Synthetic microRNA-mediated downregulation of Nogo-A in transgenic rats reveals its role as regulator of synaptic plasticity and cognitive function

Abstract

We have generated a transgenic rat model using RNAi and used it to study the role of the membrane protein Nogo-A in synaptic plasticity and cognition. The membrane protein Nogo-A is expressed in CNS oligodendrocytes and subpopulations of neurons, and it is known to suppress neurite growth and regeneration. The constitutively expressed polymerase II-driven transgene was composed of a microRNA-targeting Nogo-A placed into an intron preceding the coding sequence for EGFP, thus quantitatively labeling cells according to intracellular microRNA expression. The transgenic microRNA in vivo efficiently reduced the concentration of Nogo-A mRNA and protein preferentially in neurons. The resulting significant increase in long-term potentiation in both hippocampus and motor cortex indicates a repressor function of Nogo-A in synaptic plasticity. The transgenic rats exhibited prominent schizophrenia-like behavioral phenotypes, such as perseveration, disrupted prepulse inhibition, and strong withdrawal from social interactions. This fast and efficient microRNA-mediated knockdown provides a way to silence gene expression in vivo in transgenic rats and shows a role of Nogo-A in regulating higher cognitive brain functions.

Fig. 1.

Design and testing of the intronic miRNA/EGFP expression system. (A) Expression vectors pUTR and pINTRON containing a rationally designed miRNA targeting firefly luciferase (M) or no miRNA as controls (E). In pUTR, the miRNA is integrated between the 3′ end of EGFP and the polyadenylation signal (pA). In pINTRON, the miRNA is embedded within an artificial intron located 5′ of EGFP. Transient transfection experiments using miRNA/EGFP together with luciferase expression vectors show that relative EGFP activities and luciferase knockdown efficiencies are much higher for pINTRON than pUTR. Means (n = 9) and SEM are shown. (B) Schematic drawing of the Nogo-A miRNA insert-containing construct used for the generation of transgenic rats. Inset displays results of Nogo-A qPCR quantification relative to housekeeping genes from Nogo-A–expressing 3T3 cells transfected with either pCAG-INTRON-(miRNA Nogo-A)-EGFP (KD) or control (C) vector. ***P < 0.001.

Fig. 2.

Rat line L2 expresses transgenic EGFP and miRNA at the highest level without affecting the miRNA-processing machinery. (A) Western blot results of EGFP and GAPDH for different CNS regions derived from the transgenic rat lines L2, L3, and L18 as well as WT rats. Cb, cerebellum; Ctx, cortex; Hip, hippocampus; SC, spinal cord. (B) Absolute qPCR quantification (expressed as molecules per cell) of transgenic Nogo-A miRNA expression levels in different rat lines and CNS regions. (C) Scatter plot of qPCR expression levels of 359 abundantly expressed miRNAs from L2 and WT rats. Nonregulated miRNA levels of L2 vs. WT are identical and converge on the bisector of the angle. (D and E) Immunofluorescence of EGFP (green) and the neuronal nuclear marker neuronal nuclei (NeuN) (red) in the cortex (D) and hippocampus (E) of L2 shows a largely neuronal expression of the transgene. (Scale bar: D, 50 µm; E, 20 µm.) *P < 0.05; **P < 0.01.

Fig. 3.

