Temporal downregulation of the polyubiquitin gene Ubb affects neuronal differentiation, but not maturation, in cells cultured in vitro

Abstract

Reduced levels of cellular ubiquitin (Ub) pools due to disruption of the polyubiquitin gene Ubb lead to dysregulation of neural stem cell (NSC) differentiation and impaired neuronal maturation in cells isolated from Ubb ^-/- mouse embryonic brains. However, it is currently unknown whether Ub is required for the specific stage of neuronal development or whether it plays a pleiotropic role throughout the process. To answer this question, we aimed to downregulate Ubb expression temporally during neuronal development, which could not be achieved in Ubb ^-/- cells. Therefore, we exploited lentivirus-mediated knockdown (KD) of Ubb at different stages of neuronal development, and investigated their phenotypes. Here, we report the outcome of Ubb KD on two independent culture days in vitro (DIV): DIV1 and DIV7. We observed that NSCs did not differentiate properly via Ubb KD on DIV1, but the maturation of already differentiated neurons was intact via Ubb KD on DIV7. Intriguingly, Ubb KD activated Notch signaling when it had been suppressed, but exerted no effect when it had already been activated. Therefore, our study suggests that Ub plays a pivotal role in NSC differentiation to suppress Notch signaling, but not in the subsequent maturation stages of neurons that had already been differentiated.

Figure 1

No effect on the exit from NSC’s multipotency during culture in vitro via Ubb KD on DIV1. (a) Cells isolated from embryonic brains on 14.5 dpc (n = 3) were infected with lentivirus harboring scrambled RNA (−shUbb) or Ubb shRNA (+shUbb) on DIV1 and cultured for another 2 to 8 days (DIV3 to DIV9). Cells were fixed and subjected to immunofluorescence analysis using NSC marker nestin. DNA was visualized with DAPI. (b) To determine the percentage of nestin+ cell populations, the number of nestin+ cells was divided by the number of DAPI+ cells in three randomly selected fields for each sample (n = 3). For each sample, more than 50 DAPI+ cells were counted (>200 cells total). (c) Cells were infected with lentivirus harboring scrambled RNA (−shUbb) or Ubb shRNA (+shUbb) on DIV1 and cultured for another 4 days (DIV5). Ubb and Ubc mRNA levels in control (−shUbb) and Ubb KD (+shUbb) cells were determined by qRT-PCR (n = 3 each), normalized against Gapdh levels, and expressed as a fold change relative to control levels. Representative images of cells from three different embryonic brains are shown (a), and data are expressed as the means ± SEM from the indicated number of samples (b,c). ^**P < 0.01 between two groups as indicated by horizontal bars. NS, not significant. Scale bar, 50 μm.

Figure 2

Recapitulation of Ubb^−/− neuronal phenotypes in cells cultured in vitro via Ubb KD on DIV1. (a) Immunoblot detection of Ub conjugates (Ub_n), free Ub (Ub₁), neuronal marker Tuj1, and apoptotic marker CC3 in cells infected with lentivirus harboring scrambled RNA (−shUbb) or Ubb shRNA (+shUbb) on DIV1 and cultured for another 4 or 9 days (DIV5 or DIV10). β-Actin (β-Act) was used as a loading control. (b,c) Cells were infected with lentivirus harboring scrambled RNA (−shUbb) or Ubb shRNA (+shUbb) on DIV1 and cultured for another 2 to 11 days (DIV3 to DIV12). Cells were fixed and subjected to immunofluorescence analysis using neuronal marker Tuj1, glial marker GFAP, and apoptotic marker CC3. DNA was visualized with DAPI. (d) Cells were infected with lentivirus harboring scrambled RNA (−shUbb) or Ubb shRNA (+shUbb), and Ub (+Ub) or an empty lentiviral vector (−Ub), on DIV 1 and cultured for another 17 days (DIV18). On DIV18, cells were subjected to immunofluorescence analysis using neuronal marker Tuj1 and DNA was visualized with DAPI. (e) The percentage of GFAP+ or Tuj1+ cell populations (n = 3) was determined in a similar manner as described in Fig. 1(b). Representative images or immunoblot results of cells from three different embryonic brains are shown (a–d), and data are expressed as the means ± SEM from the indicated number of samples (e). ^**P < 0.01; ^***P < 0.001 between two groups as indicated by horizontal bars. NS, not significant. Scale bars, 50 μm.

