Multifaceted Functions of Rab23 on Primary Cilium-Mediated and Hedgehog Signaling-Mediated Cerebellar Granule Cell Proliferation

Abstract

Sonic hedgehog (Shh) signaling from the primary cilium drives cerebellar granule cell precursor (GCP) proliferation. Mutations of hedgehog (Hh) pathway repressors commonly cause medulloblastoma, the most prevalent and malignant childhood brain tumor that arises from aberrant GCP proliferation. We demonstrate that Nestin Cre-driven conditional knock-out (CKO) of a Shh pathway repressor-Rab23 in the mouse brain of both genders caused mis-patterning of cerebellar folia and elevated GCP proliferation during early development, but with no prevalent occurrence of medulloblastoma at adult stage. Strikingly, Rab23-depleted GCPs exhibited upregulated basal level of Shh pathway activities despite showing an abnormal ciliogenesis of primary cilia. In line with the compromised ciliation, Rab23-depleted GCPs were desensitized against Hh pathway activity stimulations by Shh ligand and Smoothened (Smo) agonist-SAG, and exhibited attenuated stimulation of Smo-localization on the primary cilium in response to SAG. These results implicate multidimensional actions of Rab23 on Hh signaling cascade. Rab23 represses the basal level of Shh signaling, while facilitating primary cilium-dependent extrinsic Shh signaling activation. Collectively, our findings unravel instrumental roles of Rab23 in GCP proliferation and ciliogenesis. Furthermore, Rab23's potentiation of Shh signaling pathway through the primary cilium and Smo suggests a potential new therapeutic strategy for Smo/primary cilium-driven medulloblastoma.

RAB23 loss-of-function mutation causes context-dependent ciliopathy in Carpenter syndrome

“…Notably, in the absence of SAG stimulation, Rab23- KO NPCs at basal level (DMSO) showed an up-regulated Gli activity compared to the control group ( Fig 6E , Control+DMSO versus Nes-CKO+DMSO). This observation is consistent with our previous observations in cerebellar granule progenitor cells [ 18 , 35 ], and aligned with the well-established negative regulatory role of Rab23 in the Shh signalling pathway [ 16 ]. Consistent with the Gli reporter assay results, qPCR analysis of gene expression also revealed ectopic activation of the Shh signaling pathway in the mutant NPCs, as evidenced by elevated Gli1 expression (a downstream transcriptional readout of Shh signaling activity) ( Fig 6F ).…”

“…Consistent with our previous findings in Rab23 -KO granule cell precursors, where we demonstrated that Rab23 promotes SAG-induced Smoothened localization to the ciliary axoneme, revealing a cilia-dependent mechanism of action [ 18 ], our current findings demonstrate that Rab23 exerts multi-dimensional regulatory effects on Hedgehog signaling activity in NPCs. It positively influences the pathway through promoting primary cilia-dependent Smo-mediated Hh signal transduction.…”

RAB23 loss-of-function mutation causes context-dependent ciliopathy in Carpenter syndrome

“…Notably, in the absence of SAG stimulation, Rab23- KO NPCs at basal level (DMSO) showed an up-regulated Gli activity compared to the control group ( Fig 6E , Control+DMSO versus Nes-CKO+DMSO). This observation is consistent with our previous observations in cerebellar granule progenitor cells [ 18 , 35 ], and aligned with the well-established negative regulatory role of Rab23 in the Shh signalling pathway [ 16 ]. Consistent with the Gli reporter assay results, qPCR analysis of gene expression also revealed ectopic activation of the Shh signaling pathway in the mutant NPCs, as evidenced by elevated Gli1 expression (a downstream transcriptional readout of Shh signaling activity) ( Fig 6F ).…”

“…Consistent with our previous findings in Rab23 -KO granule cell precursors, where we demonstrated that Rab23 promotes SAG-induced Smoothened localization to the ciliary axoneme, revealing a cilia-dependent mechanism of action [ 18 ], our current findings demonstrate that Rab23 exerts multi-dimensional regulatory effects on Hedgehog signaling activity in NPCs. It positively influences the pathway through promoting primary cilia-dependent Smo-mediated Hh signal transduction.…”

