An antisense-long-noncoding-RNA modulates p75NTR expression levels during neuronal polarization

Abstract

Proper polarization of newly generated neurons is a critical process for neural network formation and brain development. The pan-neurotrophin p75^NTR receptor plays a key role in this process localizing asymmetrically in one of the differentiating neurites and specifying its axonal identity in response to neurotrophins. During axonal specification, p75^NTR levels are transiently modulated, yet the molecular mechanisms underlying this process are not known. Here, we identified a previously uncharacterized natural antisense transcript, AS-p75, encoded within the p75NGFR mouse gene. Using an in vitro model of polarizing murine neurons, we found that AS-p75 and p75^NTR display divergent expression profiles and that p75^NTR expression levels increase upon competition or depletion of AS-p75, indicating that AS-p75 is a negative regulator of p75^NTR expression. Depletion of AS-p75 also results in altered p75^NTR subcellular distribution and affects the polarization process. Overall, our data uncovered AS-p75 as a modulator of p75^NTR expression, offering new insights into the regulation of this neurotrophin receptor during in vitro neuronal polarization.

Keywords: Cellular neuroscience; Molecular neuroscience.

Graphical abstract

Figure 1

A previously uncharacterized Natural Antisense RNA AS-p75 is expressed in mouse cortices (A) Schematic representation of the p75NGFR gene structure and its antisense RNA AS-p75. Blue rectangles represent p75NGFR exons, and blue lines represent introns. The green rectangle depicts AS-p75, and the dark green portion shows the region of AS-p75 complementary to the 3^rd exon of the p75NGFR gene. (B) Representative image of the electrophoretic band representing the full-length AS-p75 product amplified by RT-PCR in mouse cortices (E17). The negative control reaction (−RT) was performed omitting the reverse transcriptase enzyme. (C) Nucleotide sequence of full-length AS-p75. The antisense region complementary to the 3^rd exon of p75NGFR is shown in bold, and the putative polyadenylation signal (AATATA) is underlined.

Figure 2

Expression profile of AS-p75 and p75^NTR during in vitro neuronal polarization (A) Schematic representation of in vitro neuronal polarization in cultured mouse embryonic neurons (modified from Arimura and Kaibuchi, 2007³⁷) showing axon specification at different stages. (B–D) Expression profile of (B) AS-p75 ncRNA, (C) p75^NTR mRNA, and (D) p75^NTR protein in cortical neurons at different time points of in vitro neuronal polarization. The right panel in (D) is a representative western blot analysis for p75^NTR (the predominant band in cortical neurons, during in vitro neuronal polarization, appears around 60 kDa) and GAPDH protein profiles during the differentiation time course; three biological replicates for each time point were loaded. The original representative uncropped image of the western blot experiment is reported in Figure S7. All samples were normalized to GAPDH mRNA or protein levels. n = 7 (B), n = 5 (C), and n = 6 (D), for each experimental group. Data are represented as mean ± SEM. Student’s t test with Bonferroni correction was used to analyze the differences between means: ∗p < 0.012, ∗∗p < 0.001, ∗∗∗p < 0.0001, ∗∗∗∗p < 0.00001; ns: not significant.

Figure 3

AS-p75 subcellular localization in polarizing neurons (A) AS-p75 expression profile in whole (Wh), nuclear (Nuc), and cytoplasmic (Cyt) compartments during in vitro neuronal polarization at different time points from plating, reported as a fraction of total AS-p75 levels. n = 4 for each experimental group. (B) Representative image of electrophoretic bands of semi-quantitative analysis of endogenous AS-p75 in Wh, Nuc, and Cyt compartments of differentiated neurons at 140 h from plating. p75NTR pre-mRNA and GAPDH mRNA were used as nuclear and cytoplasmic markers, respectively. Data are represented as mean ± SEM. Student’s t test was used to analyze the differences between means; ∗∗p < 0.01, ∗∗∗∗p < 0.0001; ns: not significant.

