Primary cilia in human neuron differentiation

Primary (non-motile) cilia have long been overlooked, considered vestigial organelles lacking specific functions. In humans, all cell types except sperm, ependymal and bronchial epithelial cells, and oviduct cells extend a single primary cilium. However, it was not until the 1990s that cilia garnered significant interest in biomedical research. Cilia serve as cellular sensors for various stimuli, including mechanical, light, osmotic, and chemoreceptive cues. Their ability to detect, integrate, and transmit external information to the cell is essential for cell signaling, development, and tissue homeostasis.

Among ciliary signaling pathways, the WNT pathway is evolutionarily conserved and regulates crucial aspects of organ patterning, cell proliferation, migration, and fate determination during embryogenesis. Not surprisingly, genetic mutations in WNT pathway-related proteins have been linked to several diseases and different types of cancer. Alterations in cilia function may lead to the onset of a heterogeneous group of genetic conditions called ciliopathies. Given that cilia are nearly ubiquitous organelles, ciliopathies may affect multiple organs, including the brain. So far, dysfunction of cilia has also been implicated in a spectrum of complex neurodevelopmental disorders (NDDs) such as autism, schizophrenia, and dyslexia.

In Paper I, we established an advanced human neuronal ciliated cell model using Lund human mesencephalic (LUHMES) cells. These cells can differentiate into mature neurons in just about a week of culture, and they exhibit ciliation throughout the proliferative, differentiation, and fully differentiated phases. This in vitro cell model shared a high gene expression profile similarity with in vivo neuronal transcriptomes from humans. LUHMES neuronal cilia were shown to functionally transduce the well-known ciliary Sonic hedgehog (SHH) signaling pathway by regulating its target gene expression.

In Paper II we used the LUHMES cell model to explore the role of cilia during human neuron differentiation. Based on a dynamic differentiation ciliation pattern, we identified the functional ciliary time window and found cilia to be relevant in promoting axon outgrowth and branching. Furthermore, we generated a stable LUHMES mutant model for the ciliogenic transcription factor RFX2 and performed transient knockdowns (KDs) of essential ciliary genes IFT88 and IFT172. LUHMES RFX2 -/-, IFT88 KD and IFT172 KD neurons were not as efficient as WT counterparts in promoting the same aspects of neuron differentiation. Altered RFX2 -/- cilia showed deregulation of the ciliary WNT signaling pathway and subsequent cytoskeleton rearrangement required for proper neuron differentiation.

In Paper III, we provided an extensive compendium of how neuron differentiation is molecularly regulated. By analyzing enhancer RNAs (eRNAs) using the native elongating transcript-cap analysis of gene expression (NET-CAGE) method, we revealed nearly triple the number of new LUHMES enhancers important for neuron differentiation, as compared to what was previously known. Our analysis also showed enrichment of active enhancers among certain NDDs, including neuropsychiatric disorders. We employed capture Hi-C (HiCap) to locate target genes through promoter-enhancer interaction. Interestingly, the binding motifs of ciliogenic RFX transcription factors and the transcriptional complex activated by WNT signaling were enriched in both promoter and enhancer sequences.

Altogether, we elucidated strong implications of cilia in neuron differentiation at the cellular level unveiling a critical ciliary time window, and at the molecular level, describing the regulatory machinery promoting neuron differentiation. We speculate that cilia play an essential role in neuron maturation and brain formation, underpinning the biogenesis of aspects of certain neurodevelopmental conditions when their function is disrupted.

List of scientific papers

I. Lauter G.*, Coschiera A.*, Yoshihara M., Sugiaman-Trapman D., Ezer S., Sethurathinam S., Katayama S., Kere J., Swoboda P. Differentiation of ciliated human midbrain-derived LUHMES neurons. Journal of Cell Science. 2020 Nov 9;133(21):jcs249789. *Equal contribution - joint first authorship.
https://doi.org/10.1242/jcs.249789

II. Coschiera A.*, Yoshihara M.*, Lauter G., Ezer S., Pucci M., Li H., Kavšek A., Riedel C. G., Kere J., Swoboda P. Primary cilia promote the differentiation of human neurons through the WNT signaling pathway. BMC Biology. 2024 Feb 27;22:48. *Equal contribution - joint first authorship.
https://doi.org/10.1186/s12915-024-01845-w

III. Yoshihara M.*, Coschiera A.*, Bachmann J.*, Pucci M., Li H., Bhagat S., Murakawa Y., Weltner J., Jouhilahti E.-M., Swoboda, P., Sahlén P., Kere J. Transcriptional enhancers in human neuronal differentiation provide clues to neuropsychiatric disorders. *Equal contribution - joint first authorship. [Manuscript]