A lissencephaly-associated BAIAP2 variant causes defects in neuronal migration during brain development.

Lissencephaly is a neurodevelopmental disorder characterized by a loss of brain surface convolutions caused by genetic variants that disrupt neuronal migration. However, the genetic origins of the disorder remain unidentified in nearly one-fifth of people with lissencephaly. Using whole-exome sequencing, we identified a de novo BAIAP2 variant, p.Arg29Trp, in an individual with lissencephaly with a posterior more severe than anterior (P>A) gradient, implicating BAIAP2 as a potential lissencephaly gene. Spatial transcriptome analysis in the developing mouse cortex revealed that Baiap2 is expressed in the cortical plate and intermediate zone in an anterior low to posterior high gradient. We next used in utero electroporation to explore the effects of the Baiap2 variant in the developing mouse cortex. We found that Baiap2 knockdown caused abnormalities in neuronal migration, morphogenesis and differentiation. Expression of the p.Arg29Trp variant failed to rescue the migration defect, suggesting a loss-of-function effect. Mechanistically, the variant interfered with the ability of BAIAP2 to localize to the cell membrane. These results suggest that the functions of BAIAP2 in the cytoskeleton, cell morphogenesis and migration are important for cortical development and for the pathogenesis of lissencephaly in humans.

Novel lissencephaly-associated NDEL1 variant reveals distinct roles of NDE1 and NDEL1 in nucleokinesis and human cortical malformations.

The development of the cerebral cortex involves a series of dynamic events, including cell proliferation and migration, which rely on the motor protein dynein and its regulators NDE1 and NDEL1. While the loss of function in NDE1 leads to microcephaly-related malformations of cortical development (MCDs), NDEL1 variants have not been detected in MCD patients. Here, we identified two patients with pachygyria, with or without subcortical band heterotopia (SBH), carrying the same de novo somatic mosaic NDEL1 variant, p.Arg105Pro (p.R105P). Through single-cell RNA sequencing and spatial transcriptomic analysis, we observed complementary expression of Nde1/NDE1 and Ndel1/NDEL1 in neural progenitors and post-mitotic neurons, respectively. Ndel1 knockdown by in utero electroporation resulted in impaired neuronal migration, a phenotype that could not be rescued by p.R105P. Remarkably, p.R105P expression alone strongly disrupted neuronal migration, increased the length of the leading process, and impaired nucleus-centrosome coupling, suggesting a failure in nucleokinesis. Mechanistically, p.R105P disrupted NDEL1 binding to the dynein regulator LIS1. This study identifies the first lissencephaly-associated NDEL1 variant and sheds light on the distinct roles of NDE1 and NDEL1 in nucleokinesis and MCD pathogenesis.

Equilibrative nucleoside transporter 3 supports microglial functions and protects against the progression of Huntington's disease in the mouse model.

Huntington's disease (HD) is a hereditary neurodegenerative disorder characterized by involuntary movements, cognitive deficits, and psychiatric symptoms. Currently, there is no cure, and only limited treatments are available to manage the symptoms and to slow down the disease's progression. The molecular and cellular mechanisms of HD's pathogenesis are complex, involving immune cell activation, altered protein turnover, and disturbance in brain energy homeostasis. Microglia have been known to play a dual role in HD, contributing to neurodegeneration through inflammation but also enacting neuroprotective effects by clearing mHTT aggregates. However, little is known about the contribution of microglial metabolism to HD progression. This study explores the impact of a microglial metabolite transporter, equilibrative nucleoside transporter 3 (ENT3), in HD. Known as a lysosomal membrane transporter protein, ENT3 is highly enriched in microglia, with its expression correlated with HD severity. Using the R6/2 ENT3-/- mouse model, we found that the deletion of ENT3 increases microglia numbers yet worsens HD progression, leading to mHTT accumulation, cell death, and disturbed energy metabolism. These results suggest that the delicate balance between microglial metabolism and function is crucial for maintaining brain homeostasis and that ENT3 has a protective role in ameliorating neurodegenerative processes.

Muscleblind-like 2 knockout shifts adducin 1 isoform expression and alters dendritic spine dynamics of cortical neurons during brain development.

