Antisense oligonucleotides targeting basal forebrain ATXN2 enhances spatial memory and ameliorates sleep deprivation-induced fear memory impairment in mice

Abstract

Introduction: Regulation of brain-derived neurotrophic factor (BDNF) in the basal forebrain ameliorates sleep deprivation-induced fear memory impairments in rodents. Antisense oligonucleotides (ASOs) targeting ATXN2 was a potential therapy for spinocerebellar ataxia, whose pathogenic mechanism associates with reduced BDNF expression. We tested the hypothesis that ASO7 targeting ATXN2 could affect BDNF levels in mouse basal forebrain and ameliorate sleep deprivation-induced fear memory impairments.

Methods: Adult male C57BL/6 mice were used to evaluate the effects of ASO7 targeting ATXN2 microinjected into the bilateral basal forebrain (1 μg, 0.5 μL, each side) on spatial memory, fear memory and sleep deprivation-induced fear memory impairments. Spatial memory and fear memory were detected by the Morris water maze and step-down inhibitory avoidance test, respectively. Immunohistochemistry, RT-PCR, and Western blot were used to evaluate the changes of levels of BDNF, ATXN2, and postsynaptic density 95 (PSD95) protein as well as ATXN2 mRNA. The morphological changes in neurons in the hippocampal CA1 region were detected by HE staining and Nissl staining.

Results: ASO7 targeting ATXN2 microinjected into the basal forebrain could suppress ATXN2 mRNA and protein expression for more than 1 month and enhance spatial memory but not fear memory in mice. BDNF mRNA and protein expression in basal forebrain and hippocampus was increased by ASO7. Moreover, PSD95 expression and synapse formation were increased in the hippocampus. Furthermore, ASO7 microinjected into the basal forebrain increased BDNF and PSD95 protein expression in the basal forebrain of sleep-deprived mice and counteracted sleep deprivation-induced fear memory impairments.

Conclusion: ASOs targeting ATXN2 may provide effective interventions for sleep deprivation-induced cognitive impairments.

Keywords: ATXN2; antisense oligonucleotides; memory; sleep deprivation.

FIGURE 1

Microinjection of ASO7 into basal forebrain imposes no effect on fear memory, but enhances spatial learning and memory. (A) Schematic design. 21 days after ASO7 microinjection, fear memory was evaluated. After 30 days of microinjection, mice received Morris water maze test. RT‐PCR and Western blot were carried out after the water maze test. Box and whiskers plots (min to max) combined with scatter plots showing ATXN2 inhibition in basal forebrain has no effect on (B) short‐term and (C) long‐term fear memory. (D) Latency during the navigation training phase. (E) Representative video tracks of probe trial and histograms showing the time spent in the target quadrant. (F) Ataxin‐2 mRNA changes after ASO7 treatment. (G) Ataxin‐2 protein expression after ASO7 microinjection. Values were presented as means ± standard deviations (SDs). ^* p < 0.05 compared to the aCSF group. ^# p < .05 compared to the negative control group.

FIGURE 2

Microinjection of ASO7 into basal forebrain increases BDNF and PSD95 protein expression and synapse formation in the hippocampus. (A) Representative Western blot images and histograms showing that ASO7 increased brain‐derived neurotrophic factor (BDNF) protein expression in the basal forebrain. (B) Representative Western blot images and histograms showing ASO7 increased BDNF protein expression in the hippocampus. (C) Representative Western blot images and histograms showing ASO7 increased PSD95 protein expression in the hippocampus. Histograms (D) and representative immunohistochemical pictures (E–H) showing ASO7 increased BDNF and PSD95 expression in the hippocampus. (I–K) Spine density was quantified in the CA1 region of the dorsal hippocampus from ASO7 and aCSF group mice. (I) Representative images of the hippocampal CA1 region analyzed for spine density. (J) Representative images of spine density from aCSF and ASO7 group mice. (K) ASO7 treatment significantly increased spine density levels of mice compared to aCSF‐treated mice. *p < .05. Scale bar = 200 μm. Data were expressed as means ± SDs. Note: Western blot gel images of subpanels B and C were obtained from the same hippocampus tissues and the same GAPDH gel image was used.

FIGURE 3

ASO7 increases BDNF and PSD95 protein expression, and partly recues short‐ and long‐term fear memory impairments induced by sleep deprivation. (A) Schematic design. ASO7 (1 μg/0.5 μL/side) or aCSF (0.5 μL/side) were microinjected bilaterally into the basal forebrain of adult male mice. After 14 days, the mice were subjected to 6 h of total sleep deprivation or rest control followed by inhbitory avoidance training. The effects of basal forebrain ATXN2 inhibition on BDNF and PSD95 protein expression and short‐and long‐term memories were then detected. (B) Representative Western blot images and histograms showed incresed BDNF expression in the basal forebrain after sleep deprivation, which was enhanced by ASO7. (C) Representative Western blot images and histograms showed no change in PSD95 protein expression in basal forebrain after sleep deprivation, but increased PSD95 protein expression was detected in sleep‐deprived mice with ASO7 treatment. (D, E) Box and whiskers plots (min to max) combined with scatter plots showing the partial rescue of sleep deprivation induced (F) short‐term and (G) long‐term term fear memory impairments by ASO7. *p < .05; **p < .01 compared to the rest control group. ^# p < .05 compared to the sleep deprivation group. Data were expressed as means ± SDs for the Western blot results. n = 4 per group for the Western blot experiment. n = 8 per group for the memory test. Note: Western blot gel images of subpanels B and C were obtained from the same basal forebrain tissues and the same GAPDH gel image was used.