RACK1 Controls Cerebellum Development By Reciprocal Regulation Of Wnt/?-catenin And SHH Signaling Pathways

Cerebellum is located in the posterior cranial fossa which is below the occipital lobe and above the brainstem.Numerous studies have shown that the cerebellum is not only a motor control organ,but also participates in the regulation of multiple advanced cognitive functions including language,emotion,and social interactions.In addition,due to its unique patterned foliation,layered cortex structure and relatively simple cell types,it makes the cerebellum an ideal paradigm for studying the molecular mechanisms of neurodevelopment.But the signal mechanisms regulating cerebellar development are not yet fully elucidated.In this study,RACK1?Receptor for activated C kinase 1?was found as a novel critical gene that regulated cerebellar development.As a scaffolding protein containing 7 WD40 repetitive domains?WDR?,the expression and function of RACK1 are fairly extensive.Of the 286 WDR protein family members that have been found,part of the WDR proteins are involved in the brain development.However,the exact function of RACK1 in brain development remains absolutely unknown.Because of the lethality of RACK1 null mutation,it is necessary to prepare conditional knockout mice for investigating the exact function and molecular mechanisms of RACK1 regulating neural development.Therefore,we constructed a variety of cell specific conditional knockout mice by employing pre-established RACK1^loxP mice in this study.Firstly,we found that it would lead to embryonic lethality in specific deletion of RACK1 with the use of Nestin-Cre in the early embryonic?E9.5?neural stem cells.Meanwhile,when conditionally knock out of RACK1 in the mid embryonic?E11.5?neural stem cells with hGFAP-Cre,the mice could survive for about 3 weeks and showed typical ataxia and balance defects.The hGFAP-RACK1^-/-mutant mice were found to be accompanied by a severe cerebral and cerebellar developmental defective phenotypes.In this study,we focused on the hypoplastic phenotypes of reduced cerebellar lobule formation and disordered cortical lamination.Secondly,since hGFAP-Cre⁺neural stem cells are able to differentiate into granule neurons and Bergmann glia,it will be reasonable to separately answer the role of RACK1 in granule neurons and Bergman glial cells whichever contribute to cerebellar development.Specific knockout of RACK1 in granule neuron precursors?Math1-Cre?resulted in cerebellar developmental defects similar to those of RACK1mutant in neural stem cells(hGFAP-RACK1^-/-),both of which were manifested as a decrease in the proliferative capacity and a hindered inward migration of the granule neuron precursors.However,the conditional knockout of RACK1 in Bergmann glial cells?GLAST-Cre-ERT?did not affect the morphogenesis of Bergmann glia and cerebellum.Interestingly,knocking out RACK1 in either neural stem cells or granule neuron precursors showed obvious abnormalities in the morphology of Bergmann glia and Purkinje neurons,suggesting that RACK1 could influences the morphogenesis of Bergmann glia and Purkinje neurons through regulating the development of granule neurons in a cell non-autonomous manner.The results further confirmed the integrity of cerebellum development which was the result of different types of neural cells relying and cooperating with each other.Finally,we also conducted a preliminary exploration of the function of RACK1 in Purkinje cells.We found that in addition to regulating the development of Purkinje cells in a non-autonomous manner,RACK1 could also directly regulate its function in a cell-autonomous manner by affecting the fusion of specific cerebellum lobules and thereafter controlling the movement and balance of the mice.By ablation of RACK1 in different cell types,RACK1 was confirmed to affect cerebellar development by regulating the proliferation,migration and differentiation of neural stem/progenitor cells and granule neuron precursors.The results showed that specific knockout of RACK1 in granule neurons in vivo or in vitro both lead to aberrant activation of Wnt/?-catenin signal and severe inhibition of SHH-Gli1 signaling pathways simultaneously.Also,knockout of?-catenin in neural stem/progenitor cells,but not in granule neuron precursors,could“rescue”the deficient phenotype in the cerebellum of RACK1 knockout mice.The study also found that RACK1 could exert a positive activation on the SHH pathway by direct binding to the Smoothened receptor and that RACK1deletion would lead to a significant decrease of the HDAC1/2 protein levels.Further experimental results showed that the effect of RACK1 stabilizing HDAC1/HDAC2 protein levels was mainly dependent on reducing its ubiquitination level and thereby inhibiting its proteasomal degradation.Due to HDAC1/HDAC2 played a crucial role in regulating the transcription of SHH signaling pathway downstream factor Gli1.Thus,RACK1 could promote the activation of SHH signaling by direct activating the Smoothened receptor,or by stabilizing the protein level of HDAC1/2 to increase Gli1 transcription synergistically.Finally,treatment of HDAC1/2 pharmacological inhibitors in the early postnatal developing cerebellum fully mimicked the defective phenotype generated by RACK1deletion in granule neuron precursors.In addition,by blocking the interaction between RACK1 and PKC with overexpression of C2 domain peptide,we found that neither the development of granule neuron precursors nor the expression of key signal molecules including Wnt/?-catenin and HDAC1/HDAC2 were affected,suggesting PKC is dispensable for the signaling negotiation.Therefore,during the development of cerebella,RACK1 determines the cerebellum morphogenesis and the formation of fine structures by regulating cell fates and developmental trends at different developmental stages as well as diverse cell subtypes.For the signaling mechanisms behind this complex developmental biology phenomenon,we found that it was closely related to the function of RACK1 by inhibiting the over-activation of Wnt/?-catenin signaling pathway;on the other hand,it also relied on the activation of SHH signaling pathway,which largely depended on the role of RACK1 by regulating the stability of HDAC1/HDAC2 proteins.As a receptor for activated C kinase,the regulation of Wnt/?-catenin and SHH signaling pathway mediated by RACK1 did not require its interaction with PKC.In sum,we have systematically characterized the function and mechanisms of RACK1 in the cerebellum development at the genetic,in vivo,cellular,and molecular levels.Since the abnormal proliferation of cerebellar granule cells is closely related to the occurrence of medulloblastoma,our work not only has an important scientific significance for deep understanding and revealing the molecular mechanisms underlying cerebellum development.Moerever,it may also provide important relevant targets for molecular diagnostic subtyping and molecular targeted therapy of medulloblastoma in the clinic.