AIMS: Muscleblind-like 2 (MBNL2) plays a crucial role in regulating alternative splicing during development and mouse loss of MBNL2 recapitulates brain phenotypes in myotonic dystrophy (DM). However, the mechanisms underlying DM neuropathogenesis during brain development remain unclear. In this study, we aim to investigate the impact of MBNL2 elimination on neuronal development by Mbnl2 conditional knockout (CKO) mouse models. METHODS: To create Mbnl2 knockout neurons, cDNA encoding Cre-recombinase was delivered into neural progenitors of Mbnl2flox/flox mouse brains by in utero electroporation. The morphologies and dynamics of dendritic spines were monitored by confocal and two-photon microscopy in brain slices and live animals from the neonatal period into adulthood. To investigate the underlying molecular mechanism, we further detected the changes in the splicing and molecular interactions of proteins associated with spinogenesis. RESULTS: We found that Mbnl2 knockout in cortical neurons decreased dendritic spine density and dynamics in adolescent mice. Mbnl2 ablation caused the adducin 1 (ADD1) isoform to switch from adult to fetal with a frameshift, and the truncated ADD1 failed to interact with alpha-II spectrin (SPTAN1), a critical protein for spinogenesis. In addition, expression of ADD1 adult isoform compensated for the reduced dendritic spine density in cortical neurons deprived of MBNL2. CONCLUSION: MBNL2 plays a critical role in maintaining the dynamics and homeostasis of dendritic spines in the developing brain. Mis-splicing of downstream ADD1 may account for the alterations and contribute to the DM brain pathogenesis.

Novel lissencephaly-associated DCX variants in the C-terminal DCX domain affect microtubule binding and dynamics.

OBJECTIVE: Pathogenic variants in DCX on the X chromosome lead to lissencephaly and subcortical band heterotopia (SBH), brain malformations caused by neuronal migration defects. Its product doublecortin (DCX) binds to microtubules to modulate microtubule polymerization. How pathogenic DCX variants affect these activities remains not fully investigated. METHODS: DCX variants were identified using whole exome and Sanger sequencing from six families with lissencephaly/SBH. We examined how these variants affect DCX functions using microtubule binding, regrowth, and colocalization assays. RESULTS: We found novel DCX variants p.Val177AlafsTer31 and p.Gly188Trp, as well as reported variants p.Arg196His, p.Lys202Met, and p.Thr203Ala. Incidentally, all of the missense variants were clustered on the C-terminal DCX domain. The microtubule binding ability was significantly decreased in p.Val177AlafsTer31, p.Gly188Trp, p.Lys202Met, and previously reported p.Asp262Gly variants. Furthermore, expression of p.Val177AlafsTer31, p.Gly188Trp, p.Arg196His, p.Lys202Met, and p.Asp262Gly variants hindered microtubule growth in cells. There were also decreases in the colocalization of p.Val177AlafsTer31, p.Thr203Ala, and p.Asp262Gly variants to microtubules. SIGNIFICANCE: Our results indicate that these variants in the C-terminal DCX domain altered microtubule binding and dynamics, which may underlie neuronal migration defects during brain development.

Clinical and functional characterization of a novel STUB1 frameshift mutation in autosomal dominant spinocerebellar ataxia type 48 (SCA48).

BACKGROUND: Heterozygous pathogenic variants in STUB1 are implicated in autosomal dominant spinocerebellar ataxia type 48 (SCA48), which is a rare familial ataxia disorder. We investigated the clinical, genetic and functional characteristics of STUB1 mutations identified from a Taiwanese ataxia cohort. METHODS: We performed whole genome sequencing in a genetically undiagnosed family with an autosomal dominant ataxia syndrome. Further Sanger sequencing of all exons and intron-exon boundary junctions of STUB1 in 249 unrelated patients with cerebellar ataxia was performed. The pathogenicity of the identified novel STUB1 variant was investigated. RESULTS: We identified a novel heterozygous frameshift variant, c.832del (p.Glu278fs), in STUB1 in two patients from the same family. This rare mutation is located in the U-box of the carboxyl terminus of the Hsc70-interacting protein (CHIP) protein, which is encoded by STUB1. Further in vitro experiments demonstrated that this novel heterozygous STUB1 frameshift variant impairs the CHIP protein's activity and its interaction with the E2 ubiquitin ligase, UbE2D1, leading to neuronal accumulation of tau and α-synuclein, caspase-3 activation, and promoting cellular apoptosis through a dominant-negative pathogenic effect. The in vivo study revealed the influence of the CHIP expression level on the differentiation and migration of cerebellar granule neuron progenitors during cerebellar development. CONCLUSIONS: Our findings provide clinical, genetic, and a mechanistic insight linking the novel heterozygous STUB1 frameshift mutation at the highly conserved U-box domain of CHIP as the cause of autosomal dominant SCA48. Our results further stress the importance of CHIP activity in neuronal protein homeostasis and cerebellar functions.

