In this report, we demonstrated that the DNA NHEJ repair pathway is a mechanism, which we believe to be novel, underlying lithium-mediated protection of normal mouse hippocampal neurons from IR-induced apoptosis. We demonstrated that IR-induced chromosomal breaks were repaired with greater efficiency in lithium-treated neuronal cells, as observed by the neutral comet assay. Furthermore, lithium enhanced DNA-PK–dependent NHEJ repair activity, which was evident not only by the increased number of cells with elevated T2609 foci after IR but also by the enhanced double-stranded DNA end-joining capacity by lithium. The increase in DNA-PK–dependent NHEJ repair activity was associated with a subsequent decrease in the percentage of cells with persistent IR-induced γ-H2AX foci, which are commonly used in situ markers of DNA DSBs. The addition of the DNA-PK inhibitor IC86621 attenuated lithium-mediated protection of hippocampal cells from IR-induced apoptosis. Furthermore, this protection was abolished in NHEJ repair–deficient SCID mice. Importantly, all of these findings were not evident in the mouse glioma cancer cell line GL261. Our present findings provide direct biochemical and genetic evidence supporting the notion that lithium protects irradiated noncancerous neuronal cells from apoptosis, in part by enhancing DNA-PK–dependent NHEJ repair.
Interestingly, although our results from the 2 DSB assays both suggest an effect by lithium on repair but not induction of DSBs after IR, the kinetics of IR-induced DNA damage and/or repair as assessed by the neutral comet assay and γ-H2AX foci were slightly different: the maximal comet tail moment was detected 15 minutes after IR via the neutral comet assay versus the delayed γ-H2AX foci peak at 1 hour after IR. This observation is consistent with several reports suggesting that while 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 (10
). Additionally, the formation of γ-H2AX foci typically requires the recruitment of DNA damage response proteins by the DSB, which may delay foci formation. Alternatively, there is the potential possibility that lithium could affect H2AX phosphorylation or subcellular localization. However, our biochemical and genetic data (Figures and ) support the notion that lithium enhances repair of IR-induced DNA damage through enhancing NHEJ repair.
The mechanisms of lithium-mediated neuroprotection have been previously attributed to activation of the prosurvival PI3K/Akt pathway and inhibition of the GSK-3 enzyme (6
). In response to DNA damage, Akt is ultimately activated as part of the cellular survival response (40
). This activation has been shown to require DNA-PK, which is an efficient kinase that can phosphorylate Akt at serine 473 both in vitro and in vivo after DNA damage (42
). Regulation of these pathways in turn decreases the levels of the proapoptotic proteins p53 and Bax while enhancing expression of the antiapoptotic protein Bcl-2 (6
). Several reports have also demonstrated stabilization of the p53-dependent cell cycle checkpoint protein p21 by activation of Akt or suppression of GSK-3 (47
). Furthermore, recent studies have revealed a potential dependence of IR-induced p53 response on activation of DNA-PK and Akt and subsequent inhibition of GSK-3 and mdm2 (42
). As lithium affects the Akt and GSK-3 pathways, our present findings could potentially link lithium and inhibition of IR-induced apoptosis through regulation of DNA repair, in particular DNA-PK and NHEJ repair.
In addition to effects on cell survival/apoptotic pathways, lithium has previously been reported to alter cell cycle distribution in treated cells, in particular at the G2
/M checkpoints (51
). This is thought to be secondary to lithium-mediated activation of checkpoint kinase 1 (Chk1), a critical enzyme in DNA damage-induced G2
/M arrest. Interestingly, we did not observe any changes in cell cycle distribution in the HT-22 hippocampal neurons at the dose and time frames used in this study (Supplemental Figure 1; available online with this article; doi:10.1172/JCI34051DS1). This may be a neuron-specific effect.
Our findings suggest a role of the DNA-PK–dependent NHEJ repair pathway in lithium-mediated neuroprotection. Consistent with a role of DNA-PK in this effect, the DNA-PK inhibitor IC86621 attenuated lithium-mediated protection of hippocampal neurons from IR-induced apoptosis. DNA-PK inhibitors are well-established radiation sensitizers (33
). However, given our data suggesting a role of DNA-PK in lithium-mediated neuroprotection, the use of these inhibitors should be approached cautiously, as these compounds may decrease the therapeutic index as a result of sensitization of normal cells.
The use of lithium as a neuroprotector against brain injury has been proposed for other insults to the brain in addition to IR. Evidence suggests that lithium can protect the brain in stroke and oxidative stress, and it has been shown to reduce brain damage in animal models of neurodegenerative diseases and stroke (6
). The doses of lithium used in our study are comparable to those in previous reports. Clinically, however, lithium has a relatively low therapeutic index, requiring careful blood level monitoring in light of its well-known toxicities (55
), both acute (including gastrointestinal discomforts such as nausea, diarrhea, vomiting, and stomach pain; muscular weakness; thirst and frequent urination; feelings of being dazed, sleepy, and tired; and hand tremor) and subacute (hand tremor; constant thirst; and abundant urine excretion). In addition, the therapeutic action of lithium is delayed, requiring 5–7 days prophylaxis prior to the initiation of IR therapy to reach steady-state concentrations (56
). We are currently conducting a phase I study to evaluate the safety and toxicity of lithium as a neuroprotective agent during cranial radiotherapy.
Lithium-mediated neuroprotection, however, did not occur in mouse glioma cancer cells (Figure and ref. 6
). This differential effect of lithium in glioma cancer cells versus hippocampal neuronal cells provides the potential to improve the therapeutic index of cranial IR. Glioma cells are notoriously resistant to radiation and chemotherapy. As further evidence for the resistance of glioma cells to IR, we did not see the number of GL261 mouse glioma cells or D54 human glioma cells with IR-induced γ-H2AX foci demonstrate the vast increase seen in normal hippocampal neurons (maximum 8% in glioma cells versus maximum 30%–50% in neurons; Figure and Figure A). This low level of cells with persistent foci was associated with an efficient induction of DNA-PK in glioma cells by IR (maximum 17 fold; Figure B), which suggests that the intrinsic resistance to IR-induced DSBs in glioma cells may be, at least in part, caused by an enhanced capacity of glioma cells for DNA-PK–dependent repair. Rad51 foci were persistent and continued to increase in GL261 cells relative to hippocampal neuronal cells, particularly at 4 hours after IR (Figure C). Furthermore, IR-induced Rad51 foci formation was not affected by lithium pretreatment in these tumor cells, but was slightly suppressed in normal neuron cells. It would be interesting to determine whether glioma tumor cells also possess an enhanced capacity for HR in future studies. As HR is considered to be responsible for the repair of persistent complex lesions that cannot be resolved by simple NHEJ repair, glioma tumor cells may be capable of efficiently using both DNA repair systems to ensure survival after DNA insults. Furthermore, one could potentially take advantage of the differential induction of HR between normal and cancer cells to improve therapeutic index.
Despite its high resistance to therapy, the standard of care for gliomas still remains concurrent chemotherapy and cranial IR. In addition, CNS radiation continues to be a vital treatment modality for pediatric brain tumors and leukemia. Long-term morbidity from this treatment is devastating for these patients and their families and is of significant concern for the physician. Substantial evidence supports the idea of lithium-mediated neuroprotection from IR-induced neuronal apoptosis. Early results from our phase I trial suggest the feasibility of lithium treatment before and during cranial IR in patients with brain metastases (our unpublished observations). In addition, based on our findings in the present study, molecular-targeted therapies to enhance DNA repair may provide neuroprotection during cranial IR.