Figure 4

Inhibition of AS-p75 in neuronal progenitor cells (A) Schematic representation of ANTI-AS-p75 competition strategy. Exogenous ANTI-AS-p75 (light blue) sequesters AS-p75 (green) by annealing to its full length. (B and C) Expression profile of (B) p75^NTR mRNA and (C) p75^NTR protein levels after exogenous ANTI-AS-p75 expression in neuronal progenitor cells. Cells transfected with the pcDNA3.1 backbone plasmid served as control sample. (C) Representative image of a western blot analysis showing p75^NTR (the predominant bands in progenitor cells appear around 75–110 kDa), GAPDH, and GFP protein bands. The expression of exogenous ANTI-AS-p75 in these cells was confirmed by detection of the co-transfected GFP plasmid. n = 4 (B), n = 5 (C) for each experimental group. (D) Schematic representation of ASO1 and ASO2 sequences (red), which target the 3′ end of AS-p75 (green). The blue rectangle depicts the 3^rd exon of p75NGFR gene complementary to AS-p75. (E–G) Expression profile of (E) AS-p75 ncRNA, (F) p75^NTR mRNA, and (G) p75^NTR protein levels in neuronal progenitor cells upon AS-p75 depletion with ASO1 and ASO2, compared to a control ASO. (G) Representative image of a western blot analysis of three biological replicates for each experimental condition, showing p75^NTR (the predominant bands in progenitor cells appear around 75–110 kDa) and GAPDH proteins. n = 4 (E, F), n = 3 (G), for each experimental group. All samples were normalized to GAPDH mRNA or protein levels. The original representative uncropped images of the western blot experiments (C and G) are reported in Figure S7. Data are represented as mean ± SEM. Student’s t test was used to analyze the differences between means: ∗p < 0.05, ∗∗p < 0.01; ns: not significant.

Figure 5

Depletion of cytoplasmic AS-p75 by shRNAs (A) Schematic representation of shAS2 (red), which targets the 5′ end of AS-p75 (green). The region of the p75NGFR gene that encompasses AS-p75 is depicted with blue rectangles (exons) and blue lines (introns). (B–D) Expression profile of (B) AS-p75 ncRNA, (C) p75^NTR mRNA, and (D) p75^NTR protein in neuronal progenitor cells upon AS-p75 depletion with shAS2, compared to control shCtrl. (D) Representative western blot analysis of two biological replicates for each experimental condition, showing p75^NTR (the predominant bands in transduced progenitor cells appear around 75–110 kDa) and b-actin protein bands. n = 4 (B–D) for each experimental group. (E–G) Expression profile of (E) AS-p75 ncRNA, (F) p75^NTR mRNA, and (G) p75^NTR protein in cortical neurons at 72 h from plating after AS-p75 depletion with shAS2, compared to shCtrl. (G) Representative western blot analysis for p75^NTR (the predominant band in transduced cortical neurons appears around 70 kDa) and GAPDH protein bands. n = 4 (E, F), n = 6 (G), for each experimental group. All samples were normalized to GAPDH mRNA or protein levels. The original representative uncropped images of the western blot experiments (D and G) are reported in Figure S7. Data are represented as mean ± SEM. Student’s t test was used to analyze the differences between means: ∗p < 0.05, ∗∗p < 0.01; ns: not significant.

Figure 6

shRNA-mediated AS-p75 depletion affects p75^NTR polarization and axon specification (A) Confocal images of shCtrl or shAS2 expressing (green) cortical neurons at 72 h after plating. Smi-312 (red) and p75^NTR (white) signals are also shown. GFP⁺ neurons were analyzed to assess the presence or absence of an axon (Smi-312⁺), indicated with a yellow arrow. The blue arrow highlights a GFP⁺ shAS2-transduced neuron with no detectable Smi-312⁺ neurite. The pseudocolor plot in the inset of p75^NTR panel represents the percentage of p75^NTR expression in the soma. Scale bars represent 10 μm. (B) Percentage of shCtrl or shAS2 expressing neurons showing an axon (Smi-312⁺) or lacking an axon (Smi-312^-). A total of 22 neurons for shCtrl and 21 neurons for ShAS2 were analyzed. n = 2, for each experimental group. Data are represented as observed percentages. Statistical significance was calculated with Fisher’s exact test: ∗p < 0.05. (C) Percentage of shCtrl or shAS2expressing neurons with p75^NTR protein accumulated in one, multiple, or no neurites. For each group, a total of 19 neurons were analyzed, from two independent neuronal cultures. Data are represented as observed percentages. Statistical significance was calculated with Fisher’s exact test: ∗p < 0.05. (D and E) Mean p75^NTR intensity in (D) soma and (E) neurites of shCtrl or shAS2 expressing neurons. A total of 40 neurons for shCtrl and 35 neurons for ShAS2 were analyzed in (D) from three independent neuronal cultures. A total of 55 neurites for shCtrl and 60 neurites for shAS2 were analyzed in (E) from two independent neuronal cultures. Data are represented as mean ± SEM. Student’s t test was used to analyze the differences between means: ∗∗∗p < 0.001.