Cellular secretion and cytotoxicity of transthyretin mutant proteins underlie late-onset amyloidosis and neurodegeneration.

Transthyretin amyloidosis (ATTR) is a progressive life-threatening disease characterized by the deposition of transthyretin (TTR) amyloid fibrils. Several pathogenic variants have been shown to destabilize TTR tetramers, leading to aggregation of misfolded TTR fibrils. However, factors that underlie the differential age of disease onset amongst amyloidogenic TTR variants remain elusive. Here, we examined the biological properties of various TTR mutations and found that the cellular secretory pattern of the wild-type (WT) TTR was similar to those of the late-onset mutant (Ala97Ser, p. Ala117Ser), stable mutant (Thr119Met, p. Thr139Met), early-onset mutant (Val30Met, p. Val50Met), but not in the unstable mutant (Asp18Gly, p. Asp38Gly). Cytotoxicity assays revealed their toxicities in the order of Val30Met > Ala97Ser > WT > Thr119Met in neuroblastoma cells. Surprisingly, while early-onset amyloidogenic TTR monomers (M-TTRs) are retained by the endoplasmic reticulum quality control (ERQC), late-onset amyloidogenic M-TTRs can be secreted extracellularly. Treatment of thapsigargin (Tg) to activate the unfolded protein response (UPR) alleviates Ala97Ser M-TTR secretion. Interestingly, Ala97Ser TTR overexpression in Drosophila causes late-onset fast neurodegeneration and a relatively short lifespan, recapitulating human disease progression. Our study demonstrates that the escape of TTR monomers from the ERQC may underlie late-onset amyloidogenesis in patients and suggests that targeting ERQC could mitigate late-onset ATTR.

Gli2 modulates cell cycle re-entry through autophagy-mediated regulation of the length of primary cilia.

The primary cilium is a tiny cell protrusion known to transduce key extracellular signals, including those of the sonic hedgehog pathway, which activates Gli transcription factors for various cellular functions. To understand the significance of the Gli2 transcription factor in fibroblasts, we establish a Gli2-knockout NIH3T3 cell line by CRISPR/Cas9 technology. Surprisingly, NIH3T3 fibroblasts lacking Gli2 expression through gene knockout or RNA interference possess longer primary cilia after stimulation of ciliogenesis by serum starvation. This lengthening of primary cilia is associated with enhanced autophagy-mediated Ofd1 degradation, and can be reversed by pharmacological and genetic inhibition of autophagy. Meanwhile, flow cytometry reveals that Gli2-/- NIH3T3 fibroblasts exhibit a delay in cell cycle re-entry after serum re-stimulation. Ablation of their primary cilia through Kif3a knockdown rescues the delay in cell cycle re-entry. These results suggest that Gli2 plays an unexpected role in cell cycle re-entry through an autophagy-mediated regulation on ciliary length in fibroblasts.

Gli2 Rescues Delays in Brain Development Induced by Kif3a Dysfunction.

The primary cilium in neural stem cells plays distinct roles in different stages during cortical development. Ciliary dysfunctions in human (i.e., ciliopathy) cause developmental defects in multiple organs, including brain developmental delays, which lead to intellectual disabilities and cognitive deficits. However, effective treatment to this devastating developmental disorder is still lacking. Here, we first investigated the effects of ciliopathy on neural stem cells by knocking down Kif3a, a kinesin II motor required for ciliogenesis, in the neurogenic stage of cortical development by in utero electroporation of mouse embryos. Brains electroporated with Kif3a shRNA showed defects in neuronal migration and differentiation, delays in neural stem cell cycle progression, and failures in interkinetic nuclear migration. Interestingly, introduction of Gli1 and Gli2 both can restore the cell cycle progression by elevating cyclin D1 in neural stem cells. Remarkably, enforced Gli2 expression, but not Gli1, partially restored the ability of Kif3a-knockdown neurons to differentiate and move from the germinal ventricular zone to the cortical plate. Moreover, Cyclin D1 knockdown abolished Gli2's rescue effect. These findings suggest Gli2 may rescue neural stem cell proliferation, differentiation and migration through Cyclin D1 pathway and may serve as a potential therapeutic target for human ciliopathy syndromes through modulating the progression of neural stem cell cycle.

