Radiotherapy is the major treatment modality for primary and metastatic brain tumors which involves the exposure of brain to ionizing radiation. Ionizing radiation can induce various detrimental pathophysiological effects in the adult brain, and Alzheimer's disease and related neurodegenerative disorders are considered to be late effects of radiation. In this study, we investigated whether ionizing radiation causes changes in tau phosphorylation in cultured primary neurons similar to that in Alzheimer's disease. We demonstrated that exposure to 0.5 or 2 Gy γ rays causes increased phosphorylation of tau protein at several phosphorylation sites in a time- and dose-dependent manner. Consistently, we also found ionizing radiation causes increased activation of GSK3β, c-Jun N-terminal kinase and extracellular signal-regulated kinase before radiation-induced increase in tau phosphorylation. Specific inhibitors of these kinases almost fully blocked radiation-induced tau phosphorylation. Our studies further revealed that oxidative stress plays an important role in ionizing radiation-induced tau phosphorylation, likely through the activation of c-Jun N-terminal kinase and extracellular signal-regulated kinase, but not GSK3β. Overall, our studies suggest that ionizing radiation may cause increased risk for development of Alzheimer's disease by promoting abnormal tau phosphorylation.
Alzheimer disease is considered as a significant radiation late effect. In this study, we investigated whether ionizing radiation causes changes in tau phosphorylation and found that γ rays cause increased tau phosphorylation at multiple sites in a dose and time-dependent manner in primary neurons which are mediated by oxidative stress-dependent activation of JNK and ERK. Oxidative stress-independent activation of GSK3β is also involved. Overall, our studies suggest that ionizing radiation may cause increased risk for Alzheimer disease development by promoting abnormal tau phosphorylation.
Radiotherapy is the major treatment modality for primary and metastatic brain tumors to improve the prognosis of patients, during which, the exposure of the CNS to ionizing radiation is unavoidable. Such radiation exposures have been associated with neurological damage and cognitive/behavioral deficits, especially in children (Greene-Schloesser and Robbins 2012; Padovani et al. 2012). While the adult brain is generally considered to be resistant to ionizing radiation because of the relative radio-resistance of post-mitotic neurons to cell killing, it appears that ionizing radiation at various doses can induce various detrimental pathophysiological effects without significant anatomical/morphological abnormality in the adult brains which likely underlie the extensively reported side effects such as memory loss, cognitive dysfunction, dementia and other neurological deficits in cancer patients who received cranial radiation therapy (Greene-Schloesser and Robbins 2012; Padovani et al. 2012). In fact, studies on irradiated human populations have suggested Alzheimer's disease (AD), the leading cause of senile dementia, and related neurodegenerative disorders as significant late effects of radiation (Manton et al. 2004). For example, a retrospective study on long-term effect of radiation therapy suggested that 30% of long-term brain tumor survivors older than 50 years who received radiotherapy developed dementia and an additional 20% demonstrated significantly impaired short-term memory function and other neurological deficits (Imperato et al. 1990). In another longitudinal study, all of the 12 patients who received whole brain radiotherapy developed progressive dementia within 5–36 months (DeAngelis et al. 1989). Mild cognitive impairment, a prodromal stage of AD, is a well-documented consequence of whole brain radiation therapy that affects 40–50% of long-term brain tumor survivors (Warrington et al. 2013).
Besides cognitive deficits, there are similarities between the degenerative cellular and molecular changes induced by radiation and degenerative process during AD. Analysis of transcriptome profiles of mouse brain tissue after whole body irradiation showed that exposure to low dose (10 cGy) γ rays induces down-regulation of neural pathways associated with memory, learning and cognitive dysfunction that are also down-regulated in human aging and AD (Lowe et al. 2009). One recent study suggested that X irradiation reduces the density of dendritic spines and changes the spine morphology likely through a reduction in the levels of cytoskeletal proteins in mature hippocampal neurons in vitro (Shirai et al. 2013). Most recent studies from two groups further demonstrated that clinically relevant γ radiation induces significant reductions in dendritic complexity and in the number and density of dendritic spines on hippocampal neurons of the dentate gyrus in vivo one month after radiation, resembling similar types of changes in AD (Chakraborti et al. 2012; Parihar and Limoli 2013). Most importantly, it was demonstrated that exposure to 56Fe particle radiation can increase amyloid pathology and exacerbate cognitive deficits in an amyloid protein precursor/presenilin 1 mouse model of AD (Cherry et al. 2012).