Expression of Nogo-A is reduced in L2 rats. (A) Western blot analysis of Nogo-A (∼200 kDa) and Nogo-B (∼50 kDa) expression compared with GAPDH (∼40 kDa) in different CNS tissues. Whereas Nogo-A is significantly down-regulated in the cortex of L2 rats (P = 0.0173), no cortical knockdown can be seen in animals of L3. In addition, Nogo-B remains unchanged in both L2 and L3. Nogo-A knockdown can also be observed in the hippocampus (P = 0.0195) of L2 rats, whereas only a trend is visible in whole spinal cord (P = 0.0659). Averaged Nogo-A/GAPDH ratios from three animals per transgenic line or WT are reported as a percentage of WT mean ± SEM. (B) Densitometric quantification of Nogo-A immunoreactivity as determined by epifluorescence microscopy in a Zeiss Axiophot microscope. Down-regulation of Nogo-A is evident in the cingulate (CC; P = 0.0499) and motor cortex (MC; P = 0.0041) of L2 rats. In addition, Nogo-A immunoreactivity is decreased in the cell layers of the hippocampus (Hip; P = 0.0080) and the caudate putamen (CPu; P = 0.0008). Down-regulation can also be observed in the cerebellar white matter (WM; P = 0.0166), whereas only a trend is seen in the molecular layer (ML; P = 0.2155), Purkinje cell layer (PCL; P = 0.1675), and granular cell layer (GCL; P = 0.2310). In the spinal cord, whereas a marked reduction of Nogo-A immunoreactivity is detected in motoneurons (MNs; P < 0.0001), no significant change can be observed in spinal oligodendrocytes (ODCs; P = 0.1135). Similar results were obtained after ethanol/acetic acid treatment (EtOH/AcOH) of the spinal cord sections (MN, P = 0.0002; ODC, P = 0.2449). (C) Representative confocal images show the decrease of Nogo-A immunoreactivity in L2 rats in different CNS regions. Images of the spinal cord show sections after EtOH/AcOH treatment. (Scale bars: 50 μm.) Data are presented as mean + SEM. Asterisks represent P values obtained by comparing L2 and WT rats with unpaired t test: *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4.

L2 rats exhibit schizophrenia-like phenotypes. (A) Locomotor activity in the open field arena. No difference on distance traveled between WT and Nogo-A knockdown rats (L2). (B) PPI. Two-way ANOVA revealed a significant reduction in PPI in L2 rats compared with WT rats [F_Genotype (1,54) = 4.97; P = 0.039]. However, Bonferroni posthoc testing revealed that, only at a PPI of 70 dB (PPI-70), startle amplitudes of L2 rats were significantly lower than those startle amplitudes of WT rats. (C) Effects of Nogo-A knockdown on novel object recognition memory in the novel object recognition task. L2 and WT rats showed no significant differences in percent of time spent in exploration of the identical objects (IOs) during the training phase of the test. In contrast, L2 rats have a significant impairment in discriminating between the novel and the familiar object during testing (NO). (D) Novel object relocation task. L2 and WT rats showed no significant differences in exploration of the IOs during the training phase of the test. During the test phase, L2 rats had a significant impairment in discriminating the relocated object (RO). (E–G) Behavioral performance during social interaction with an unknown social partner (social interaction). (E) Significant differences between WT and L2 rats were found for nonanogenital exploration (non-AG), whereas no differences were observed for anogenital exploration (AG) and following/approach (FA). (F) A strong trend was found for a decrease in the number of social contact behaviors (grooming/crawling). (G) L2 rats show significantly more social withdrawal behavior than WT littermates. (H) Reversal learning in the water T maze. Two-way ANOVA revealed that there was no significant difference between animals of the two genotypes in the reversal of their escape strategy in the water T maze, which was seen from their percentage of correct trails within the task [L, F_Genotype(1.108) = 1.22; P = 0.284]. In contrast, Bonferroni posthoc testing revealed that, in trial 2, L2 rats have a significant impairment in finding the escape platform. All data are mean values ± SEM. Asterisks represent P values obtained by comparing L2 and WT rats with either unpaired t or Bonferroni posthoc test after two-way ANOVA of repeated measures: *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5.

Hippocampal as well as cortical LTP are increased in Nogo-A knockdown rats. (A) Input–output relationships (I/O curves) from WT (n = 6) and L2 Nogo-A knockdown rats (n = 6) recorded in the hippocampal CA3-CA1 Schaffer collateral pathway. I/O curves indicate no significant difference in synaptic strength across stimulation intensities. (B) Paired pulsed ratio measured at different interstimulus intervals did not significantly differ between WT and L2 (n = 6 each). (C) LTP was induced by TBS (arrow) in L2 and WT rats. At 60 min after TBS, a significant difference between L2 and WT rats could be observed. Inset shows original traces from representative individual experiments; numbers correspond to the time point when traces were taken. (D) I/O curves from WT (n = 15) and L2 Nogo-A knockdown rats (n = 15) recorded in layer II/III horizontal connections in the M1 forelimb area of brain slices. I/O curves indicate no significant difference in synaptic strength across stimulation intensities. (E) Maximum synaptic strength (LTP saturation) was determined by repeated induction of LTP (multiple arrows). Peak amplitudes were significantly larger in L2 (n = 16) compared with WT rats (n = 15). Each field potential trace represents an average of 10 individual responses at times indicated by numbers.