Constitutive Inactivation of the PRRT2 Gene Alters Short-Term Synaptic Plasticity and Promotes Network Hyperexcitability in Hippocampal Neurons.

Mutations in PRoline-Rich Transmembrane protein 2 (PRRT2) underlie a group of paroxysmal disorders including epilepsy, kinesigenic dyskinesia and migraine. Most of the mutations lead to impaired PRRT2 expression and/or function, emphasizing the pathogenic role of the PRRT2 deficiency. In this work, we investigated the phenotype of primary hippocampal neurons obtained from mouse embryos in which the PRRT2 gene was constitutively inactivated. Although PRRT2 is expressed by both excitatory and inhibitory neurons, its deletion decreases the number of excitatory synapses without significantly affecting the number of inhibitory synapses or the nerve terminal ultrastructure. Analysis of synaptic function in primary PRRT2 knockout excitatory neurons by live imaging and electrophysiology showed slowdown of the kinetics of exocytosis, weakened spontaneous and evoked synaptic transmission and markedly increased facilitation. Inhibitory neurons showed strengthening of basal synaptic transmission, accompanied by faster depression. At the network level these complex synaptic effects resulted in a state of heightened spontaneous and evoked activity that was associated with increased excitability of excitatory neurons in both PRRT2 knockout primary cultures and acute hippocampal slices. The data indicate the existence of network instability/hyperexcitability as the possible basis of the paroxysmal phenotypes associated with PRRT2 mutations.

WNT3A Promotes Neuronal Regeneration upon Traumatic Brain Injury.

The treatment of traumatic brain injury (TBI) remains a challenge due to limited knowledge about the mechanisms underlying neuronal regeneration. This current study compared the expression of WNT genes during regeneration of injured cortical neurons. Recombinant WNT3A showed positive effect in promoting neuronal regeneration via in vitro, ex vivo, and in vivo TBI models. Intranasal administration of WNT3A protein to TBI mice increased the number of NeuN+ neurons without affecting GFAP+ glial cells, compared to control mice, as well as retained motor function based on functional behavior analysis. Our findings demonstrated that WNT3A, 8A, 9B, and 10A promote regeneration of injured cortical neurons. Among these WNTs, WNT3A showed the most promising regenerative potential in vivo, ex vivo, and in vitro.

PRRT2 missense mutations cluster near C-terminus and frequently lead to protein mislocalization.

OBJECTIVE: Variants in human PRRT2 cause paroxysmal kinesigenic dyskinesia (PKD) and other neurological disorders. Most reported variants resulting in truncating proteins failed to localize to cytoplasmic membrane. The present study identifies novel PRRT2 variants in PKD and epilepsy patients and evaluates the functional consequences of PRRT2 missense variations. METHODS: We investigated two families with PKD and epilepsies using Sanger sequencing and a multiple gene panel. Subcellular localization of mutant proteins was investigated using confocal microscopy and cell surface biotinylation assay in Prrt2-transfected cells. RESULTS: Two novel PRRT2 variants, p.His232Glnfs*10 and p.Leu298Pro, were identified, and functional study revealed impaired localization of both mutant proteins to the plasma membrane. Further investigation of other reported missense variants revealed decreased protein targeting to the plasma membrane in eight of the 13 missense variants examined (p.Trp281Arg, p.Ala287Thr, p.Ala291Val, p.Arg295Gln, p.Leu298Pro, p.Ala306Asp, p.Gly324Glu, and p.Gly324Arg). In contrast, all benign variants we tested exhibited predominant localization to the plasma membrane similar to wild-type Prrt2. Most likely pathogenic variants were located at conserved amino acid residues near the C-terminus, whereas truncating variants spread throughout the gene. SIGNIFICANCE: PRRT2 missense variants clustering at the C-terminus often lead to protein mislocalization. Failure in protein targeting to the plasma membrane by PRRT2 variants may be a key mechanism in causing PKD and related neurological disorders.

Lis1 dysfunction leads to traction force reduction and cytoskeletal disorganization during cell migration.