RAB23 loss-of-function mutation causes context-dependent ciliopathy in Carpenter syndrome

“…Notably, in the absence of SAG stimulation, Rab23- KO NPCs at basal level (DMSO) showed an up-regulated Gli activity compared to the control group (Figure 5C, Control+DMSO versus Nes-CKO+DMSO). This observation is consistent with our previous observations in cerebellar granule progenitor cells (C. H. Hor & Goh, 2019; C. H. H. Hor et al, 2021), and aligned with the well-established negative regulatory role of Rab23 in the Shh signalling pathway (Eggenschwiler et al, 2001). Consistent with the Gli reporter assay results, qPCR analysis of gene expression also revealed ectopic activation of the Shh signaling pathway in the mutant NPCs, as evidenced by elevated Gli1 expression (a downstream transcriptional readout of Shh signaling activity) (Figure 5D).…”

“…Our previous study (C. H. H. Hor et al, 2021) and our current findings demonstrate that Rab23 exerts bi-directional functions in regulating Hedgehog signaling activity. It can positively influence the pathway through promoting primary cilia-dependent Hh signal transduction (Figure 5A-C), and negatively affect it by acting on Gli transcription factors in the cytoplasm (Figure 5E).…”

“…Two independent groups have reported a reduced ciliation (Gerondopoulos et al, 2019; Yoshimura et al, 2007), whereas another study observed no discernible change in the percentage of ciliation, despite using the same siRNA-mediated knockdown approach to silence Rab23 in hTERT-RPE1 cells (Lim & Tang, 2015). However, in our previous study on Rab23- knockout cerebellar primary granule progenitor cells culture (C. H. H. Hor et al, 2021), we observed a reduction in the length of primary cilia, albeit with an unperturbed rate of ciliation. Given these inconsistent observations on the effect of Rab23 silencing on the primary cilia formation and its structural integrity, more extensive investigations are necessary to elucidate the precise functions of Rab23 within the primary cilium.…”

RAB23 loss-of-function mutation causes context-dependent ciliopathy in Carpenter syndrome

“…Notably, in the absence of SAG stimulation, Rab23- KO NPCs at basal level (DMSO) showed an up-regulated Gli activity compared to the control group ( Fig 6E , Control+DMSO versus Nes-CKO+DMSO). This observation is consistent with our previous observations in cerebellar granule progenitor cells [ 18 , 35 ], and aligned with the well-established negative regulatory role of Rab23 in the Shh signalling pathway [ 16 ]. Consistent with the Gli reporter assay results, qPCR analysis of gene expression also revealed ectopic activation of the Shh signaling pathway in the mutant NPCs, as evidenced by elevated Gli1 expression (a downstream transcriptional readout of Shh signaling activity) ( Fig 6F ).…”

“…Consistent with our previous findings in Rab23 -KO granule cell precursors, where we demonstrated that Rab23 promotes SAG-induced Smoothened localization to the ciliary axoneme, revealing a cilia-dependent mechanism of action [ 18 ], our current findings demonstrate that Rab23 exerts multi-dimensional regulatory effects on Hedgehog signaling activity in NPCs. It positively influences the pathway through promoting primary cilia-dependent Smo-mediated Hh signal transduction.…”

RAB23 loss-of-function mutation causes context-dependent ciliopathy in Carpenter syndrome

“…Notably, in the absence of SAG stimulation, Rab23- KO NPCs at basal level (DMSO) showed an up-regulated Gli activity compared to the control group (Figure 5C, Control+DMSO versus Nes-CKO+DMSO). This observation is consistent with our previous observations in cerebellar granule progenitor cells (C. H. Hor & Goh, 2019; C. H. H. Hor et al, 2021), and aligned with the well-established negative regulatory role of Rab23 in the Shh signalling pathway (Eggenschwiler et al, 2001). Consistent with the Gli reporter assay results, qPCR analysis of gene expression also revealed ectopic activation of the Shh signaling pathway in the mutant NPCs, as evidenced by elevated Gli1 expression (a downstream transcriptional readout of Shh signaling activity) (Figure 5D).…”