In addition to amyloid pathology and selective neuronal death in hippocampus and cortex, neurofibrillary tangles are also the hallmark pathology of AD which mainly contain fibrillar structures of hyperphosphorylated and aggregated tau protein, a microtubule-binding protein (Wang et al. 2013a; Zimmer et al. 2014). In fact, tau pathology correlates better than amyloid pathology with the degree of dementia in AD patients (Braak and Braak 1994). In vitro and in vivo studies suggested that tau is needed for amyloid-β neurotoxicity (Park and Ferreira 2005; Roberson et al. 2007; Iijima et al. 2010; Shipton et al. 2011). The critical role of tau alterations in the pathogenesis of AD is further supported by the recognition of so-called ‘tau-only’ dementia (Heutink 2000). As a microtubule-binding protein, tau regulates the assembly and stability of the microtubules. It has more than 40 known phosphorylation sites and becomes hyperphosphorylated in neurofibrillary tangles (Wang et al. 2013a). Although it is still up for debate as to whether tau phosphorylation drives its aggregation or is a consequence of it, it is known that, upon abnormal phosphorylation, tau reduces affinity for and dissociates from microtubules, leading to the destabilization of microtubules which likely represents a critical step leading to abnormal axonal transport and synaptic degeneration in the pathogenesis of AD (Wang et al. 2013a). The objective of this study is to investigate whether ionizing radiation causes changes in tau phosphorylation in cultured primary neurons similar to that in AD and explores the underlying molecular mechanism with a focus on oxidative stress-induced signaling transduction pathways.
Embryonic primary cortical neuron isolation and culture
Timed pregnant Sprague–Dawley rats (Harlan Inc., Indianapolis, IN, USA or Charles River Laboratories Inc., Wilmington, MA, USA) were killed following the protocol approved by the Institutional Animal Care and Use Committee at Case Western Reserve University. Primary cortical neurons were isolated from E18 rats as previously described (Kaech and Banker 2006) with some modifications. Taken briefly, brains were dissected out in Hank's balanced salt solution (Invitrogen, Carlsbad, CA, USA) and stored in Hibernate E (BrainBits) supplemented with 2% B27 (Invitrogen). Under a dissecting microscope, the meninges were removed completely with fine forceps and cortices were dissected out. Cortices were then digested in 0.25% trypsin for 15 min at 23°C followed by brief incubation in Opti-MEM (Invitrogen) supplemented with 10% fetal bovine serum and 50 units/mL DNAse I (Worthington Biochemical, Lakewood, NJ, USA). Digested cortices were further dissociated by gentle trituration with pipette until the cell suspension was homogenous and no large pieces of tissue were visible. Cortical neurons were finally collected and seeded on poly-d-lysine (Sigma, St Louis, MO, USA) 24 well plates and cultured as we described before (Wang et al. 2008b, 2013b).
Western blot analysis
Neurons were lysed with 1×Cell Lysis Buffer (Cell Signaling Technology, Beverly, MA, USA) or radio-immunoprecipitation assay buffer, plus 1 mM phenylmethylsulfonyl fluoride (Sigma) and Protease Inhibitor Cocktail (Sigma). Equal amounts of total protein extract (5 μg or 20 μg) were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to Immobilon-P (Millipore Corporation, Bedford, MA, USA). Following blocking with 10% non-fat dry milk, primary and secondary antibodies were applied as previously described (Wang et al. 2008a) and the blots developed with Immobilon Western Chemiluminescent horseradish peroxidase Substrate (Millipore). Primary antibodies used included mouse anti-phospho-tau (AT8, Pierce, Rockford, IL, USA), rabbit anti-phospho-tau (Ser262, Abcam, Cambridge, MA, USA), rabbit anti-tau (tau5, Millipore), rabbit anti-phospho-GSK3β (Ser9) (Cell signaling), rabbit anti-phospho-ERK (Cell Signaling), rabbit anti-JNK (Cell Signaling), mouse anti-actin (Millipore), and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling Technology, Danvers, MA, USA).
Irradiation and treatment
Primary neurons at days in vitro 7 were irradiated with 0, 0.5 or 2 Gy of 137Cs -generated γ-rays using a Shephard Mark I 137Cs irradiator (J. L. Shepherd). The mean dose rate of the γ-rays was 3.1 Gy/min. To evaluate the radioprotective effects of different inhibitors and antioxidant, including LiCl (Sigma), SP600125 (Cell signaling), U0126 (1 Cell signaling) and N-Acetyl-l-cysteine (NAC, Sigma), neurons were pre-treated at 1 h before irradiation. To confirm the damaging effect of ionizing radiation in the condition used in this study, radiation-induced DNA damage was determined by comet assays using Comet Assay Kits (Cell Biolabs, San Diego, CA, USA) following manufacture's instruction.