Cell migration is a critical process during development, tissue repair, and cancer metastasis. It requires complex processes of cell adhesion, cytoskeletal dynamics, and force generation. Lis1 plays an important role in the migration of neurons, fibroblasts and other cell types, and is essential for normal development of the cerebral cortex. Mutations in human LIS1 gene cause classical lissencephaly (smooth brain), resulting from defects in neuronal migration. However, how Lis1 may affect force generation in migrating cells is still not fully understood. Using traction force microscopy (TFM) with live cell imaging to measure cellular traction force in migrating NIH3T3 cells, we showed that Lis1 knockdown (KD) by RNA interference (RNAi) caused reductions in cell migration and traction force against the extracellular matrix (ECM). Immunostaining of cytoskeletal components in Lis1 KD cells showed disorganization of microtubules and actin filaments. Interestingly, focal adhesions at the cell periphery were significantly reduced. These results suggest that Lis1 is important for cellular traction force generation through the regulation of cytoskeleton organization and focal adhesion formation in migrating cells.

Cdk12 Regulates Neurogenesis and Late-Arising Neuronal Migration in the Developing Cerebral Cortex.

DNA damage response (DDR) pathways are critical for ensuring that replication stress and various types of DNA lesion do not perturb production of neural cells during development. Cdk12 maintains genomic stability by regulating expression of DDR genes. Mutant mice in which Cdk12 is conditionally deleted in neural progenitor cells (NPCs) die after birth and exhibit microcephaly with a thinner cortical plate and an aberrant corpus callosum. We show that NPCs of mutant mice accumulate at G2 and M phase, and have lower expression of DDR genes, more DNA double-strand breaks and increased apoptosis. In addition to there being fewer neurons, there is misalignment of layers IV-II neurons and the presence of abnormal axonal tracts of these neurons, suggesting that Cdk12 is also required for the migration of late-arising cortical neurons. Using in utero electroporation, we demonstrate that the migrating mutant cells remain within the intermediate zone and fail to adopt a bipolar morphology. Overexpression of Cdk5 brings about a partially restoration of the neurons reaching layers IV-II in the mutant mice. Thus, Cdk12 is crucial to the repair of DNA damage during the proliferation of NPCs and is also central to the proper migration of late-arising neurons.

Asymmetric centrosome inheritance maintains neural progenitors in the neocortex.

Asymmetric divisions of radial glia progenitors produce self-renewing radial glia and differentiating cells simultaneously in the ventricular zone (VZ) of the developing neocortex. Whereas differentiating cells leave the VZ to constitute the future neocortex, renewing radial glia progenitors stay in the VZ for subsequent divisions. The differential behaviour of progenitors and their differentiating progeny is essential for neocortical development; however, the mechanisms that ensure these behavioural differences are unclear. Here we show that asymmetric centrosome inheritance regulates the differential behaviour of renewing progenitors and their differentiating progeny in the embryonic mouse neocortex. Centrosome duplication in dividing radial glia progenitors generates a pair of centrosomes with differently aged mother centrioles. During peak phases of neurogenesis, the centrosome retaining the old mother centriole stays in the VZ and is preferentially inherited by radial glia progenitors, whereas the centrosome containing the new mother centriole mostly leaves the VZ and is largely associated with differentiating cells. Removal of ninein, a mature centriole-specific protein, disrupts the asymmetric segregation and inheritance of the centrosome and causes premature depletion of progenitors from the VZ. These results indicate that preferential inheritance of the centrosome with the mature older mother centriole is required for maintaining radial glia progenitors in the developing mammalian neocortex.

Analysis of Neuronal Morphology by Two-Photon Microscopy.

During the development of mammalian brains, pyramidal neurons in the cerebral cortex form highly organized six layers with different functions. These neurons undergo developmental processes such as axon extension, dendrite outgrowth, and synapse formation. A proper integration of the neuronal connectivity through dynamic changes of dendritic branches and spines is required for learning and memory. Disruption of these crucial developmental processes is associated with many neurodevelopmental and neurodegenerative disorders. To investigate the complex dendritic architecture, several useful staining tools and genetic methods to label neurons have been well established. Monitoring the dynamics of dendritic spine in a single neuron is still a challenging task. Here, we provide a methodology that combines in vivo two-photon brain imaging and in utero electroporation, which sparsely labels cortical neurons with fluorescent proteins. This protocol may help elucidate the dynamics of microstructure and neural complexity in living rodents under normal and disease conditions.

Oscillatory DeltaC Expression in Neural Progenitors Primes the Prototype of Forebrain Development.

