Limits...
In utero gene therapy rescues microcephaly caused by Pqbp1-hypofunction in neural stem progenitor cells.

Ito H, Shiwaku H, Yoshida C, Homma H, Luo H, Chen X, Fujita K, Musante L, Fischer U, Frints SG, Romano C, Ikeuchi Y, Shimamura T, Imoto S, Miyano S, Muramatsu SI, Kawauchi T, Hoshino M, Sudol M, Arumughan A, Wanker EE, Rich T, Schwartz C, Matsuzaki F, Bonni A, Kalscheuer VM, Okazawa H - Mol. Psychiatry (2014)

Bottom Line: Human mutations in PQBP1, a molecule involved in transcription and splicing, result in a reduced but architecturally normal brain.Exogenous Apc4, a hub protein in the network of affected genes, recovered the cell cycle, proliferation, and cell phenotypes of NSPCs caused by Pqbp1-cKO.These data reveal a mechanism of brain size control based on the simple reduction of the NSPC pool by cell cycle time elongation.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuropathology, Medical Research Institute and Center for Brain Integration Research, Tokyo Medical and Dental University, Tokyo, Japan.

ABSTRACT
Human mutations in PQBP1, a molecule involved in transcription and splicing, result in a reduced but architecturally normal brain. Examination of a conditional Pqbp1-knockout (cKO) mouse with microcephaly failed to reveal either abnormal centrosomes or mitotic spindles, increased neurogenesis from the neural stem progenitor cell (NSPC) pool or increased cell death in vivo. Instead, we observed an increase in the length of the cell cycle, particularly for the M phase in NSPCs. Corresponding to the developmental expression of Pqbp1, the stem cell pool in vivo was decreased at E10 and remained at a low level during neurogenesis (E15) in Pqbp1-cKO mice. The expression profiles of NSPCs derived from the cKO mouse revealed significant changes in gene groups that control the M phase, including anaphase-promoting complex genes, via aberrant transcription and RNA splicing. Exogenous Apc4, a hub protein in the network of affected genes, recovered the cell cycle, proliferation, and cell phenotypes of NSPCs caused by Pqbp1-cKO. These data reveal a mechanism of brain size control based on the simple reduction of the NSPC pool by cell cycle time elongation. Finally, we demonstrated that in utero gene therapy for Pqbp1-cKO mice by intraperitoneal injection of the PQBP1-AAV vector at E10 successfully rescued microcephaly with preserved cortical structures and improved behavioral abnormalities in Pqbp1-cKO mice, opening a new strategy for treating this intractable developmental disorder.

Show MeSH

Related in: MedlinePlus

Nestin-Cre Pqbp1-cKO (conditional Pqbp1-knockout) delays the cell cycle but does not affect neurogenesis of neural stem progenitor cells (NSPCs). (a) The M phase was specifically elongated in NSPCs of Pqbp1-cKO mice (Nes-Cre; XFloxY) in vivo. Cumulative labeling of NSPCs at E14 in vivo showed an increase in the total cell cycle length (Tc) of +2.2 h, +12% (upper left panel). G2/M phase time was evaluated using phosphorylated histone H3 (upper right panel). The middle panels show immunostaining data corresponding to the upper panels. pH3+/BrdU+ cells were reduced in number in cKO embryos (arrows), indicating elongation of the G2/M phase (middle right panel). The summary of cumulative labeling and G2/M analyses is shown in the lower table. A significant extension of the G2/M phase (+67%) and a slight extension of G1 (+6%) were observed. The crossing point between the plot line and the x-axis indicates the length of G2 phase (upper right panel). Thus, the M phase was remarkably elongated while the length of the G2 phase was unchanged in Pqbp1-cKO mice. (b) Neurogenesis from the stem cell pool was analyzed by co-staining for BrdU and Ki67. At 12 or 72 h after intraperitoneal injection of BrdU, E15 embryonic brains were analyzed to calculate the numbers of cells after neurogenesis (BrdU+/Ki67−), cells remaining in the stem cell pool (BrdU+/Ki67+) and non-labeled stem/progenitor cells (BrdU−/Ki67+). The bar graphs show the relative percentages of the three groups (left graph) and the neurogenesis percentage of BrdU-labeled cells (right graph). No difference was detected at 24 and 72 h after BrdU injection by the Student's t-test or Welch's t-test. (c) Levels of cell death in the cerebral cortex were evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining at E10, E15, E18, P0 and P60 of Pqbp1-cKO mice (Nes-Cre; XFloxY) and nestin-Cre mice (Nes-Cre; XY). Quantitative analysis of apoptotic cells did not reveal any differences (Student's t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4378255&req=5

