Decreased proliferation of cortical neural progenitor cells

Cell proliferation refers to the process of increasing the total number of cells through growth and division (Homem et al., 2015). In tissues, the rate of proliferation is influenced by multiple factors such as the initial pool of progenitor cells, the number and frequency of divisions they undergo, and the proportion of daughter cells that retain the proliferative potential (Homem et al., 2015).

During brain development, the regulation of cell proliferation is important for the production of all cell types, including neurons which assemble into functional neural circuits (Ohnuma and Harris, 2003). Neurons in the brain originate from the neuroepithelial cell (NEC) population initially undergoing symmetric, self-renewing divisions at the luminal surface of the neural tube (Malatesta et al., 2008). NECs can be identified by expression of Sox2 and Nestin, or apical surface markers like Occludin and Zonula Occludens 1 (ZO-1) (Götz and Huttner, 2005). Subsequently, NECs undergo asymmetric divisions, generating radial glial cells (RGCs), a proliferative cell population which represents the main pool of neural progenitors for all regions of the developing brain (Anthony et al., 2004; Malatesta et al., 2008).  RGCs continue to express Nestin, and additionally express glial markers such as the glutamate transporter GLAST, glial fibrillary acidic protein (GFAP) and brain-lipid-binding protein (BLBP) (Anthony et al., 2004; Götz and Huttner, 2005).

During neocortical histogenesis, RGCs divide symmetrically and asymmetrically. The majority of asymmetric divisions are neurogenic, whereby RGC division yields a new RGC and a postmitotic neuron (Noctor et al., 2004). Other asymmetric divisions generate intermediate progenitors, which can then divide symmetrically to produce two neurons (Noctor et al., 2004). Following neurogenesis, a fraction of RGCs transition to gliogenesis to give rise to glial cells, including astrocytes and oligodendrocytes, whereas the remaining RGCs exit the cell cycle through a terminal neurogenic division (Gao et al., 2014).

Proliferation in neural progenitor cells can be measured using various experimental approaches.

1. Incorporation of thymidine analogues

In preparation of cell division, eukaryotic cells duplicate their genetic material. During this process, the nucleoside subunits inserted into the newly synthesised DNA can be labelled and quantified, thus providing a means to measure cell proliferation. This is typically done by using various analogues of the nucleoside thymidine, such as tritiated thymidine (3H-thymidine), bromodeoxyuridine (BrdU) or 5-ethynyl-2'-deoxyuridine (EdU) (Cavanagh et al., 2011). These thymidine analogues can be detected by autoradiography or scintillation techniques (3H-thymidine), immunofluorescence (BrdU) and reaction with fluorescent azides (EdU) (Cavanagh et al., 2011). These approaches have been successfully applied to quantify NPC proliferation (Koch et al., 2022; Liu et al., 2018; Wu et al., 2009; Wang et al., 2005).

2. Immunostaining or flow cytometric analysis of proteins associated with the cell cycle, such as cell cycle regulators or proteins with important functions during mitosis. 

Examples: 

• Ki-67 is a nuclear protein expressed in actively dividing cells during all phases of the cell cycle, except for the resting phase (G0) (Gerdes et al., 1984). Immunofluorescent imaging of Ki-67 can be used to identify and quantify proliferating NPCs (Zhou et al., 2020).

• Proliferating cell nuclear antigen (PCNA) is an important component for DNA replication and repair, and is present throughout the cell cycle (Strzalka and Ziemienowicz, 2011; Essers et al., 2005; Kurki et al., 1986). PCNA immunofluorescence can be used to assess proliferating NPCs (Arai et al., 2011).

• Minichromosome maintenance protein 2 (MCM2) is part of the DNA replication initiation complex and is expressed throughout the cell cycle (Kearsey and Labib, 1998). MCM2 can be detected in developing and adult NPCs (Fauser et al., 2020; Dougherty et al., 2005; Sun et al., 2018).

• Phosphohistone H3 (PH3) is present during the G2 and M phases of cell division (Hendzel 1997) and has been employed to measure NPC proliferation (Xing et al., 2020; Fietz et al., 2020; Hashimoto-Torii et al., 2014; Kim et al., 2010).

3. In-situ hybridization (ISH) techniques can be applied to quantify mRNA transcripts of various markers of cycling NPCs (Yeh et al., 2013).

4. Time-lapse Imaging: Live-cell imaging techniques allow tracking of neural progenitor cells over time and assessment of their proliferation rates and patterns (Bestman et al., 2012; Keenan et al., 2010).

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