mSEL-1L (Suppressor/enhancer Lin12-like) protein levels influence murine neural stem cell self-renewal and lineage commitment

Abstract

Murine SEL-1L (mSEL-1L) is a key component of the endoplasmic reticulum-associated degradation pathway. It is essential during development as revealed by the multi-organ dysfunction and in uterus lethality occurring in homozygous mSEL-1L-deficient mice. Here we show that mSEL-1L is highly expressed in pluripotent embryonic stem cells and multipotent neural stem cells (NSCs) but silenced in all mature neural derivatives (i.e. astrocytes, oligodendrocytes, and neurons) by mmu-miR-183. NSCs derived from homozygous mSEL-1L-deficient embryos (mSEL-1L(-/-) NSCs) fail to proliferate in vitro, show a drastic reduction of the Notch effector HES-5, and reveal a significant down-modulation of the early neural progenitor markers PAX-6 and OLIG-2, when compared with the wild type (mSEL-1L(+/+) NSCs) counterpart. Furthermore, these cells are almost completely deprived of the neural marker Nestin, display a significant decrease of SOX-2 expression, and rapidly undergo premature astrocytic commitment and apoptosis. The data suggest severe self-renewal defects occurring in these cells probably mediated by misregulation of the Notch signaling. The results reported here denote mSEL-1L as a primitive marker with a possible involvement in the regulation of neural progenitor stemness maintenance and lineage determination.

FIGURE 1.

mSEL-1L expression in murine embryonic (ES46C), neural-induced (NEP46C), and NS46C mouse stem cells. A, ES46C cells, OCT-4-positive and Nestin-negative, after 7 days of exposure to neuralizing conditions, generated a heterogeneous population of neural progenitors (NEP46C), OCT-4-negative and Nestin-positive. Radial glial-like NSCs (NS46C) did not express OCT-4 but were characterized by a high Nestin immunoreactivity. The process of neuralization was distinguished by the persistence of mSEL-1L expression. Photomicrographs were acquired with Leica 60× objective. B, Western blot analysis of undifferentiated ES46C lysates revealed two forms of the mSEL-1L protein: 95 and 105 kDa. Although the 105-kDa form disappeared completely during the process of differentiation, the canonical 95-kDa form remained unaffected. β-Tubulin was used as protein loading control. C, Western blot analysis performed on total cell lysates during trilineage maturation showed the down-modulation followed by the disappearance of both mSEL-1L and Nestin during astrocyte (at 1, 4, and 7 days), oligodendrocyte (at 4 and 7 days), and neuronal (at 3 and 21 days) differentiation. β-Tubulin was used as protein loading control. D, NS46C cells cultured in proliferation medium were immunostained for mSEL-1L and Sox-2 and showed good expression of both markers in all cells. Upon exposure to appropriate differentiation conditions, astrocytes, oligodendrocytes, and neurons became mSEL-1L immunonegative. Photomicrographs were acquired with Leica 100× oil-immersion objective.

FIGURE 2.

mSEL-1L deficiency induces NSCs apoptosis, loss of stemness, and astrocyte differentiation. Embryonic day 11.5 mSEL-1L wild type (mSEL-1L^+/+) and heterozygous (mSEL-1L^+/−) mutant fetuses exhibited a normal brain morphology, whereas the homozygous mutant embryo (mSEL-1L^−/−) showed a reduced brain mass and thinning of the cerebral cortex. A and B, SCs directly derived from the telencephalic cortex of embryonic day 11.5 mSEL-1L^+/+ (A) and mSEL-1L^+/− (B) embryos showed immunopositivity for Nestin but not for the activated form of caspase III, whereas no or very few astrocytic, neuronal, and oligodendroglial differentiated cells were observed. The cell population expressed high levels of Sox-2. C, mSEL-1L^−/− NSCs were completely devoid of Nestin, but close to half of the cell population was Sox-2-immunopositive. Cells exhibited premature GFAP expression, no βIII-tubulin and CNPase staining, and a discrete rate of apoptosis. D, histogram represents the number of immunopositive cells scored in a total of five random fields for a total of 800 cells/slide for mSEL-1L^+/+ and mSEL-1L^+/− NSCs. For the mSEL-1L^−/− cell line, the entire population consisted of ∼100 cells. Although Nestin and Sox-2 were expressed by the entire mSEL-1L^+/+ and mSEL-1L^+/− cell population, the mSEL-1L-deficient line exhibited no or very few Nestin-positive cells and only 40% of SOX-2 immunopositivity. GFAP was expressed in 50% of mSEL-1L^−/− NSCs, but it was undetectable in both the wild type and heterozygous cell lines. Very few βIII-tubulin- and CNPase-expressing cells were observed in the three cell lines. Unlike wild type and heterozygous cell lines, 50% of mSEL-1L^−/− cells displayed the cleaved form of caspase III. Photomicrographs were acquired with a Leica 60× objective. E, HES-5, PAX-6, and OLIG-2 expression in mSEL-1L^+/+, mSEL-1L^+/−, and mSEL-1L^−/− NSCs was analyzed by qRT-PCR, using SYBR-green detection. HES-5 levels were drastically affected by mSEL-1L depletion, whereas PAX-6 and OLIG-2 expression was only partially down-modulated. The experiments were performed in triplicate, the results were normalized for GAPDH expression using the ΔΔC_t method, and the standard deviation was calculated.

