Role Of Gamma-secretase In Cortical Development And The Transition Of Neural Progenitors

During the development of the mammalian central nervous system,all types of cells,including neurons,astrocytes and oligodendrocytes,are derived from neural stem cells(NSCs).The six-layer cortical structure depends on proliferation of neural progenitor cells(NPCs),neurogenesis and neuronal migration.Incorrect formation of cortical development will cause psychiatric and neurological diseases,such as primary microcephaly.As a subunit of?-secretase,presenilin enhancer 2(Pen-2)has been proved to play important roles in the function of the catalytic subunit presenilin(PS).The well-known?-secretase can cleave type I integral membrane proteins,including Notch.Once activated after ligand-receptor binding,?-secretase cleaves Notch and releases Notch intracellular domain(NICD).NICD will translocate to nucleus to induce the expression of Hes1 and Hes5.The Hes genes repress the expression of the proneural genes,such as Mash1 and Ngn2,to maintain the stemness.Pen-2 gene knockout mice died during embryonic period and shared similar phenotypes with Notch deficient mice.Notch signaling was also impaired in Pen-2 deficient mice.Moreover,Pen-2 has been reported to be related in human 19q13 microdeletion syndrome manifesting microcephaly.All of the above suggest the importance of Pen-2 in cerebral cortex development.We used Emx1^IREScre which expresses since E10.5 to knockout Pen-2 specifically in our study.We generated Pen-2 conditional knockout mice(cKO)by utilizing Cre-lox P system.The cortex was dramatically thinner in Pen-2 cKO than in control at P21.Nissl staining revealed smaller cortex and abnormal laminar pattern in Pen-2 cKO mice than that in controls.IHC analysis showed that,cortical layers positive for Cux1,Ctip2or Tbr1 were well-organized in control mice but completely mixed-up in Pen-2 cKOs,indicating severe developmental defect.There are mainly two types of NPCs(Pax6+radial glial progenitors,RGPs and Tbr2+intermediate progenitors,IPs)in the developing cerebral cortex.Both of them can self-renew through symmetric cell division.We wonder whether the defects in Pen-2 cKO cortices comes from abnormal NPCs.In the dorsal telencephalon of Pen-2 cKO mice,the number of Pax6+cells and the number of Tbr2+cells showed a dynamic change.To study whether the changes in Pax6+cells and Tbr2+cells were due to altered proliferation,we conducted intraperitoneal injection of Brd U into pregnant mice at E12.5 and then collected brain sections 30 minutes after.Cell counting results indicated that the average number of Pax6+/Brd U+cells in the dorsal telencephalon was comparable between two groups.Moreover,the percentage of the number of Pax6+/Brd U+cells to that of Pax6+cells was normal in Pen-2 cKO,suggesting normal proliferation ratio for RGP cells.The ratio at E13.5 was also normal in Pen-2 cKO.Whereas the average number of Brd U+/Tbr2+cells was increased in Pen-2 cKO embryos,the proliferation ratio of Tbr2+cells there was also comparable between two groups,indicating normal proliferation of IPs.We excluded the possibility that depletion of RGPs(since E13.5)was due to abnormal apoptosis in Pen-2 cKO cortices by TUNEL staining and IHC on cleaved caspase 3.We concluded that Pen-2 depletion could cause dynamic changes of NPCs,but these changes were not due to altered proliferation ability of RGPs or IPs,and not from altered apoptosis.Due to RGPs can generate IPs through asymmetric division,we wanted to know whether the increased IPs at E12.5 came from enhanced transition from RGPs to IPs.Cell counting results revealed that the number of Sox2+/Tbr2+cells in Pen-2 cKO mice was significantly increased than controls,indicating accelerated switch of RGPs to IPs.We also observed more RGPs in cKO underwent asymmetric cell division.Enhanced transition of RGPs to IPs may be responsible for the altered NPC population.We generated NCT cKO mice by crossing NCT^f/f with Emx1^IREScre.NCT cKO mice showed similar phenotypes with Pen-2 cKO mice,including disorganized layers after birth,reduced RGPs and increased IPs at E13.5.Therefore,Pen-2 regulates cortical layer development through?-secretase-dependent manner.Western blot analysis confirmed that knockout of Pen-2 caused decreased?-secretase activity,repressed downstream transcription factor Hes1,and increased Ngn2 and Neuro D1.We used fluorescence activated cell sorting(FACs)to purify Pen-2 KO cells.Molecular analyses confirmed impaired?-secretase activity and downregulated Notch-Hes signaling pathway in Pen-2 KO cells.We demonstrate that reintroduction of Notch1 intracellullar domain(NICD)restores NPC population and partially rescues cortical morphology in Pen-2 mutant animals.NICD also rescue relative molecules,such as Hes1,Dll1,Ngn2and Neuro D1.Therefore,Pen-2 regulates the switch of RGPs to IPs via?-secretase-Notch signaling pathway.In addition,birthdating experiments showed that migration defect in Pen-2 cKO was not rescued by the reintroduction of NICD.Taken together,these findings highlight the role of Pen-2/?-secretase-Notch axis in cortical development,especially the transition from RGPs to IPs.