fig2: Nestin-Cre Pqbp1-cKO (conditional Pqbp1-knockout) delays the cell cycle but does not affect neurogenesis of neural stem progenitor cells (NSPCs). (a) The M phase was specifically elongated in NSPCs of Pqbp1-cKO mice (Nes-Cre; XFloxY) in vivo. Cumulative labeling of NSPCs at E14 in vivo showed an increase in the total cell cycle length (Tc) of +2.2 h, +12% (upper left panel). G2/M phase time was evaluated using phosphorylated histone H3 (upper right panel). The middle panels show immunostaining data corresponding to the upper panels. pH3+/BrdU+ cells were reduced in number in cKO embryos (arrows), indicating elongation of the G2/M phase (middle right panel). The summary of cumulative labeling and G2/M analyses is shown in the lower table. A significant extension of the G2/M phase (+67%) and a slight extension of G1 (+6%) were observed. The crossing point between the plot line and the x-axis indicates the length of G2 phase (upper right panel). Thus, the M phase was remarkably elongated while the length of the G2 phase was unchanged in Pqbp1-cKO mice. (b) Neurogenesis from the stem cell pool was analyzed by co-staining for BrdU and Ki67. At 12 or 72 h after intraperitoneal injection of BrdU, E15 embryonic brains were analyzed to calculate the numbers of cells after neurogenesis (BrdU+/Ki67−), cells remaining in the stem cell pool (BrdU+/Ki67+) and non-labeled stem/progenitor cells (BrdU−/Ki67+). The bar graphs show the relative percentages of the three groups (left graph) and the neurogenesis percentage of BrdU-labeled cells (right graph). No difference was detected at 24 and 72 h after BrdU injection by the Student's t-test or Welch's t-test. (c) Levels of cell death in the cerebral cortex were evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining at E10, E15, E18, P0 and P60 of Pqbp1-cKO mice (Nes-Cre; XFloxY) and nestin-Cre mice (Nes-Cre; XY). Quantitative analysis of apoptotic cells did not reveal any differences (Student's t-test).

Mentions: Hence, we first evaluated the cell cycle time of NSPCs in vivo using the cumulative labeling method.23 The data showed that the total cell cycle time (Tc) was increased (+2.2 h, +12%) (Figure 2a). The S phase (Ts) was unaltered compared to the strict control nestin-Cre XY mice (Figure 2a). The G1 phase was slightly longer (Figure 2a). However, G1 elongation in the Pqbp1-cKO was minimal (5.2%) compared to other reports of cell cycle length regulating brain size.33,34 G2 phase time was not changed (Figure 2a). In contrast to the G1, S, and G2 phases, the M phase was remarkably increased (Figure 2a) and the increase in Tc was principally due to a longer M phase (+1.4 h, +67%). This cell cycle change in Pqbp1-cKO mice is quite unique among various disease models.


In utero gene therapy rescues microcephaly caused by Pqbp1-hypofunction in neural stem progenitor cells.

Ito H, Shiwaku H, Yoshida C, Homma H, Luo H, Chen X, Fujita K, Musante L, Fischer U, Frints SG, Romano C, Ikeuchi Y, Shimamura T, Imoto S, Miyano S, Muramatsu SI, Kawauchi T, Hoshino M, Sudol M, Arumughan A, Wanker EE, Rich T, Schwartz C, Matsuzaki F, Bonni A, Kalscheuer VM, Okazawa H - Mol. Psychiatry (2014)

Nestin-Cre Pqbp1-cKO (conditional Pqbp1-knockout) delays the cell cycle but does not affect neurogenesis of neural stem progenitor cells (NSPCs). (a) The M phase was specifically elongated in NSPCs of Pqbp1-cKO mice (Nes-Cre; XFloxY) in vivo. Cumulative labeling of NSPCs at E14 in vivo showed an increase in the total cell cycle length (Tc) of +2.2 h, +12% (upper left panel). G2/M phase time was evaluated using phosphorylated histone H3 (upper right panel). The middle panels show immunostaining data corresponding to the upper panels. pH3+/BrdU+ cells were reduced in number in cKO embryos (arrows), indicating elongation of the G2/M phase (middle right panel). The summary of cumulative labeling and G2/M analyses is shown in the lower table. A significant extension of the G2/M phase (+67%) and a slight extension of G1 (+6%) were observed. The crossing point between the plot line and the x-axis indicates the length of G2 phase (upper right panel). Thus, the M phase was remarkably elongated while the length of the G2 phase was unchanged in Pqbp1-cKO mice. (b) Neurogenesis from the stem cell pool was analyzed by co-staining for BrdU and Ki67. At 12 or 72 h after intraperitoneal injection of BrdU, E15 embryonic brains were analyzed to calculate the numbers of cells after neurogenesis (BrdU+/Ki67−), cells remaining in the stem cell pool (BrdU+/Ki67+) and non-labeled stem/progenitor cells (BrdU−/Ki67+). The bar graphs show the relative percentages of the three groups (left graph) and the neurogenesis percentage of BrdU-labeled cells (right graph). No difference was detected at 24 and 72 h after BrdU injection by the Student's t-test or Welch's t-test. (c) Levels of cell death in the cerebral cortex were evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining at E10, E15, E18, P0 and P60 of Pqbp1-cKO mice (Nes-Cre; XFloxY) and nestin-Cre mice (Nes-Cre; XY). Quantitative analysis of apoptotic cells did not reveal any differences (Student's t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4378255&req=5

