This study provides biochemical and genetic evidence supporting that inhibition of GSK3 enhances the repair of IR-induced DSBs in mouse hippocampal neurons and, importantly, not in glioma cancer cells. Specifically, we found that inhibition of GSK3 accelerated the resolution of persistent IR-induced DSBs as measured by the neutral comet assay and IR-induced γ-H2AX foci both in cultured hippocampal neurons and in mice hippocampus. This was associated with an enhanced DSB end-joining capacity in GSK3-inhibited neurons. These results were validated via genetic manipulation of GSK3β activity using a dominant-negative kinase inactive GSK3β mutant and shRNA-mediated silencing of GSK3β expression. These findings demonstrate that one of the potential underlying mechanisms by which GSK3β inhibition protects IR-induced apoptosis of hippocampal neurons and attenuates neurocognitive function in irradiated mice, as demonstrated previously,9
may be through enhanced end-joining-mediated repair of IR-induced DSBs.
GSK3β is enriched in the brain and has been implicated in a number of prevalent disorders, such as Alzheimer disease, schizophrenia, Parkinson disease, and bipolar disorder.44–47
Inhibition of GSK3 has been shown to alleviate some of these processes. The enhanced repair of IR-induced DSBs by GSK3 inhibition may provide a novel mechanism by which neurons can survive from insults to the brain. It remains to be seen whether the neuroprotective effects of GSK3β inhibition outside of IR-induced apoptosis is similarly due to enhanced repair of DNA damage induced by other insults.
The GSK3 family consists of both GSK3α and GSK3β. The inhibitor used in this study, SB216763 (Tocris Biosciences), inhibits both isoforms of GSK. It is possible that the effects on repair observed in this study may also include a GSK3α-mediated response. However, our experiment using a kinase inactive GSK3β mutant and specific shRNA targeting GSK3β result in a 2–3-fold increase in end-joining capacity, which is similar to inhibitor-treated cells and suggests that the effects are most likely due to inhibition of GSK3β. Nevertheless, a role of GSK3α cannot be entirely ruled out. Whether GSK3α also plays a role in regulation DNA damage repair is an interesting question and needs to be investigated in future studies.
Although the present data from the neutral comet assay and γ-H2AX foci both suggest an enhanced repair by GSK3β inhibition, the kinetics of IR-induced DNA damage/repair are slightly different (ie, maximal comet tail moment detected 15 min after IR via the neutral comet assay versus delayed γ-H2AX foci peak at 1 h after IR). However, this may be explained by the fact that, although the neutral comet assay allows for the direct measurement of IR-induced DSBs, persistent or low-level (<50 strand breaks) DNA damage can go undetected, especially at lower radiation doses.39,40,48
In addition, the comet assay can be affected by changes in chromatin structure independent of DSBs. Futhermore, γ-H2AX foci formation typically requires the recruitment of the DNA damage response proteins by the DSB, which may “delay” foci formation. Alternatively, an effect on H2AX phosphorylation or subcellular localization cannot be ruled out. However, results from the in vivo plasmid-based NHEJ assay supports the notion that the observed effects of GSK3 inhibition on comet tails and γ-H2AX foci more likely represent persistent DSBs due to suppressed NHEJ-mediated DSB repair in cells without GSK3 inhibition.
The enhanced end-joining capacity of GSK3β inhibited neurons was shown via an end-joining assay of episomes. This assay has been shown to represent the end-joining capacity of cells and to be compatible with recently developed chromosomally integrated reporter substrates.42,43
This is in contrast to homologous recombination-mediated repair (HR), which is largely dependent on DNA replication processes and thus cannot be readily measured in nonreplicating episomal plasmids. Both biochemical and genetic data support a role of GSK3β inhibition and enhanced end-joining repair of DSBs. In contrast, however, GSK3β overexpression did not reduce end-joining capacity (Figure B). Because GSK3β is ubiquitously expressed and control of its function is not dictated by its expression level, this result is not unexpected.
It has been well established that radiation induces DSBs which is lethal if not repaired.13–15
We have recently observed that lithium-mediated neuroprotection from IR-induced apoptosis also occurs by increasing the NHEJ pathway.12
Our results support the hypothesis that GSK3β may be the target by which lithium enhances the repair capacity of neurons. GSK3β has also been reported to regulate the transcriptional activity of p53 through phosphorylation, which is an important regulator of DNA damage–induced apoptosis.49
Interestingly, the priming event of this regulation involves DNA-PK–mediated phosphorylation of p53. Additional investigation to dissect the role of GSK3 in the cross-talk between DNA repair pathways and apoptotic pathways is warranted. In addition, whether the enhanced repair capacity through GSK3β inhibition depends on cellular p53 status is an interesting avenue of future studies.
