HDAC inhibition–mediated differentiation of RGC-5 cells was synergistic with neurotrophic factors that increase RGC survival. This combination resulted in increased viability, differentiated proportion, longest neurite length, and number of neurites extending from the soma. Only 50% of differentiated and neurotrophin-treated RGC-5 cells were viable on day 3, similar to what was seen in primary P1 RGCs treated with neurotrophic factors but less than the 90% survival rate of neurotrophic factor–treated P8 RGCs.10
The likely reason for this difference is that rat RGCs undergo target deprivation-induced cell death during P1 to P415
and RGC-5 cells are immortalized at P1, at the major peak of developmental cell death. Surprisingly, RGC neurotrophic factors also had noticeable effects on HDAC-inhibitor–treated cell neurite outgrowth as early as 24 hours (before significant cell death occurred). This finding strongly suggests that adding RGC survival factors to HDAC inhibition had an effect beyond simply increasing cell viability; it also had an effect on morphology, even though the survival factors themselves showed no differentiating activity when used alone. Unlike what was seen with staurosporine-differentiated RGC-5 cells, the length of the longest neurite and the primary neurite count did not change over time with HDAC inhibition, in the presence or absence of RGC survival factors. This lack of effect on neurite extension was similar to what others have observed for primary cultures of RGCs, where neurotrophic factors alone do not induce significant axon elongation in the absence of suitable chemoattractive cues, such as growth on laminin or localized gradients of attractive/repulsive neurotrophic factors.16,17
Similarly, the fact that the number of differentiated cells had a bimodal distribution after HDAC inhibition but a graded distribution with staurosporine supports the notion that HDAC inhibition results in differentiation similar to the in vivo differentiation process. Cellular differentiation is similar to a switch, with cells passing rapidly from undifferentiated to differentiated states in a transcriptionally regulated process.18,19
The finding of a bimodal distribution of cells, clustered in undifferentiated and differentiated states, is consistent with turning of an all-or-nothing differentiation switch, a pattern seen only with transcription-dependent HDAC inhibition.
More support for HDAC inhibition– differentiated RGC-5 cells behaving similarly to RGCs is the effect of conditioned media. RGCs in vitro exhibit conditioned media effects, whereby survival is significantly enhanced by plating at higher rather than lower densities.10
Our results with HDAC inhibition (but not differentiated mediated by staurosporine) were similar in that differentiation was enhanced by increasing densities at all levels except the highest.
The observation that tau localizes predominantly to the distal portions of single neurites in RGC-5 cells differentiated by HDAC inhibition suggests that true neuritogenesis occurred, not process formation caused by actin stress fibers, as has been reported in v-ras
–transformed NIH 3T3 cells treated with TSA.20
Although tau staining of distal aspects of neurites is actin dependent, the localization of tau specifically to one neurite is actin independent.11
In other words, actin stress fiber formation cannot explain the enrichment of tau observed localized to one particular neurite, which is indicative of axons. Furthermore, the observed diffuse staining of MAP2 in all other neurites is consistent with these neurites being dendrites.
A likely mechanism for RGC-5 differentiation with HDAC inhibition is removal of the neuron gene–specific transcription repressor neuron-restrictive silencing factor (NRSF) from neuron-restrictive silencing element (NRSE). This process is a necessary, though not necessarily sufficient, component of in vivo central nervous system neuronal differentiation.7
NRSEs are located in the promoter regions of many neuron-specific genes. NRSF, when bound to NRSE, sterically prevents transcription of these genes. TSA, however, inhibits the complexing of NRSE with NRSF.21
NRSF recruitment to NRSE is mediated by the HDAC 1– and 2–containing transcription corepressor Sin3/HDAC1,2.22,23
When HDAC activity is inhibited, NRSE is released from NRSF, permitting transcription factor access to genes downstream of NRSE-containing promoters. This mechanism would also explain why, in our preliminary screening of HDAC inhibitors, the class 3 HDAC inhibitor nicotinamide24
did not induce differentiation in RGC-5 cells. Sin3/HDAC1,2 is composed of only TSA-sensitive class 1 and class 2 HDACs. Our observations that Brn-3a and Brn-3b, transcription factors necessary for RGC differentiation,25
were upregulated on TSA treatment fits this model because both are regulated by Math5 and NRSF and are upregulated during in vivo RGC differentiation.26
Although staurosporine-differentiation is not dependent on transcription, in contrast to HDAC inhibition-mediated differentiation, a subthreshold concentration of staurosporine (1 nM) caused longer longest neurites and increased the number of neurites in the presence of HDAC inhibition. These data imply that though these two mechanisms differ, some common downstream transduction processes likely take place.
In summary, we found that HDAC inhibition of a committed retinal neuronal cell line, RGC-5, results in differentiation similar to that occurring during development and different from that observed with staurosporine-mediated differentiation. HDAC inhibition could potentially be used to differentiate RGC progenitors for use in repopulating the retina in optic neuropathies, resulting in RGCs that are more physiologically neurotrophic factor dependent.