Spatial centrosome proteome of human neural cells uncovers disease-relevant heterogeneity

The centrosome provides an intracellular anchor for the cytoskeleton, regulating cell division, cell migration, and cilia formation. We used spatial proteomics to elucidate protein interaction networks at the centrosome of human induced pluripotent stem cell–derived neural stem cells (NSCs) and neurons. Centrosome-associated proteins were largely cell type–specific, with protein hubs involved in RNA dynamics. Analysis of neurodevelopmental disease cohorts identified a significant overrepresentation of NSC centrosome proteins with variants in patients with periventricular heterotopia (PH). Expressing the PH-associated mutant pre-mRNA-processing factor 6 (PRPF6) reproduced the periventricular misplacement in the developing mouse brain, highlighting missplicing of transcripts of a microtubule-associated kinase with centrosomal location as essential for the phenotype. Collectively, cell type–specific centrosome interactomes explain how genetic variants in ubiquitous proteins may convey brain-specific phenotypes. Description RNA modification hub at the centrosome The centrosome acts as the cell’s microtubule organizing center, supporting cell division and the extension of cilia and neurites. Newly born neurons require the microtubule organizing activity of centrosomes to migrate away from their birthplace at the ventricle. O’Neill et al. analyzed the centrosome proteome of human induced pluripotent stem cell–derived neural stem cells and neurons. The neural centrosome proteome contains a variety of RNA-binding/modifying proteins, including an RNA-splicing factor mutation that is linked to periventricular heterotopia. —PJH Cell-type-specific centrosome interactomes explain how genetic variants in ubiquitous proteins may convey brain-specific phenotypes. INTRODUCTION The centrosome is an interaction hub composed of two centrioles surrounded by pericentriolar material that collectively exerts many pancellular functions, such as cell division, cell migration, and cilia formation. The centrosome acts as the main microtubule-organizing center (MTOC) in many cells, including stem and progenitor cells, but loses this activity often during differentiation. Very little is known, however, about the extent of its cell type–specific composition and function. Individual proteins have been found to be specific to the centrosome of, for example, neural stem cell subtypes, but whether these are exceptions or the rule is unknown. RATIONALE To assess any potential cell type–specific functions of the centrosome, its composition needs to be further investigated. However, no comprehensive proteome of neural centrosomes exists to date, and hence, the differences in centrosome composition between neural and other cell types are unknown. Likewise, the extent of the changes in this organelle’s distinct makeup during the differentiation of neural stem cells to neurons has not been explored. Because centrosome dysfunction is also linked to many neurodevelopmental conditions, information from such analysis could identify yet unknown disease associations. RESULTS To map the centrosome proteome of human neural stem cells and neurons, we chose a spatial proteomic approach to identify not only which proteins are present at this organelle but also where they are localized. Specifically, we selected 10 bait proteins known to localize to distinct sites of the centrosome, immunoprecipitated them from induced pluripotent stem cell–derived neural stem cells and neurons, and reproducibly determined their interactome with mass spectrometry. Interrogation of their interacting partners revealed diversity at this organelle, in which around 60% of the centrosome proteins had not yet been detected at the centrosome in other cell types. Furthermore, upon neuronal differentiation, more than half of these proteins become exchanged for new interactions at specific localizations within the centrosome. The neural centrosome proteomes comprise significantly enriched Gene Ontology terms of RNA-interacting proteins that were not observed in other cell types. Overlapping the neural stem cell and neuron centrosome proteomes with gene variants observed in patients with neurodevelopmental conditions of unknown etiology highlights specific and significant enrichment in epilepsy patients for the neuronal and, in periventricular heterotopia (PH), for the neural stem cell centrosome proteome. With respect to PH, we explored the effect of one candidate variant within the ubiquitously expressed gene that encodes the pre-mRNA processing factor 6 (PRPF6). We chose this candidate because several members of the PRPF6 complex were detected at the neural stem cell centrosome and had variants associated with PH. We show that the specific mutation of PRPF6 recapitulates aspects of the disease phenotype with ectopic cell localization in the periventricular region of the developing mouse cortex. Expression of the mutated form of PRPF6 results in misregulated splicing of, among others, the microtubule-associated protein kinase Brsk2. Coexpression of the correctly spliced form—but not the misspliced form, which lacks exon 19—with the mutant PRPF6 rescued the aberrant cell accumulation at the ventricle. The localization of Brsk2 mRNA at the centrosome is consistent with a role for PRPF6 in bringing its splicing targets to the centrosome for local translation and fine tuning of microtubule function at the centrosome for proper migration out of the periventricular region. CONCLUSION Centrosome composition differs between cell types, offering a diversity that is important for development and disease. The ubiquitously expressed protein PRPF6 is enriched at the centrosome in neural stem cells but not neurons, which causes, when mutated, a PH-like phenotype. The extensive characterization of centrosome proteins unraveled in this study provides a rich resource with which to explore further disease associations and cell type– and stage-specific functions. Neural centrosome proteome identifies disease candidates. Spatial proteomics of human neural stem cell and neuronal centrosomes uncovers cell type–specific protein hubs. Overlapping the proteomes with de novo mutations identified in patients with neurodevelopmental diseases revealed cell type–specific disease associations, enabling prioritization of disease variants. Among those, the expression of the PH-associated mutant R23W [in which arginine (R) at position 23 is replaced with tryptophan (W); red] PRPF6 (blue) recapitulated the periventricular cellular misplacement in the developing mouse brain by missplicing of brain-specific serine/threonine kinase 2 (Brsk2).