Induction of axon and dendrite formation during early RGC-5 cell differentiation

Abstract

The retinal ganglion cell (RGC)-like RGC-5 line can be differentiated with staurosporine to stop dividing, extend neurites, and increase levels of several ganglion cell markers. This allows study of regulation of neurite development on a single cell basis. However, it is unclear whether the neurites induced by differentiation have features characteristic of dendrites or axons. To address this question, RGC-5 cells were differentiated with staurosporine and then immunoblotted for microtubuleassociated protein 2 (MAP2) and actin, or stained immunocytochemically for different MAP2 isoforms, tau, growth-associated protein 43 (GAP-43), or the neuronal marker β-III-tubulin. We found that staurosporine-induced differentiation led to an upregulation of MAP2c, a MAP2 isoform expressed in developing neurons. Some neurites expressed MAP2c but not the dendritic markers MAP2a and b, consistent with an axonal phenotype. Some neurites expressed the axonal marker tau in a characteristic proximal-to-distal gradient, and had GAP-43 labeling characteristic of axonal growth cones. The presence of MAP2c in differentiated RGC-5 cells is indicative of RGC-like neurite development, and the pattern of staining for the different MAP2 isoforms, as well as positivity for tau and GAP-43, indicates that differentiation induces axon-like and dendrite-like neurites.

Distinct nuclear localization patterns of DNA methyltransferases in developing and mature mammalian retina

“…This clone (AP-20) of a-MAP-2 antibody detects HMW isoforms of MAP2 on immunoblots from immature rat hippocampus (Jalava et al, 2007) and immortalized RGC progenitor line RGC-5 (Lieven et al, 2007;Wood et al, 2010). In control adult retinal lysate (6-week-old retina), we observed a band of the expected size (control lane: mouse IgG) upon immunoblot analysis ( Supplementary Fig.…”

What is the Nature of the RGC-5 Cell Line?

“…STSN represents a reliable agent to induce neuronal differentiation of RGC-5. The most commonly used protocol is 316 nM STSN in normal growth medium for usually 24 h, which can be extended for up to 5 days [10,18,24].…”

Staurosporine-induced differentiation of the RGC-5 cell line leads to apoptosis and cell death at the lowest differentiating concentration

“…The main finding of the present study is that staurosporine even at 50 nM, which is the lowest concentration that led to RGC-5 cell differentiation in our settings, induces apoptosis Since the publication of Frassetto et al [5] in 2006, which demonstrated that staurosporine differentiates RGC-5 cells to cells with numerous neuronal properties, in the absence of apoptosis induction, staurosporine has been considered to be the agent of choice to differentiate RGC-5 cells [4,[7][8][9].…”

“…Later on, the group of Levin [7] published that only a minor percentage of RGC-5 cells would be propidium iodide (PI)-positive, indicative for death, when cultured for 120 h with a staurosporine concentration of 316 nM. Since then, staurosporine has become the agent of choice for differentiating RGC-5 cells at a concentration of 316 nM for 24 h [7][8][9].…”