FIGURE 3.

mSEL-1L^+/− NSCs preferentially differentiate in astrocytes and exhibit impairment of oligodendroglial commitment. A, mSEL-1L^+/+ NSCs were nucleofected with 250 pmol of siRNA against mSEL-1L and of a negative control. After 48 h, mSEL-1L and GFAP expression was evaluated by qRT-PCR. The experiments were performed in triplicate, the results were normalized for GAPDH expression using the ΔΔC_t method, and the standard deviation was calculated. mSEL-1L down-modulation was associated to GFAP overexpression. B, the histogram represents mSEL-1L and GFAP expression in mSEL-1L^+/− NSCs, related to the wild type line. The analysis was performed by qRT-PCR using SYBR-green detection method in triplicate, and the data were normalized to GAPDH expression, using the ΔΔC_t method. The heterozygous cells obviously exhibited low levels of mSEL-1L and were characterized by an increased expression of GFAP when compared with control cells. C and D, mSEL-1L^+/+ and mSEL-1L^+/− NSCs, when subjected to neuronal differentiation, generated approximately the same number of βIII-tubulin immunopositive cells. E and F, when induced to astrocytic maturation, the heterozygous cells were more GFAP immunoreactive than the wild type differentiated cells. G and H, during oligodendroglial maturation, the wild type population produced more O4-expressing cells with respect to mSEL-1L^+/− NSCs. Photomicrographs were acquired with Leica 60 × objective. I, histogram represents the number of immunopositive cells scored in five random fields of ∼600 mSEL-1L^+/+ and mSEL-1L^+/− NSCs/slide, and the standard deviation was calculated. GFAP intense immunopositive cells represented ∼10 and 20% of the astrocyte differentiated wild type and heterozygous cell lines, respectively. During oligodendroglial maturation mSEL-1L^+/+ NSCs originated 3-fold more O4-positive cells than those detected in mSEL-1L^+/−. The number of βIII-tubulin-positive neurons was approximately the same in mSEL-1L^+/+ and mSEL-1L^+/− cell lines.

FIGURE 4.

mSEL-1L protein levels inversely correlate with mmu-miR-183 expression. A, RT-PCR showed that during NS46C trilineage maturation, whereas GFAP and βIII-tubulin mRNAs were up-regulated, mSEL-1L expression did not change during astrocyte (7 days), oligodendrocyte (7 days), and neuron (21 days) maturation. GAPDH was used as loading control. B, TaqMan assay performed on the same differentiated cells described in A showed an increased expression of mmu-miR-183 when compared with undifferentiated cells. The values were normalized relatively to snoRNA202 levels; the data were presented as the averages of three independent experiments. C, dissected tissue fragments collected from different adult mouse brain areas (olfactory bulbs, hippocampus, thalamus, and cortex) were analyzed for mmu-miR-183 expression by specific TaqMan assay, using snoRNA202 as normalizer; the microRNA was highly expressed in all adult brain sections. The data reported the averages of three independent experiments and are presented as fold increase compared with undifferentiated cells. For all the experiments, the standard deviation was calculated, and the significant differences were indicated with asterisks (p < 0.05, t test). D, mSEL-1L 95-kDa protein expression detected in the same adult brain areas described for C did not reflect the mRNA levels (E) and was expressed only in undifferentiated NS cells. β-Tubulin was used as protein loading control. mSEL-1L transcript was evaluated in the brain total RNA by RT-PCR, using actin as a housekeeping gene. F, the histogram represents the expression levels of mmu-miR-183 in mSEL-1L^+/− NSCs compared with the wild type cell line. The same experiment was performed in triplicate by TaqMan assay, using snoRNA202 as normalizer.

FIGURE 5.

mmu-miR-183 overexpression induces mSEL-1L protein decrease and a premature GFAP expression. NS46C cells were nucleofected with pre-miR-183 and pre-miR-negative control. A, after 48 h, close to 70% reduction in mSEL-1L protein levels was observed in pre-miR-183 transfected cells, as assayed by Western blot analysis. B, histogram shows data obtained from qRT-PCR. pre-miR-183 overexpression did not affect mSEL-1L transcript levels, as well as SOX-2 and βIII-tubulin mRNAs, but determined an increase of ∼2.5-fold of GFAP messenger and a significant down-modulation of PAX-6 and OLIG-2 levels. Gene expression was analyzed using the SYBR-green detection method. The experiments were performed in triplicate, and the data were normalized to GAPDH expression using the ΔΔC_t method and were compared with values obtained from negative control nucleofected cells. The standard deviation was appositively calculated. C, NS46C overexpressing pre-miR-183 exhibited more cells positive for the activated form of the caspase III when compared with pre-miR-negative control transfected cells. Pre-miR-183 and pre-miR-negative control nucleofected cells showed the same expression of Nestin and Sox-2. D, βIII-tubulin and especially GFAP-positive cells increased in mmu-miR-183 overexpressing population, whereas no CNPase-positive cells were detected. E, histogram represents the number of immunopositive cells scored in a total of five random fields of ∼1000 cells/slide, and the standard deviation was calculated. Although Nestin and Sox-2 were not affected by pre-miR-183 nucleofection, the number of βIII-tubulin cells increased ∼2-fold, and the cells positive for the cleaved form of the caspase III were augmented 3-fold. Interestingly, GFAP immunopositive cells increased up to 4-fold in pre-miR-183-overexpressing population. All of the experiments were performed in triplicate. Photomicrographs were acquired with Leica 60× objective.