Newly generated cells are increased in hippocampus of adult mice lacking a serine protease inhibitor

Abstract

Background: Neurogenesis in the hippocampal dentate gyrus and the subventricular zone occurs throughout the life of mammals and newly generated neurons can integrate functionally into established neuronal circuits. Neurogenesis levels in the dentate gyrus are modulated by changes in the environment (enrichment, exercise), hippocampal-dependent tasks, NMDA receptor (NMDAR) activity, sonic hedgehog (SHH) and/or other factors.

Results: previously, we showed that Protease Nexin-1 (PN-1), a potent serine protease inhibitor, regulates the NMDAR availability and activity as well as SHH signaling. Compared with wild-type (WT), we detected a significant increase in BrdU-labeled cells in the dentate gyrus of mice lacking PN-1 (PN-1 -/-) both in controls and after running exercise. Patched homologue 1 (Ptc1) and Gli1 mRNA levels were higher and Gli3 down-regulated in mutant mice under standard conditions and to a lesser extent after running exercise. However, the number of surviving BrdU-positive cells did not differ between WT and PN-1 -/- animals. NMDAR availability was altered in the hippocampus of mutant animals after exercise.

Conclusion: All together our results indicate that PN-1 controls progenitors proliferation through an effect on the SHH pathway and suggest an influence of the serpin on the survival of newly generated neurons through modulation of NMDAR availability.

Figure 1

PN-1 expression increases after running wheel exercise. (A-B) PN-1 expression detected by X-Gal staining in the brain of a reporter knock-in mouse (PN-1KI) without (CTR) and after 12 days of running exercise (RUN). (C, D) Enlargement of the dentate gyrus in control and exercised animals, respectively. (E, F) Enlargement of the thalamus in control and exercise animals, respectively. (G, H) BrdU and X-Gal staining in controls and after running, respectively. BrdU-positive cells (brown) lack PN-1 (black arrows) or are positive for PN-1 (white arrows). Enlargements are shown on the upper left. Cx: Cortex; DG: Dentate gyrus; Th: Thalamus. Scale bars: A, B, G and H 800 μm (insert in H 400 μm), C, D 400 μm, E, F 100 μm.

Figure 2

PN-1 ^-/- mice show increased BrdU incorporation in the dentate gyrus. (A) BrdU-positive cells in dentate gyrus sections of WT and mutant mice without and with 12 days of running wheel exercise. (B) Number of BrdU-positive cells in WT and PN-1 ^-/- mice without and after running. All data are the means of 4 animals/condition/genotype, *P < 0.05, **P < 0.01. Scale bar: A 100 μm. (C) Scheme illustrating the timeline of the running injection protocol. WT and PN 1^-/- mice kept without and with running wheel, were injected with BrdU daily for 12 days. BrdU analysis was performed either one day after the last BrdU injection or 4 weeks later. BrdU A: BrdU analysis.

Figure 3

Increase of Gli1 and Ptc1 and decrease of Gli3 expression in PN-1 ^-/- mice. (A) In situ hybridization of Ptc1, Gli1 and Gli3 in WT and PN-1 ^-/- mice without (CTR) and with 12 days of running wheel exercise (RUN). (B) Quantification of the in situ hybridization of Ptc1, Gli1 and Gli3 in WT and PN-1 ^-/- mice without and with 12 days of running wheel exercise (C) Changes in Ptc1, Gli1, and Gli3 expression detected by RT-PCR in wild type and mutant mice without and with running exercise. All data are the means of 3 animals/condition/genotype. *P < 0.05, **P < 0.01; ***P < 0.001. Scale bar: A 100 μm.

Figure 4

Reduced survival of Brdu positive cells generated by running exercise in PN-1 ^-/- dentate gyrus. (A) Number of BrdU-positive cells (brown) in dentate gyrus 4 weeks after the end of BrdU injection in WT and PN-1 ^-/- mice without (CTR) and with running wheel exercise (RUN). (B) Lack of differences in the number of BrdU-positive cells in WT and PN-1 ^-/- mice without and with exercise. (C) Imaging of double labeled BrdU/NeuN and BrdU/S100 cells in hippocampus of WT and PN-1 KO mice with and without running condition. BrdU: green (Alexa 488), NeuN: red (Alexa 594), S100: violet-cy5 (Jackson 633). (D) Phenotypes of surviving cells determined by immunofluorescent triple-labeling for BrdU, NeuN (neurons) and S100 (astrocytes). The percentages of BrdU-positive cells double-labeled for either S100 or NeuN are shown. All data are the means of 3 animals/condition/genotype.

Figure 5

Effect of running exercise on NMDAR availability in wild type and PN-1 ^-/- mice. (A) Representative immunoblot detecting NR1, NR2A and NR2B NMDAR subunits in hippocampal synaptosomal fraction of WT and PN-1 ^-/- mice without (CTR) and with running exercise (RUN). (B-E) Quantification of similar immunoblots including AMPA receptor as control. All data are the means of 3 animals. For each treatment, values in the WT without exercise were taken as 100% relative to the actin level.