B) A summary graph showing the field input/output relationships for the P7 saline- and 5-AzaC-treated adult mice. Insert: An example of traces taken from representative experiments showing the input/output relationships for the saline group. Although it is not shown, the other groups also exhibited comparable patterns. (C) The time course of the average fEPSP slope obtained from the P7 saline- and 5-AzaC-treated adult mice. The fEPSP slopes from each LTP recording were normalized to the mean value of 10 min recordings obtained before stimulation. The arrows indicate the time of TBS (4 pulses at 100 Hz, with bursts repeated at 5 Hz, and each tetanus including 3 different 10-burst trains separated by 15 s). Representative traces of the fEPSPs before (trace 1) and after (trace 2) the induction of LTP in hippocampal slices from P7 saline- and 5-AzaC-treated adult mice. (C) A combined plot of the average of the fEPSP slopes at several time points. Error bars, SEM (n = 5 mice/group; 10 slices/group). *p < 0.01 vs. saline.

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