Knockdown of long non-coding RNA SOX2OT downregulates SOX2 to improve hippocampal neurogenesis and cognitive function in a mouse model of sepsis-associated encephalopathy

Abstract

Background: Aberrant hippocampal neurogenesis is an important pathological feature of sepsis-associated encephalopathy. In the current study, we examined the potential role of the long noncoding RNA (lncRNA) sex-determining region Y-box 2 (SOX2) overlapping transcript (SOX2OT), a known regulator of adult neurogenesis in sepsis-induced deficits in hippocampal neurogenesis and cognitive function.

Methods: Sepsis was induced in adult C57BL/6 J male mice by cecal ligation and perforation (CLP) surgery. Randomly selected CLP mice were transfected with short interfering RNAs (siRNAs) against SOX2OT or SOX2, or with scrambled control siRNA. Cognitive behavior was tested 8-12 days post-surgery using a Morris water maze. Western blotting and RT-qPCR were used to determine expression of SOX2, Ki67, doublecortin (DCX), nestin, brain lipid-binding protein, and glial fibrillary acidic protein (GFAP) in the hippocampus. The number of bromodeoxyuridine (BrdU)⁺/DCX⁺ cells, BrdU⁺/neuronal nuclei (NeuN)⁺ neurons, and BrdU⁺/GFAP⁺ glial cells in the dentate gyrus were assessed by immunofluorescence.

Results: CLP mice showed progressive increases in SOX2OT and SOX2 mRNA levels on days 3, 7, and 14 after CLP surgery, accompanied by impaired cognitive function. Sepsis led to decrease in all neuronal markers in the hippocampus, except GFAP. Immunofluorescence confirmed the decreased numbers of BrdU⁺/DCX⁺ cells and BrdU⁺/NeuN⁺ neurons, and increased numbers of BrdU⁺/GFAP⁺ cells. SOX2OT knockdown partially inhibited the effects of CLP on levels of SOX2 and neuronal markers, neuronal populations in the hippocampus, and cognitive function. SOX2 deficiency recapitulated the effects of SOX2OT knockdown.

Conclusion: SOX2OT knockdown improves sepsis-induced deficits in hippocampal neurogenesis and cognitive function by downregulating SOX2 in mice. Inhibiting SOX2OT/SOX2 signaling may be effective for treating or preventing neurodegeneration in sepsis-associated encephalopathy.

Keywords: Cognitive dysfunction; Hippocampal neurogenesis; SOX2; SOX2OT; Sepsis-associated encephalopathy.

Fig. 1

Schematic illustration of the experimental design. a Timeline for naive, sham-operated, or CLP-operated mice. b Timeline for mice injected with scrambled control siRNA or SOX2OT siRNA. c Timeline for mice injected with scrambled control siRNA or SOX2 siRNA. Abbreviations: CLP, cecal ligation and perforation; d, day; MWM, Morris water maze; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; siRNA, short interfering RNA; SOX2, sex-determining region Y-box 2; SOX2OT, SOX2 overlapping transcript

Fig. 2

A schematic illustration of CLP procedure and i.c.v. injection. a CLP procedure. The basis of the cecum is indicated by the yellow line. The cecum is ligated (indicated by dotted green line) at half the distance between distal pole and the base of the cecum. b Injection site of the mouse lateral ventricle. The injection site is at 1.0 mm lateral and 0.5 mm posterior to the bregma over the left hemisphere. c A 27 G needle with a 10-μl syringe is used for the injection into the lateral ventricle 2.5-mm deep, perpendicular to the skull surface. Abbreviations: CLP, cecal ligation and perforation; i.c.v., intracerebroventricular

Fig. 3

Cognitive impairment and SOX2OT and SOX2 upregulation in a mouse model of sepsis-associated encephalopathy. a Escape latency time, b swimming speed, c time spent in the target quadrant, and d number of crossings over the target quadrant in the Morris water maze. e Freezing time in context and f freezing time in response to a cue in the fear conditioning test (n = 15/group). Relative levels of g SOX2OT and h SOX2 mRNAs were measured by RT-qPCR on days 3, 7, and 14 after sham or CLP surgery (n = 5/group). ^&p < 0.05 vs. day 1 of Morris water maze test; ^#p < 0.05 vs. Sham; **p < 0.05. Abbreviations: CLP, cecal ligation and perforation; MWM, Morris water maze; SOX2, sex-determining region Y-box 2; SOX2OT, SOX2 overlapping transcript

