The retinoblastoma protein is essential for survival of postmitotic neurons

Abstract

The retinoblastoma protein (Rb) family members are essential regulators of cell cycle progression, principally through regulation of the E2f transcription factors. Growing evidence indicates that abnormal cell cycle signals can participate in neuronal death. In this regard, the role of Rb (p105) itself has been controversial. Germline Rb deletion leads to massive neuronal loss, but initial reports argue that death is non-cell autonomous. To more definitively resolve this question, we generated acute murine knock-out models of Rb in terminally differentiated neurons in vitro and in vivo. Surprisingly, we report that acute inactivation of Rb in postmitotic neurons results in ectopic cell cycle protein expression and neuronal loss without concurrent induction of classical E2f-mediated apoptotic genes, such as Apaf1 or Puma. These results suggest that terminally differentiated neurons require Rb for continuous cell cycle repression and survival.

Figure 1.

Acute Rb removal results in neuronal apoptosis independent of classical E2f-regulated apoptotic genes. A, Cortical neurons were fixed at the indicated DIV and condensed nuclei were examined by DAPI staining (n = 3). B, Western blot analysis of total protein extracted from control and Rb-deficient cortical neurons at the indicated DIV. C, Cortical neurons were infected with the indicated constructs, and condensed nuclei were examined by DAPI staining at 8 DIV (n = 3). D, Western blots on total protein extracted at 6 DIV. E, qRT-PCR on RNA extracted at 6 DIV. RNA levels were normalized to GAPDH (n = 3). *p < 0.05,***p < 0.001. Error bars indicate SEM.

Figure 2.

Loss of Rb upregulates genes associated with cell cycle function. A, Heat map representing top 200 genes significantly upregulated by microarray upon acute Rb deletion (fold increase of ≥1.5, FDR <5%, n = 3). B, Gene functional classification of upregulated transcripts following Rb loss. C, qRT-PCR validation of targets identified by microarray analysis. D, Analysis of distribution of cell cycle regulatory genes induced upon Rb deletion. Error bars indicate SEM.

Figure 3.

Rb-deficient neurons display cell cycle and DNA damage markers. A, B, Cortical neurons were fixed at 8 DIV and stained for Ki67. C, Western blots were performed on total protein extracted at 8 DIV. D, E, Cortical neurons were fixed at the indicated DIV and quantified for the percentage of double labeling (Ki67⁺/GFP+ over GFP+, γ-H2AX/GFP+ over GFP+) (n = 3 or 4). **p < 0.01,***p < 0.001. Scale bar, 50 μm. Error bars indicate SEM.

Figure 4.

Acute Rb removal in adult neurons triggers neuronal loss. A, TAM injection paradigm. B, Western blot of cortex lysates confirming the decrease of Rb levels in CamKCreERT2; Rb^flox/floxmice (Rb^f/f) 1 week after the last TAM injection. C, Rb loss results in neuronal loss in the cerebral cortex 4 weeks following TAM treatment (n = 3). D, E, Representative pictures of NeuN immunofluorescence displaying loss of neurons in the cortex of CamKCreERT2; Rb^flox/floxmice (E) compared with CamKCreERT2; Rb^flox/+mice (D). F, G, Ectopic expression of the proliferative marker Ki67 in NeuN⁺ neurons (arrows) of CamKCreERT2; Rb^flox/flox mice 1 week after the last TAM injection. H, Ectopically proliferating Ki67⁺ cells exhibit γ-H2AX immunoreactivity (arrows) following Rb deficiency. **p < 0.01,***p < 0.001. Scale bars: D, E, 100 μm; F–H, 50 μm. Error bars indicate SEM.