In preprints: unraveling a new non-canonical role of Cyclin D1 in corticogenesis Free

The precise regulation of the cell cycle is fundamental to neural development, ensuring the proper proliferation, differentiation and survival of neural progenitor cells (NSPCs). Cyclins, as key regulators of cell cycle progression, play a crucial role in these processes. Traditionally, cyclins function within the nucleus, forming complexes with cyclin-dependent kinases (CDKs) to regulate cell cycle transitions. However, a recent preprint by Pedraza et al. (2024 preprint) presents a previously undescribed role for cyclin D1 (Ccnd1) that extends beyond its conventional function in cell cycle regulation.

The mammalian neocortex originates from the neural plate, a sheet-like structure of neuroepithelium. During early development, the neural plate folds up to form the neural tube, which subsequently expands in both width (by proliferation) and thickness (by neurogenesis). NSPCs, especially those at the neurogenic stage, exhibit a unique morphology; they are thin, elongated cells that extend processes both apically (toward the ventricular side of the neural tube) and basally (toward the pial surface). These NSPCs are termed radial glial cells (RGCs), and their morphology and function has been instrumental in the evolution of the highly complex mammalian neocortex.

RGC nuclei are densely packed at the inner side of the neural tube, within the ventricular zone (VZ), a primary germinal layer. As neocortical expansion progresses, RGCs extend further, developing a highly polarized morphology. This polarity is accompanied by specific molecular distributions: centrosome-related proteins, such as ninein, localize to the apical endfeet (Shinohara et al., 2013), while basal endfeet contain molecules such as integrins that anchor the cells to the basal membrane (Fietz et al., 2010).

RGCs serve a dual role: they proliferate to maintain the NSPC pool, and they generate neurons. Newly produced neurons migrate from the VZ toward the cortical plate (CP), using RGC basal fibers as scaffolding. The timing of neurogenic versus mitotic divisions is tightly regulated by the duration of the G1 phase of the cell cycle, and prolonging the G1 phase promotes neurogenesis (Lukaszewicz et al., 2005).

Cyclins, particularly D-type cyclins (Ccnd1-3), are key nuclear proteins that regulate the cell cycle by forming complexes with CDK4 and CDK6, thereby promoting cell cycle entry (Sherr, 1993). However, non-canonical functions of Ccnd/CDK complexes have been recognized in recent years. Eloi Garí’s group, one of the co-authors of the preprint, previously demonstrated using cultured fibroblasts and keratinocytes that Ccnd1 associates with the plasma membrane, regulating cytoplasmic targets such as β1-integrin and its downstream effector paxillin, which influence cell migration and adhesion (Fernández-Hernández et al., 2013; Fusté et al., 2016). Additionally, cytoplasmic Ccnd1 has been implicated in neuronal communication by modulating neurotransmitter signaling in rat hippocampal neurons (Pedraza et al., 2024 preprint).

The new findings by Pedraza et al. extend this concept, demonstrating a role for cytoplasmic Ccnd1 in RGC behavior during cortical development. Using molecular, genetic and imaging approaches, the authors show that Ccnd1 localizes specifically within the basal endfeet of RGCs, overlapping with β1-integrin at the plasma membrane. Knockout (KO) mice lacking Ccnd1 show abnormal cortical layering, a phenotype that is not linked to impairment in proliferation. Instead, cortical layering defects can be partially reproduced by overexpressing a cytoplasmic dominant-negative Ccnd1 via in utero electroporation on embryos at the neurogenic stage. This suggests that cytoplasmic Ccnd1 plays a role in cortical layer formation.

To investigate the underlying mechanism, the authors performed in utero electroporation for a short period (12 h). They found that cytoplasmic Ccnd1 induced detachment of the basal process of RGCs from the basement membrane, while the cytoplasmic dominant-negative Ccnd1 resulted in the formation of more numerous and longer processes in RGCs. These findings suggest that membrane-associated cytoplasmic Ccnd1 is essential for the proper detachment of the basal process from the basement membrane at the pial surface. After 3 days, electroporated cells migrated basally toward the upper region of the CP. In control brains, most cells exhibited a unipolar or bipolar morphology or attached to the basement membrane. However, a greater population of cells electroporated with cytoplasmic Ccnd1 adopted a multipolar morphology, while those expressing cytoplasmic dominant-negative Ccnd1 were more frequently attached to the basement membrane. This process is mediated by phosphorylation of paxillin, an effector of the integrin signaling complex. Thus, cytoplasmic Ccnd1 appears to play a distinct role in cortical development, separate from its traditional function in cell cycle regulation.

The discovery of the function of cytoplasmic Ccnd1 in RGCs is intriguing. However, previous studies have reported that Ccnd1 KO mice develop normally without gross morphological defects and exhibit no major alterations in proliferation markers (Glickstein et al., 2007, 2009). Mild proliferation defects have been noted in postnatal granule progenitor cells, leading to a reduction in cerebellar size (Pogoriler et al., 2006). Given the redundancy among Ccnd family members (Satyanarayana and Kaldis, 2009), Ccnd2, which has overlapping expression in the developing cortex, may compensate for the loss of Ccnd1 in cortical proliferation (Glickstein et al., 2007, 2009). Future studies should reanalyze cortical layering defects in greater detail to clarify the unique contribution of Ccnd1.

One of the most intriguing questions is how Ccnd1 localizes specifically to the basal endfoot of RGCs. A similar localization has been observed for Ccnd2, the mRNA of which is basally transported via specific cis-acting sequences within the 3′ untranslated region (UTR) (Tsunekawa et al., 2012; Kikkawa et al., 2023). However, the authors of the preprint state that Ccnd1 lacks equivalent sequences within its 3′ UTR, suggesting an alternative transport mechanism. Ccnd2 localization plays a crucial role in asymmetrical inheritance during neurogenesis: daughter cells receiving Ccnd2 continue proliferating, while those without it undergo neuronal differentiation (Tsunekawa et al., 2012). During late neurogenesis, as RGCs elongate, the time required for Ccnd2 protein to return to the cytoplasm increases, possibly leading to a prolonged G1 phase and enhanced neurogenesis as extrapolating from the findings of other papers (Lange et al., 2009; Pilaz et al., 2009).

Currently, the transport machinery for Ccnd2 mRNA remains unknown – key questions include identifying the RNA-binding proteins responsible for carrying Ccnd2 mRNA and the motor proteins that transport RNA granules along microtubules. Similarly, uncovering the mechanisms that govern Ccnd1 localization at the basal tip of RGCs is crucial for furthering our understanding of cortical development.