Fig. 4

SOX2OT knockdown attenuated sepsis-induced cognitive dysfunction in mice with sepsis-associated encephalopathy. a Escape latency, b swimming speed, c time spent in the target quadrant, d number of times mice crossing the target quadrant, and e representative swim paths on the test day in the Morris water maze. The white circle indicates the location of the training platform, which was removed during the test. f Freezing time in context and g freezing time in response to a cue in the fear conditioning test (n = 16/group). ^&p < 0.05 vs. day 1 of Morris water maze test; *p < 0.05 vs. Sham; ^#p < 0.05 vs. CLP; ^^p < 0.01. Abbreviations: CLP, cecal ligation and perforation; si-CRTL, scrambled control siRNA; si-SOX2OT, SOX2OT siRNA

Fig. 5

SOX2OT knockdown using siRNA attenuated the loss of cell proliferation and mature neurons, and reduced glial differentiation in the hippocampus of mice with sepsis-associated encephalopathy. Representative immunofluorescence micrographs showing (a) BrdU⁺/DCX⁺ cells, (b) BrdU⁺/GFAP⁺ glial cells and (c) BrdU⁺/NeuN⁺ neurons in the dentate gyrus of the hippocampus from CLP mice injected with SOX2OT siRNA. Magnification: × 200. Scale bar = 100 μm. Quantification of immunofluorescent cells staining positive for (d) BrdU/DCX, (e) BrdU/GFAP or (f) BrdU/NeuN. *p < 0.05; **p < 0.01. Abbreviations: CLP, cecal ligation and perforation; si-CRTL; scrambled control siRNA; si-SOX2OT, SOX2OT siRNA

Fig. 6

SOX2OT knockdown attenuated changes in neuronal marker levels in the hippocampus of mice with sepsis-associated encephalopathy. a Representative images showing protein bands of Ki67, DCX, Nestin, BLBP, and GFAP from CLP mice injected with SOX2OT siRNA or scrambled control. Densitometry-based protein quantitation of b Ki67, c DCX, d Nestin, e BLBP, and f GFAP. β-actin was used as an internal control (n = 8/group). *p < 0.05; **p < 0.01. Abbreviations: CLP, cecal ligation and perforation; si-CRTL; scrambled control siRNA; si-SOX2OT, SOX2OT siRNA

Fig. 7

SOX2OT knockdown downregulated SOX2 and OCT4 in the hippocampus of mice with sepsis-associated encephalopathy. a Representative images showing SOX2 and OCT4 protein bands in hippocampal tissue from CLP mice injected with SOX2OT siRNA or scrambled control. b Densitometry quantitation of SOX2 and OCT4 expression. β-actin was used as an internal control. c Levels of SOX2OT, SOX2, and OCT4 transcripts (n = 8/group). **p < 0.01. Abbreviations: CLP, cecal ligation and perforation; si-CRTL, scrambled control siRNA; si-SOX2OT, SOX2OT siRNA

Fig. 8

SOX2 knockdown using siRNA alleviated sepsis-induced cognitive dysfunction in sepsis-associated encephalopathy mice. a Escape latency, b swimming speed, c time spent in the target quadrant, and d number of times mice crossed the target quadrant on the test day in the Morris water maze. e Freezing time to context and f freezing time to cue in the fear conditioning test (n = 16/group). ^&p < 0.05 vs. day 1 of Morris water maze test; *p < 0.05 vs. Sham; ^#p < 0.05 vs. CLP; ^p < 0.05; ^^p < 0.01. Abbreviations: CLP, cecal ligation and perforation; si-CRTL, scrambled control siRNA; si-SOX2, SOX2 siRNA

Fig. 9

SOX2 siRNA attenuated the loss of cell proliferation and mature neurons and reduced glial differentiation in the hippocampus from sepsis-associated encephalopathy mice. Representative immunofluorescence micrographs showing (a) BrdU⁺/DCX⁺ cells, (b) BrdU⁺/GFAP⁺ glial cells and (c) BrdU⁺/NeuN⁺ neurons in hippocampal dentate gyrus of CLP mice injected with SOX2 siRNA. Magnification: × 200. Bar = 100 μm. Quantification of immunofluorescent cells staining positive for (d) BrdU/DCX, (e) BrdU/GFAP or (f) Brdu/NeuN. *p < 0.05; **p < 0.01. Abbreviations: CLP, cecal ligation and perforation; si-SOX2, SOX2 siRNA