All data presented here are presented as Mean ± SEM. Data are analyzed by either student t test or one-way anova followed by Tukey's multiple comparison test. Statistical difference was considered significant if p < 0.05.
Radiation induces increased tau phosphorylation in primary cortical neurons
Both hippocampal and cortical neurons degenerate in AD (Hong-Qi et al. 2012). To investigate the effects of γ radiation on tau phosphorylation, fully differentiated mouse primary cortical neurons (days in vitro 7) were exposed to low (0.5 Gy) or high doses (2 Gy) of γ rays for various period of time. To confirm the damaging effect of γ rays, alkaline comet assay was used to detect DNA damages after ionizing radiation (Calini et al. 2002). Even at the lower dosage (Fig. 1), DNA damage was apparent 3 h post-irradiation, which was almost completely repaired 24 h post-irradiation, consistent with previous study (Calini et al. 2002).
We first measured tau phosphorylation in cells harvested 24 h post-irradiation. Phosphorylation status of several specific sites on tau known to be important in the regulation of tau function and hyperphosphorylated in AD such as Ser202/Thr205 (i.e., AT8 site) and Ser262 were determined by western blot. Both high (2 Gy) and low doses (0.5 Gy) of γ rays used in this study caused a significant increase in the phosphorylation of tau protein at both AT8 (Ser202/Thr205) and Ser262 sites (Fig. 2). The total tau level detected by tau5, which recognizes all the tau proteins, was only increased in neurons treated with 2 Gy γ rays.
We then performed a time course study to investigate tau phosphorylation in primary neurons exposed to 0.5 Gy γ rays at various post-radiation times (2 h to 48 h) (Fig. 3). While there is a trend toward increased phosphorylation at the AT8 sites at 2 h and 8 h post-radiation, it became significant only after 24 h post-radiation which lasted until 48 h post-radiation. Consistent with the dose-dependent study, there was no change in total tau levels at 24 h after exposure to 0.5 Gy γ rays. However, significant increase in the total tau level was found 48 h post-radiation. On the basis of these studies, we chose to perform detailed studies on tau phosphorylation induced by 0.5 Gy γ rays at 24 h post-radiation in later studies.
GSK3β, ERK and JNK are involved in radiation-induced tau phosphorylation
To explore the pathways involved in increased tau phosphorylation after exposure to γ rays, we first determined the activation status of several kinases (Fig. 4). Glycogen synthase kinase 3β (GSK3β) is a major tau kinase and its activity is inhibited by phosphorylation at Ser9 (Hernandez et al. 2013). We assessed GSK3β phosphorylation at Ser9 by western blot with a phospho-specific antibody in primary neurons at various time points after exposure to 0.5 Gy γ rays. As shown in Fig. 4a, the level of GSK3β phosphorylation at Ser9 was significantly reduced by more than 60% at 12 h post-radiation which persisted until 24 h, suggesting that GSK3β is activated before the significant increase in tau phosphorylation at 24 h. c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) are also activated in AD brain and believed to be involved in tau phosphorylation in AD (Perry et al. 1999; Zhu et al. 2001). We also assessed their activation status by phospho-specific antibodies (Fig. 4b). Similar to GSK3β, ERK phosphorylation was significantly increased since 12 h post-radiation and persisted until 24 h. While there is a trend toward increased JNK phosphorylation/activation as early as 1 h post-radiation, it became significant only after 24 h post-radiation.
We next determined whether radiation-induced activation of these kinases is causally involved in increased tau phosphorylation by applying inhibitors that specifically inhibit these kinases (Fig. 5a–c). Lithium is a direct inhibitor of GSK-3 and has been widely used to test the potential role of GSK-3 in multiple settings (Zhang et al. 2003). Pre-treatment of LiCl was found to decrease levels of both total tau and phosphorylated tau at basal condition. We found that it also prevented radiation-induced changes in tau phosphorylation at AT8 sites (Fig. 5a). SP600125 is a specific inhibitor for JNK. It had no effect on total tau levels but decreased tau phosphorylation levels at basal condition. It also completely prevented radiation-induced tau phosphorylation (Fig. 4b). U0126, a specific inhibitor for MEK1/2 which are the MAPKKs upstream of ERK1/2, had no effect on either total tau or tau phosphorylation levels under basal condition, but specifically prevented radiation-induced increase in tau phosphorylation (Fig. 5c).