Figure 3

Observation of neuronal maturation in cells cultured in vitro via Ubb KD on DIV7. (a) Immunoblot detection of Ub conjugates (Ub_n), free Ub (Ub₁), neuronal marker Tuj1, and apoptotic marker CC3 in cells infected with lentivirus harboring scrambled RNA (−shUbb) or Ubb shRNA (+shUbb) on DIV7 and cultured for another 4 or 9 days (DIV11 or DIV16). β-Actin (β-Act) was used as a loading control. (b) Cells were infected with lentivirus harboring scrambled RNA (−shUbb) or Ubb shRNA (+shUbb) on DIV7 and cultured for another 4 days (DIV11). Ubb and Ubc mRNA levels in control (−shUbb) and Ubb KD (+shUbb) cells were determined by qRT-PCR (n = 3 each), normalized against Gapdh levels, and expressed as a fold change relative to control levels. (c) Cells were infected with lentivirus harboring scrambled RNA (−shUbb) or Ubb shRNA (+shUbb) on DIV7, cultured for another 11 days (DIV18), and subjected to immunofluorescence analysis using neuronal marker Tuj1, glial marker GFAP, and neuronal maturation marker MAP2. DNA was visualized with DAPI. (d) The percentage of GFAP+, MAP2+, or Tuj1+ cell populations (n = 3) was determined in a similar manner as described in Fig. 1(b). Representative images or immunoblot results of cells from three different embryonic brains are shown (a,c), and data are expressed as the means ± SEM from the indicated number of samples (b,d). ^***P < 0.001 between two groups as indicated by horizontal bars. NS, not significant. Scale bar, 50 μm.

Figure 4

Activation of Notch signaling and its consequences via Ubb KD on DIV1. (a,d,e) Cells isolated from embryonic brains on 14.5 dpc (n = 3) were infected with lentivirus harboring scrambled RNA (−shUbb) or Ubb shRNA (+shUbb) on DIV1 and cultured for another 4 days (DIV5). Hes5, Hey1, NeuroD1, and Lcn2 mRNA levels in control (−shUbb) and Ubb KD (+shUbb) cells were determined by qRT-PCR (n = 3 each), normalized against Gapdh levels, and expressed as a fold change relative to control levels. (b) Cells were infected with lentivirus harboring scrambled RNA (−shUbb) or Ubb shRNA (+shUbb) on DIV1, treated with 10 μM DAPT on DIV4 to repress Notch signaling, and cultured for another 3 days (DIV7). Hes5 and Hey1 mRNA levels were determined by qRT-PCR (n = 3 each), normalized against Gapdh levels, and expressed as a fold change relative to control (−shUbb, −DAPT) levels. (c) Immunoblot detection of cleaved NICD in cells infected with lentivirus harboring scrambled RNA (−shUbb) or Ubb shRNA (+shUbb) on DIV1 and cultured for another 4 days (DIV5). β-Actin (β-Act) was used as a loading control. To prevent potential degradation of NICD, cells were also treated with 10 μM MG-132 for 2 hrs (+MG-132) before harvest on DIV5. (f,g,h) Cells isolated from embryonic brains on 14.5 dpc (n = 3) were infected with lentivirus harboring scrambled RNA (−shUbb), Ubb shRNA (+shUbb), NICD1 (+NICD1), or an empty lentiviral vector (−NICD1) on DIV1 and cultured for another 4 days (DIV5). Hes5, Hey1, Tubb3, and Lcn2 mRNA levels in control (−shUbb or −NICD1), Ubb KD (+shUbb), and NICD1-overexpressing (+NICD1) cells were determined by qRT-PCR (n = 3 each), normalized against Gapdh levels, and expressed as a fold change relative to control levels. Representative immunoblot results of cells from three different embryonic brains are shown (c), and data are expressed as the means ± SEM from the indicated number of samples (a,b,d–h). ^#P < 0.1; ^*P < 0.05; ^**P < 0.01; ^***P < 0.001 between two groups as indicated by horizontal bars.

Figure 5

No effect on already activated Notch signaling via Ubb KD on DIV7. (a,c,d) Cells isolated from embryonic brains on 14.5 dpc (n = 3) were infected with lentivirus harboring scrambled RNA (−shUbb) or Ubb shRNA (+shUbb) on DIV7 and cultured for another 4 days (DIV11). Hes5, Hey1, NeuroD1, and Lcn2 mRNA levels in control (−shUbb) and Ubb KD (+shUbb) cells were determined by qRT-PCR (n = 3 each), normalized against Gapdh levels, and expressed as a fold change relative to control levels. (b) Cells isolated from embryonic brains on 14.5 dpc (n = 3) were infected with lentivirus harboring scrambled RNA (−shUbb) or Ubb shRNA (+shUbb) on DIV1 or DIV7 and cultured for another 4 days (DIV5 or DIV11). Hes5 mRNA levels in control (−shUbb) and Ubb KD (+shUbb) cells on DIV5 and DIV11 were determined by qRT-PCR (n = 3 each), normalized against Gapdh levels, and expressed as a fold change relative to control levels on DIV5. All data are expressed as the means ± SEM from the indicated number of samples. ^**P < 0.01 between two groups as indicated by horizontal bars. NS, not significant.