fig2: Nestin-Cre Pqbp1-cKO (conditional Pqbp1-knockout) delays the cell cycle but does not affect neurogenesis of neural stem progenitor cells (NSPCs). (a) The M phase was specifically elongated in NSPCs of Pqbp1-cKO mice (Nes-Cre; XFloxY) in vivo. Cumulative labeling of NSPCs at E14 in vivo showed an increase in the total cell cycle length (Tc) of +2.2 h, +12% (upper left panel). G2/M phase time was evaluated using phosphorylated histone H3 (upper right panel). The middle panels show immunostaining data corresponding to the upper panels. pH3+/BrdU+ cells were reduced in number in cKO embryos (arrows), indicating elongation of the G2/M phase (middle right panel). The summary of cumulative labeling and G2/M analyses is shown in the lower table. A significant extension of the G2/M phase (+67%) and a slight extension of G1 (+6%) were observed. The crossing point between the plot line and the x-axis indicates the length of G2 phase (upper right panel). Thus, the M phase was remarkably elongated while the length of the G2 phase was unchanged in Pqbp1-cKO mice. (b) Neurogenesis from the stem cell pool was analyzed by co-staining for BrdU and Ki67. At 12 or 72 h after intraperitoneal injection of BrdU, E15 embryonic brains were analyzed to calculate the numbers of cells after neurogenesis (BrdU+/Ki67−), cells remaining in the stem cell pool (BrdU+/Ki67+) and non-labeled stem/progenitor cells (BrdU−/Ki67+). The bar graphs show the relative percentages of the three groups (left graph) and the neurogenesis percentage of BrdU-labeled cells (right graph). No difference was detected at 24 and 72 h after BrdU injection by the Student's t-test or Welch's t-test. (c) Levels of cell death in the cerebral cortex were evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining at E10, E15, E18, P0 and P60 of Pqbp1-cKO mice (Nes-Cre; XFloxY) and nestin-Cre mice (Nes-Cre; XY). Quantitative analysis of apoptotic cells did not reveal any differences (Student's t-test).
Mentions: Hence, we first evaluated the cell cycle time of NSPCs in vivo using the cumulative labeling method.23 The data showed that the total cell cycle time (Tc) was increased (+2.2 h, +12%) (Figure 2a). The S phase (Ts) was unaltered compared to the strict control nestin-Cre XY mice (Figure 2a). The G1 phase was slightly longer (Figure 2a). However, G1 elongation in the Pqbp1-cKO was minimal (5.2%) compared to other reports of cell cycle length regulating brain size.33,34 G2 phase time was not changed (Figure 2a). In contrast to the G1, S, and G2 phases, the M phase was remarkably increased (Figure 2a) and the increase in Tc was principally due to a longer M phase (+1.4 h, +67%). This cell cycle change in Pqbp1-cKO mice is quite unique among various disease models.

Bottom Line: Human mutations in PQBP1, a molecule involved in transcription and splicing, result in a reduced but architecturally normal brain.Exogenous Apc4, a hub protein in the network of affected genes, recovered the cell cycle, proliferation, and cell phenotypes of NSPCs caused by Pqbp1-cKO.These data reveal a mechanism of brain size control based on the simple reduction of the NSPC pool by cell cycle time elongation.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuropathology, Medical Research Institute and Center for Brain Integration Research, Tokyo Medical and Dental University, Tokyo, Japan.

ABSTRACT
Human mutations in PQBP1, a molecule involved in transcription and splicing, result in a reduced but architecturally normal brain. Examination of a conditional Pqbp1-knockout (cKO) mouse with microcephaly failed to reveal either abnormal centrosomes or mitotic spindles, increased neurogenesis from the neural stem progenitor cell (NSPC) pool or increased cell death in vivo. Instead, we observed an increase in the length of the cell cycle, particularly for the M phase in NSPCs. Corresponding to the developmental expression of Pqbp1, the stem cell pool in vivo was decreased at E10 and remained at a low level during neurogenesis (E15) in Pqbp1-cKO mice. The expression profiles of NSPCs derived from the cKO mouse revealed significant changes in gene groups that control the M phase, including anaphase-promoting complex genes, via aberrant transcription and RNA splicing. Exogenous Apc4, a hub protein in the network of affected genes, recovered the cell cycle, proliferation, and cell phenotypes of NSPCs caused by Pqbp1-cKO. These data reveal a mechanism of brain size control based on the simple reduction of the NSPC pool by cell cycle time elongation. Finally, we demonstrated that in utero gene therapy for Pqbp1-cKO mice by intraperitoneal injection of the PQBP1-AAV vector at E10 successfully rescued microcephaly with preserved cortical structures and improved behavioral abnormalities in Pqbp1-cKO mice, opening a new strategy for treating this intractable developmental disorder.

Show MeSH
Related in: MedlinePlus