“…Our previous study (C. H. H. Hor et al, 2021) and our current findings demonstrate that Rab23 exerts bi-directional functions in regulating Hedgehog signaling activity. It can positively influence the pathway through promoting primary cilia-dependent Hh signal transduction (Figure 5A-C), and negatively affect it by acting on Gli transcription factors in the cytoplasm (Figure 5E).…”

“…Two independent groups have reported a reduced ciliation (Gerondopoulos et al, 2019; Yoshimura et al, 2007), whereas another study observed no discernible change in the percentage of ciliation, despite using the same siRNA-mediated knockdown approach to silence Rab23 in hTERT-RPE1 cells (Lim & Tang, 2015). However, in our previous study on Rab23- knockout cerebellar primary granule progenitor cells culture (C. H. H. Hor et al, 2021), we observed a reduction in the length of primary cilia, albeit with an unperturbed rate of ciliation. Given these inconsistent observations on the effect of Rab23 silencing on the primary cilia formation and its structural integrity, more extensive investigations are necessary to elucidate the precise functions of Rab23 within the primary cilium.…”

RAB23 loss-of-function mutation causes context-dependent ciliopathy in Carpenter syndrome

“…Notably, in the absence of SAG stimulation, Rab23- KO NPCs at basal level (DMSO) showed an up-regulated Gli activity compared to the control group ( Fig 6E , Control+DMSO versus Nes-CKO+DMSO). This observation is consistent with our previous observations in cerebellar granule progenitor cells [ 18 , 35 ], and aligned with the well-established negative regulatory role of Rab23 in the Shh signalling pathway [ 16 ]. Consistent with the Gli reporter assay results, qPCR analysis of gene expression also revealed ectopic activation of the Shh signaling pathway in the mutant NPCs, as evidenced by elevated Gli1 expression (a downstream transcriptional readout of Shh signaling activity) ( Fig 6F ).…”

“…Consistent with our previous findings in Rab23 -KO granule cell precursors, where we demonstrated that Rab23 promotes SAG-induced Smoothened localization to the ciliary axoneme, revealing a cilia-dependent mechanism of action [ 18 ], our current findings demonstrate that Rab23 exerts multi-dimensional regulatory effects on Hedgehog signaling activity in NPCs. It positively influences the pathway through promoting primary cilia-dependent Smo-mediated Hh signal transduction.…”

RAB23 loss-of-function mutation causes context-dependent ciliopathy in Carpenter syndrome

“…Notably, in the absence of SAG stimulation, Rab23- KO NPCs at basal level (DMSO) showed an up-regulated Gli activity compared to the control group (Figure 5C, Control+DMSO versus Nes-CKO+DMSO). This observation is consistent with our previous observations in cerebellar granule progenitor cells (C. H. Hor & Goh, 2019; C. H. H. Hor et al, 2021), and aligned with the well-established negative regulatory role of Rab23 in the Shh signalling pathway (Eggenschwiler et al, 2001). Consistent with the Gli reporter assay results, qPCR analysis of gene expression also revealed ectopic activation of the Shh signaling pathway in the mutant NPCs, as evidenced by elevated Gli1 expression (a downstream transcriptional readout of Shh signaling activity) (Figure 5D).…”

“…Our previous study (C. H. H. Hor et al, 2021) and our current findings demonstrate that Rab23 exerts bi-directional functions in regulating Hedgehog signaling activity. It can positively influence the pathway through promoting primary cilia-dependent Hh signal transduction (Figure 5A-C), and negatively affect it by acting on Gli transcription factors in the cytoplasm (Figure 5E).…”

“…Two independent groups have reported a reduced ciliation (Gerondopoulos et al, 2019; Yoshimura et al, 2007), whereas another study observed no discernible change in the percentage of ciliation, despite using the same siRNA-mediated knockdown approach to silence Rab23 in hTERT-RPE1 cells (Lim & Tang, 2015). However, in our previous study on Rab23- knockout cerebellar primary granule progenitor cells culture (C. H. H. Hor et al, 2021), we observed a reduction in the length of primary cilia, albeit with an unperturbed rate of ciliation. Given these inconsistent observations on the effect of Rab23 silencing on the primary cilia formation and its structural integrity, more extensive investigations are necessary to elucidate the precise functions of Rab23 within the primary cilium.…”