Notch signaling plays a pivotal role in regulating various developmental processes, particularly in controlling the timing of neuronal production within the developing neocortex. Central to this regulatory mechanism is the oscillatory pattern of Delta, which functions as a developmental clock modulator. Its deficiency profoundly impairs mammalian brain formation, highlighting its fundamental role in brain development. However, zebrafish carrying a mutation in the functional ortholog DeltaC (dlc) within their functional ortholog exhibit an intact forebrain structure, implying evolutionary variations in Notch signaling within the forebrain. In this study, we unveil the distinct yet analogous expression profiles of Delta and Her genes in the developing vertebrate forebrain. Specifically, for the first time, we detected the oscillatory expression of the Delta gene dlc in the developing zebrafish forebrain. Although this oscillatory pattern appeared irregular and was not pervasive among the progenitor population, attenuation of the dlc-involved Notch pathway using a γ-secretase inhibitor impaired neuronal differentiation in the developing zebrafish forebrain, revealing the indispensable role of the dlc-involved Notch pathway in regulating early zebrafish neurogenesis. Taken together, our results demonstrate the foundational prototype of dlc-involved Notch signaling in the developing zebrafish forebrains, upon which the intricate patterns of the mammalian neocortex may have been sculpted.

Pathogenic SHQ1 variants result in disruptions to neuronal development and the dopaminergic pathway.

BACKGROUND: Compound heterozygous variants of SHQ1, an assembly factor of H/ACA ribonucleoproteins (RNPs) involved in critical biological pathways, have been identified in patients with developmental delay, dystonia, epilepsy, and microcephaly. We investigated the role of SHQ1 in brain development and movement disorders. METHODS: SHQ1 expression was knocked down using short-hairpin RNA (shRNA) to investigate its effects on neurons. Shq1 shRNA and cDNA of WT and mutant SHQ1 were also introduced into neural progenitors in the embryonic mouse cortex through in utero electroporation. Co-immunoprecipitation was performed to investigate the interaction between SHQ1 and DKC1, a core protein of H/ACA RNPs. RESULTS: We found that SHQ1 was highly expressed in the developing mouse cortex. SHQ1 knockdown impaired the migration and neurite morphology of cortical neurons during brain development. Additionally, SHQ1 knockdown impaired neurite growth and sensitivity to glutamate toxicity in vitro. There was also increased dopaminergic function upon SHQ1 knockdown, which may underlie the increased glutamate toxicity of the cells. Most SHQ1 variants attenuated their binding ability toward DKC1, implying SHQ1 variants may influence brain development by disrupting the assembly and biogenesis of H/ACA RNPs. CONCLUSIONS: SHQ1 plays an essential role in brain development and dopaminergic function by upregulating dopaminergic pathways and regulating the behaviors of neural progenitors and their neuronal progeny, potentially leading to dystonia and developmental delay in patients. Our study provides insights into the functions of SHQ1 in neuronal development and dopaminergic function, providing a possible pathogenic mechanism for H/ACA RNPs-related disorders.

Regulation of primary cilia disassembly through HUWE1-mediated TTBK2 degradation plays a crucial role in cerebellar development and medulloblastoma growth.

Development of the cerebellum requires precise regulation of granule neuron progenitor (GNP) proliferation. Although it is known that primary cilia are necessary to support GNP proliferation, the exact molecular mechanism governing primary cilia dynamics within GNPs remains elusive. Here, we establish the pivotal roles for the centrosomal kinase TTBK2 (Tau tubulin kinase-2) and the E3 ubiquitin ligase HUWE1 in GNP proliferation. We show that TTBK2 is highly expressed in proliferating GNPs under Sonic Hedgehog (SHH) signaling, coinciding with active GNP proliferation and the presence of primary cilia. TTBK2 stabilizes primary cilia by inhibiting their disassembly, thereby promoting GNP proliferation in response to SHH. Mechanistically, we identify HUWE1 as a novel centrosomal E3 ligase that facilitates primary cilia disassembly by targeting TTBK2 degradation. Disassembly of primary cilia serves as a trigger for GNP differentiation, allowing their migration from the external granule layer (EGL) of the cerebellum to the internal granule layer (IGL) for subsequent maturation. Moreover, we have established a link between TTBK2 and SHH-type medulloblastoma (SHH-MB), a tumor characterized by uncontrolled GNP proliferation. TTBK2 depletion inhibits SHH-MB proliferation, indicating that TTBK2 may be a potential therapeutic target for this cancer type. In summary, our findings reveal the mechanism governing cerebellar development and highlight a potential anti-cancer strategy for SHH-MB.