GSK3β is an enzyme that regulates glycogen synthesis in response to insulin. It is a ubiquitously expressed serine/threonine protein kinase that is a critical downstream element of the PI3 kinase/Akt cell survival pathway. Activation of this PI3 kinase/Akt pathway results in inhibition of GSK3β and subsequent cell survival or proliferation. The results from this study suggest a potential and interesting link between metabolic pathways and response to DNA damage. It is intriguing to hypothesize a model whereby cells under metabolic stress (nutrient deprivation) will activate the prosurvival Akt pathway to not only inhibit cell death but also activate DNA damage repair pathways. In addition, whether the mechanism by which neuroprotection occurs is through direct inhibition of GSK3β versus inhibition of downstream insulin or wnt signaling pathways is an interesting future study.
Radiation treatment approaches have been designed to spare the anatomic regions in the brain—namely, the hippocampus—which contain normal neural progenitor cells.50–54
These include tomotherapy and intensity-modulated radiotherapy techniques to specifically protect the hippocampus and reduce the neurocognitive toxicity following cranial irradiation.50–54
Many have proposed that the hippocampal neuron progenitor cells are the most affected during radiation-induced neurocognitive decline.55
Consistent with these findings, IR-induced cognitive deficit involves apoptosis in the neurogenic region of the hippocampus and the subsequent diminished neurogenesis in the hippocampus.56–60
Specifically, inhibition of GSK3β has been shown to protect neural progenitor cells from apoptosis induced by oxidative and other stresses.31,32
It would be interesting to investigate whether the radioprotective effects of GSK3ß inhibition occurs in normal neural progenitor cells.
Importantly, we did not observe enhanced repair in GL261 and D54 glioma tumor cells following GSK3 inhibition. This is consistent with our previous findings that lithium and inhibitors of GSK3 did not protect cancer cells from IR-induced apoptosis.7,9,12
One possible explanation may be the already enhanced repair capacity of cancer cells. In support of this notion, glioblastoma cells with hyperactivated Akt signaling, which can inhibit GSK3β activity, exhibit increased DSB repair capacity and radioresistance.61
In addition, the downstream targets of GSK3β in glioblastoma cells were not further altered after IR or GSK3β inhibition (Figure ), suggesting a potential explanation for the differential effect of GSK3 inhibition between hippocampal neurons and cancer. In addition, several recent studies demonstrate the heterogeneity that exists in glioma cells with regard to their response to GSK3 inhibition, from blockade of glioma cell differentiation to induction of cell death.62,63
This differential response clearly illustrates the need for further investigation of the effects of GSK3 inhibition in a larger panel of gliomas, including glioma stem-like cells, which have been implicated to contribute to treatment resistance, to determine whether cancer cells are resistant to GSK3 inhibition–mediated radioprotection.
Another possible explanation may be the p53 status in these cancer cells. We previously reported that inhibition of GSK3β in irradiated neurons prevented IR-induced p53 accumulation, suggesting a potential link between p53, GSK3β, and neuroprotection.9
Interestingly, genetic knockout of NHEJ proteins in mice has also been shown to increase p53-dependent cell death of neurons and results in deficits in neurogenesis.64–67
In the cancer cells examined in this study, p53 was dysfunctional. It is possible that GSK3β inhibition can protect p53 proficient cancer cells. Future investigations to address the contributing factors and the underlying mechanisms for the lack of protection in cancer cells are warranted. In addition, it may be important to determine the p53 status of each individual patient's cancer prior to the use of these neuroprotective compounds for protection of normal tissues.
Our previous studies reveal similar effects with lithium. Lithium, however, requires a long 7-day prophylaxis, has a narrow therapeutic window, and lacks specificity. Because of these reasons, the use of GSK3β inhibitors as neuroprotectors provides obvious advantages over lithium. Prophylaxis with GSK3β inhibitors can start as early as 16 h before commencement of cranial IR, eliminating the need to wait 1 week before initializing radiation treatment that is necessary with lithium. In addition, a theoretical increased specificity can be achieved using these inhibitors, potentially decreasing the unwanted side effects of neuroprotectors from off-target effects.
Taken together, this study provides direct biochemical and genetic evidence supporting the notion that inhibition of GSK3β increases DNA repair in irradiated noncancerous neuronal cells. This not only links GSK3β signaling to DNA repair pathways but also generate novel targets for the development of neuroprotective drugs for use during whole brain radiation. Furthermore, these findings warrant future clinical investigations of neuroprotection with GSK3β inhibitors during cranial irradiation, especially in the pediatric population.
Conflict of interest statement. None declared.