Role of oxidative stress in radiation-induced tau phosphorylation
It is known that ionizing radiation induced DNA damage (Fig. 1) and oxidative stress (Azzam et al. 2012). Previous studies demonstrated that oxidative stress could lead to increased tau phosphorylation in neuronal cells (Zhu et al. 2005; Su et al. 2010). We, therefore, tested whether radiation-induced oxidative stress is involved in tau phosphorylation, primary neurons were pre-treated with NAC, a widely used antioxidant, and tau phosphorylation was determined 24 h after exposure to 0.5 Gy γ rays. Clearly, NAC decreased both total tau and phosphorylated tau levels under basal condition and it also prevented radiation-induced tau phosphorylation (Fig. 5d).
Because GSK3β, JNK and ERK are involved in radiation-induced tau phosphorylation, and it is known that GSK3β, JNK and ERK could be activated by oxidative stress (Zhu et al. 2002, 2004, 2007; Dal Santo et al. 2012), we then determined whether the activation of these kinases is downstream to radiation-induced oxidative stress. Indeed, as shown in Fig. 6, NAC almost completely prevents radiation-induced JNK phosphorylation and ERK phosphorylation, which places JNK and ERK activation downstream of oxidative stress. However, NAC had little effect on radiation-induced reduction in GSK3β phosphorylation, suggesting that NAC could not prevent radiation-induced GSK3β activation.
While mature neurons are resistant to radiation-induced cell death, prior studies demonstrated that they were sensitive to radiation-induced cellular and molecular changes such as reduced cytoskeletal proteins associated with reduced dendritic spines (Shirai et al. 2013). In this study, we demonstrated that exposure to 0.5 or 2 Gy γ rays causes increased phosphorylation of tau protein at several phosphorylation sites in a time and dose-dependent manner. To the best of our knowledge, this is the first study to examine the effect of ionizing radiation on tau phosphorylation. Consistently, we also found ionizing radiation causes increased activation of several candidate tau kinases such as GSK3β, JNK and ERK before radiation-induced increase in tau phosphorylation. Studies with specific inhibitors of these kinases further confirmed that activation of GSK3β, JNK and ERK contributed to radiation-induced tau phosphorylation. Our studies revealed that oxidative stress plays an important role in ionizing radiation-induced tau phosphorylation, however, it appears that it is the activation of JNK and ERK, but not GSK3β, that directly mediates the effects of oxidative stress on radiation-induced tau phosphorylation.
The major finding of this study is that ionizing radiation, even at relatively low levels (0.5 and 2 Gy), causes increased tau phosphorylation at multiple sites in primary neurons in vitro. It is of importance to note that these sites are known to be hyperphosphorylated in AD (Wang et al. 2013a). Since increased tau phosphorylation occurs during the course of disease and likely plays a critical role in the formation of neurofibrillary pathology in AD, this study demonstrated that ionizing radiation induces biochemical changes in neurons which resemble similar types of changes in AD, thus providing new evidence to support the notion that ionizing radiation may increase the risk for AD development. Nevertheless, we must admit that only three AD-relevant sites out of the many potential phosphorylation sites were determined in this study, the phosphorylation status of other sites may need to be further determined. Although the brain is able to tolerate significant radiation doses (~ 50–60 Gy) without obvious anatomical/morphological injury or tissue toxicity (Greene-Schloesser and Robbins 2012; Padovani et al. 2012), pre-clinical studies suggested radiation around 10 Gy, much lower than the clinically used doses, can induce acute and late effects on cognitive dysfunction (Chakraborti et al. 2012; Parihar and Limoli 2013). Given the even lower doses we used (as low as 0.5 Gy to 2 Gy), our studies suggested that the radiation doses that can be safely administered may need to be further limited to avoid biochemical/molecular changes that may eventually manifest as AD-type pathology. During radiotherapy, large doses were usually given in 1–2 Gy fractions repeatedly over a period of time (Greene-Schloesser and Robbins 2012; Padovani et al. 2012), although the effects of repeated exposure to 0.5–2 Gy ionizing radiation should be further determined, our studies suggested that such clinical practice will very likely cause unwanted deleterious biochemical sequelae in the neuronal populations that are vulnerable to AD. Dose limits for various organs are imposed by NASA to limit or prevent degenerative tissue disease that could occur post-mission. The 30-day-limit for the CNS is 0.5 Gy-equivalent and one-year limit is 1 Gy-equivalent. Our results call for more detailed study of whether an accumulative dose of 0.5 or 1 Gy with repeated lower dose factions could elicit similar tau changes.
