Neutralization of LINGO-1 during in vitro differentiation of neural stem cells results in proliferation of immature neurons

Abstract

Identifying external factors that can be used to control neural stem cells division and their differentiation to neurons, astrocytes and oligodendrocytes is of high scientific and clinical interest. Here we show that the Nogo-66 receptor interacting protein LINGO-1 is a potent regulator of neural stem cell maturation to neurons. LINGO-1 is expressed by cortical neural stem cells from E14 mouse embryos and inhibition of LINGO-1 during the first days of neural stem cell differentiation results in decreased neuronal maturation. Compared to neurons in control cultures, which after 6 days of differentiation have long extending neurites, neurons in cultures treated with anti-LINGO-1 antibodies retain an immature, round phenotype with only very short processes. Furthermore, neutralization of LINGO-1 results in a threefold increase in βIII tubulin-positive cells compared to untreated control cultures. By using BrdU incorporation assays we show that the immature neurons in LINGO-1 neutralized cultures are dividing neuroblasts. In contrast to control cultures, in which no cells were double positive for βIII tubulin and BrdU, 36% of the neurons in cultures treated with anti-LINGO-1 antibodies were proliferating after three days of differentiation. TUNEL assays revealed that the amount of cells going through apoptosis during the early phase of differentiation was significantly decreased in cultures treated with anti-LINGO-1 antibodies compared to untreated control cultures. Taken together, our results demonstrate a novel role for LINGO-1 in neural stem cell differentiation to neurons and suggest a possibility to use LINGO-1 inhibitors to compensate for neuronal cell loss in the injured brain.

Figure 1. LINGO-1 expression increases during neural stem cell differentiation.

A) Western blot analysis was used to study LINGO-1 expression during NSPC differentiation. Cell lysates from proliferating cells (Day 0) and NSPCs differentiating for 1–9 days was immunoprecipitated with anti-LINGO-1 antibodies (Novartis) and blotted with LINGO-1 ab (Abcam). LINGO-1 is present in both proliferating and differentiated NSPCs, but the expression increases during the differentiation. B) Protein quantification show a 9-fold increase in LINGO-1 expression in cultures differentiated for 9 days compared to proliferating NSPCs (Day 0). Double immunostainings with specific antibodies against LINGO-1 (C–N) and nestin (C–E), βIIItubulin (F–H), GFAP (I–K) or CNPase (L–N) show that NSPCs, neurons and oligodendrocytes express LINGO-1, but not astrocytes. The NSPC cultures were fixed at day 0 (C–E) and the differentiated cultures were fixed at day 6 after mitogen withdrawal (F–N). Scale bars = 20 µm.

Figure 2. LINGO-1 neutralization during NSCP differentiation results in immature neurons.

To investigate the effect of LINGO-1 neutralization on NSPC differentiation, NSPCs were cultured for 6 days in the absence (A, C and E) or presence (B, D and F) of LINGO-1 ab (Novartis) following mitogen withdrawal. The cells were fixed and stained with specific antibodies against βIIItubulin (A–B), GFAP (C–D) or CNPase (E–F). In control cultures (A) neurons were rather mature with multiple, long extending processes, but in cultures treated with LINGO-1 antibodies (B) the neurons had a more immature phenotype with only one or two short processes. There was no distinct difference in astrocyte staining (C–D) or oligodrocyte staining (E–F) between control cultures and LINGO-1 inhibited cultures. Scale bars = 20 µm.

Figure 3. Neutralization of LINGO-1 leads to an increased percentage of neurons.

A) The number of neurons, astrocytes and oligodendrocytes was counted and plotted as the ratio of specific marker-positive cells to the total cell number (DAPI). After 6 days of differentiation there was a 3-fold increase in the percentage of neurons in LINGO-1 ab treated cultures compared to untreated controls. There was only a modest, but significant, increase in the percentage of GFAP positive cells and no difference was found in the percentage of CNPase positive cells. B) Comparison of the percentage of mature and immature neurons of the total number of βIIItubulin positive cells show a 7-fold increase of immature cells in LINGO-1 ab treated cultures compared to untreated control cultures after 6 days of differentiation. *** denotes p<0,001, ** denotes p<0,01 and * denotes p<0,05.

Figure 4. LINGO-1 neutralization promotes cell proliferation.

BrdU labeling was used to investigate the effect of LINGO-1 neutralization on cell proliferation (A–D). Cells were exposed to BrdU for 16 hours prior to fixation and stained with specific antibodies against BrdU (red) and DAPI (blue). E) The number of cells that had incorporated BrdU was counted and plotted as the ratio of BrdU-positive cells to the total cell count. F) The total cell number in anti-LINGO-1 antibody treated and untreated control cultures were measured at day 0, 1, 3 and 6 days of differentiation. The cells in each dish were harvested using a cell scraper and counted using a NucleoCounter™. The mean values of the total cell count/dish were plotted. *** denotes p<0,001, ** denotes p<0,01 and * denotes p<0,05 and scale bars = 20 µm.

Figure 5. Neutralization of LINGO-1 leads to an increased percentage of proliferating neuroblasts.

To investigate if the immature neurons in LINGO-1 neutralized cultures proliferate, we double-labeled the cells with specific antibodies against BrdU (red), βIIItubulin (green) and DAPI (blue). NSPCs were fixed at day 0 (A–B) or differentiated in only medium for 3 days (C–D) or for 6 days (E–F) medium containing anti-LINGO-1 antibodies (G–J). BrdU was added to the cultures 16 hours prior to fixation. Scale bars = 20 µm.

Figure 6. Decreased number of TUNEL positive cells in cultures treated with anti-LINGO-1 antibodies.

TUNEL assay was performed on NSPC and parallel cultures of cells differentiated for 1 and 3 days in the absence or presence of LINGO-1 ab (A–D). Representative photos of control cultures (A–B) and LINGO-1 neutralized cultures (C–D) at day 3 of differentiation, TUNEL (green) and DAPI (blue). E) Cells going through apoptosis were counted and plotted as the ratio of the total number of cells. *** denotes p<0,001, ** denotes p<0,01 and * denotes p<0,05 and scale bars = 20 µm.

Figure 7. LINGO-1 neutralization has no effect on PKB/c-Akt phosphorylation.

A) Western blot analysis was used to study PKB/c-Akt phosphorylation in differentiating NSPCs in the absence or presence of LINGO-1 ab. Total cell lysates were used for immunoblotting with anti-PKB/c-Akt antibody (Akt) and anti-phosphorylated PKB/c-Akt antibody (p-Akt). B) The ratio of phosphorylated Akt was measured and plotted.