Ser262 locates within the first microtubule-binding repeats of tau protein. Its phosphorylation drastically reduced the binding of tau to microtubule and was suggested to play a key role in the regulation of microtubule stabilization (Biernat et al. 1993). Phosphorylation at Ser202/Thr205 (i.e., AT8 site) has a similar effect which may help tau to convert into a toxic molecule (Wang et al. 2013a). Indeed, it has been shown by several groups both in vitro and in vivo that tau expression and phosphorylation are required for, and directly mediate, the toxicity of amyloid-β (Park and Ferreira 2005; Roberson et al. 2007; Iijima et al. 2010; Shipton et al. 2011). Nevertheless, recent studies demonstrated that neurons with neurofibrillary tangles may function normally (Kuchibhotla et al. 2014) and can survive for decades (Morsch et al. 1999; de Calignon et al. 2009), suggesting that the formation of neurofibrillary tangles may represent a necessary, or even critical, adaptation for neurons to continue their survival in an adverse environment. Along this line of reasoning, it was reported that oxidative damage is reduced by the formation of neurofibrillary lesions (Nunomura et al. 2001), suggesting that tau phosphorylation and/or aggregation may serve antioxidant function. Therefore, the possibility that ionizing radiation-induced tau phosphorylation may be an adaptation process in response to increased DNA damage or oxidative stress (induced by ionizing radiation) to ensure the neuronal survival and/or preserve neuronal structure/function cannot be ruled out.
Tau phosphorylation is regulated by several protein kinases including GSK3β and MAPKs (Zhu et al. 2002; Takashima 2006). We performed a time course study to investigate the temporal sequence of events between tau phosphorylation and kinases activation, hoping to reveal the major kinases involved in ionizing radiation-induced tau phosphorylation. We found that both GSK3β and ERK became significantly activated 12 h post-radiation, preceding the significant increase in tau phosphorylation at 24 h. JNK also demonstrated a time-dependent activation which reached significance around 24 h post-radiation, the time when increased tau phosphorylation became significant. Furthermore, treatment with LiCl (inhibitor of GSK3β), SP600125 (inhibitor of JNK) or U0126 (inhibitor of ERK pathway) could almost completely prevent increased tau phosphorylation. These findings demonstrated that these three kinases are causally involved in ionizing radiation-induced increased tau phosphorylation. It is a bit surprising that each of these specific inhibitors could almost completely prevent the increased tau phosphorylation induced by ionizing radiation. Such a fact likely suggests that these three kinases work synergistically on the AT8 site. One possibility is that some of these kinases may be involved in the phosphorylation of sites other than AT8 that has the priming effects on the phosphorylation of AT8 site by other kinases (Wang et al. 2013a). Our results also demonstrated that GSK3β and JNK play an important role in the phosphorylation of tau at basal levels. In fact, GSK3β activity is also important for the tau expression levels, although the mechanism is not clear. While we focused on GSK3β, ERK and JNK in this study, it does not necessarily exclude the possibility that changes in other potential tau kinases and phosphatases may also contribute to ionizing radiation-induced tau phosphorylation, which merits further investigation, especially if additional phosphorylation sites are involved.
It has been demonstrated that ionizing radiation induces DNA damage and oxidative stress in cultured cells (Azzam et al. 2012). We further determined the involvement of oxidative stress and demonstrated that oxidative stress likely mediates the increased tau phosphorylation induced by ionizing radiation since antioxidant (i.e., NAC) pre-treatment could completely prevent increased tau phosphorylation. This study also demonstrated the usefulness of antioxidants in preventing ionizing radiation-induced damaging effect (Sun et al. 2013). Interestingly, NAC pre-treatment blocked radiation-induced activation of JNK and ERK but had no effect on the activation of GSK3β, suggesting that it is likely that oxidative stress mediates radiation-induced tau phosphorylation through activation of the JNK and ERK pathway but not GSK3β pathway in our system.
In conclusion, we demonstrated that exposure to 0.5 and 2 Gy γ rays caused increased tau phosphorylations at several sites resembling tau changes in AD. This is likely mediated by oxidative stress-induced activation of the JNK and ERK pathway along with some parallel mechanism(s) acting through the activation of GSK3β. These studies suggest that ionizing radiation may cause increased risk for AD development by promoting abnormal tau phosphorylation, implying that doses that can be safely administered to human brain during radiotherapy may be further limited to avoid late effects of dementia.
Acknowledgments and conflict of interest disclosure
This study is supported by grants from National Aeronautics and Space Administration (NNX11AC27G to X. Z.). L.L. is supported by Advanced Studies and Visiting Program for Young College Teachers at Shanghai Municipal Education Commission and T. Z. is supported by the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Municipal Education Commission and Key Disciplines of Clinical Integrative Chinese and Western Medicine at State Administration of Traditional Chinese Medicine of the People's Republic of China.
All experiments were conducted in compliance with the ARRIVE guidelines. The authors have no conflict of interest to declare.