The mammalian CNS, particularly the brain, represents an important target-organ that might be injured following radiation exposure (e.g. iatrogenic origins, nuclear accidents, war/terrorist attacks) resulting in a drastic reduction of either survival or long-term quality of life. The detrimental effects of brain radiation exposure might occur despite the implementation of preventative measures (as in the clinical setting) or prophylactic protocols (as after radiation contamination due to nuclear disaster or attack)1,2. While most radiation research investigations focus on minimizing long-term neurological consequences in the context of specific radiotherapy protocols (for example, using low-dose fractionated radiation for primary brain tumors) or on looking for prophylactic tools to minimize systemic injurious effects of radiation (e.g. using a compound such as captopril immediately after radiation), few studies have actually investigated the possible role of low-dose brain radiation to beneficially interfere with pathological processes occurring in neurodegenerative disorders such as Alzheimer’s disease (AD) for example - a disorder associated with pathological accumulation of intracellular (pTau) and extracellular (β-amyloid) misfolded proteins across different vulnerable regions of the brain37. Interestingly, though, a recent study in transgenic rodents described reduction of β-amyloid plaque loads in the radiation-treated animals38. However, no earlier pTau or APP expression level changes across different brain regions have ever been described after a low-dose of total-body radiation using a large normal mammalian brain.
We have focused on the identification of some possible early molecular (protein expression level changes) and neuropathological consequences of low-dose total-body radiation (1.79 Gy total dose, which is a dose comparable to the ones commonly administered in the current radiotherapeutics protocols for brain tumors) after a relatively short period of time (~ 4 weeks) in a large mammalian brain (swine), and we have measured:
- Lower levels of pTau (CP13) in the FC and H region of RAD- vs. SH-animals;
- Lower levels of APP and GAP43 in the CRB of RAD- vs. SH-animals;
- Higher level of GFAP in the H of RAD- vs. SH-animals;
- Higher level of DNA-polymerase-β in the FC of RAD- vs. SH-animals.
No significant changes were found in microglial activation signals (as detected by either soluble IBA–1 levels or immunohistochemistry/morphological changes) or loss of myelin (as detected by levels of soluble MBP or through LFB stain assessment). Importantly, these findings on early molecular changes in a normal (non-transgenic) large mammalian brain in the absence of obvious neurohistological changes or brain lesions support the hypothesis that early specific molecular changes occur and are detectable after 4 weeks post-radiation across different brain regions exposed to the same fractionated low-dose of total-body radiation. Remarkably, the lower levels of pTau, APP and GAP43 observed across the different examined regions of the swine brain were not associated with apparent histological or morphological changes or activation signs of microglial cells or demyelination phenomena, which are two processes normally observed at a later stage during the progression of the post-radiation process (>6 months/years after post-radiation)33. In addition, these novel findings show that the neuroanatomical regions involved in radiation-induced reduction of the examined protein expression levels are not uniformly altered along the same direction across different regions of the brain. Rather, these new data indicate a cellular response that involves different brain regions at different degrees and rates of changes along the varying post-radiation time points. This differential post-radiation timing is likely due to the intrinsic radiosensitivity to γ-rays in each specific cerebral region (based, for example, on their intrinsic genetically-determined metabolic rate) as well as possibly related to the total radiation dose administered39–41. These differential radiosensitivity-related aspects across different brain regions as well as their possible controlled modulation could lead to precise and effective neuroanatomical-based brain radiation protocols selectively tuned for a specific region of the brain, or a group of disease-related neuroanatomical regions, in the context of a specific brain disease and local brain tissue vulnerability.
Notably, the reduction of pTau levels induced by total-body radiation was observed in the absence of corresponding significant changes of all-Tau forms (HT7) across all the examined regions. The unchanged levels of total-Tau protein were also indirectly confirmed by the unchanged levels of β-tubulin (Figure 2),, a protein strictly associated with Tau for the stabilization of microtubules42. It appears that one of the early effects of the low-dose radiation on intracellular Tau levels is the change of the ratios between phosphorylated and dephosphorylated forms. Intriguingly, the observed phenomenon of decreased pTau levels induced by low-dose radiation contrasts with the opposite phenomenon observed in AD pathogenesis (and other tauopathies), where a progressive process of Tau hyperphosphorylation is instead associated with increased levels of insoluble pTau and consequent accumulation in neurons and astroglial cells across the cerebral cortex43,44. Similarly, although through different biochemical mechanisms, the observed decrease of APP levels could also represent another pathway through which low-dose brain radiation could be employed as a tool to reduce the biochemical substrates of β-amyloid plaques and consequently reduce the related toxic effects45.
Importantly, these new findings need to be confirmed in terms of safety and long-term efficacy by using different types of animal experiments and clinical trials in humans. Some clinical trials employing radiation in the context of a specific neurodegenerative disorder such AD have been recently proposed and approved (https://clinicaltrials.gov).
If confirmed at a larger scale, these new experimental data could have a major clinical and societal impact for the use of low-dose brain radiation as new neuro-radiotherapeutic tool for different neurodegenerative conditions, including, but not limited to, tauopathies such as AD, progressive supranuclear palsy (PSP) and chronic traumatic encephalopathy (CTE).
Intriguingly, the hyperphosphorylation process of Tau (as measured, for example, by CP13 levels) is a process normally present during normal brain development, and it is one of the earliest events that occur during the pathogenesis of different neurodegenerative disorders46. Tau hyperphosphorylation is hypothesized to be a complex molecular mechanism whereby a single and then multiple amino acid sites across the entire amino acid sequence of the protein are consecutively and progressively hyperphosphorylated leading to abnormally high levels of insoluble pTau followed by its intracellular pathological accumulation ultimately affecting normal neuronal and non-neuronal (e.g. glia) cellular functions43,44. In addition, our findings show that, at least three of the possible enzymes normally activated during Tau phosphorylation/dephosphorylation processes (GSK3β, pGSKβ [Y216], PP2A- βα),, remained unchanged after 4 weeks from the radiation time point in brain regions examined in this study. These data seem to suggest that both kinase and phosphatase enzymes could have reached a steady-state equilibrium in those examined regions at that specific post-radiation time point (4 weeks). Nonetheless, we cannot exclude that other phosphorylation/dephosphorylation enzymes or biochemical mechanisms might be involved in the interactions between pTau and the effects of radiation or that other chemical reactions (e.g. methylation) may occur.
Furthermore, our data show that lower levels of Ser202-phosphorylated-Tau (as detected by CP13 levels) in the FC and H regions were accompanied by concomitant lower levels of APP and GAP43 in the CRB. Intriguingly, a series of studies described close and complex interactions between APP and GAP43 in different regions of the rodent brain47,48. APP is the precursor protein for 1–40 β-amyloid protein, one of the two proteins (pTau being the other), that pathologically accumulates (in the form of extracellular 1–42 β-neuritic plaques) in subjects diagnosed with AD and it has also been shown to interact with GAP43 in mechanisms of axonal generation and neuroplasticity49. On the other hand, GAP43 is a protein known to be involved in multiple structural and functional aspects of axonal formation during neosynaptogenic processes (during developmental period) and reparative neuronal mechanisms (during adult life)50. In our study, a decreased level of APP and GAP43 was unexpected, but it was even more surprising to observe a post-radiation effect in only the CRB. This latter finding may be related to the well-established notion that the CRB is one of the most resistant regions of the CNS to AD pathology in comparison to other regions of the brain (for example, the H or the FC). Remarkably, this AD-pathology resistance is maintained even during the late stages of the AD natural progression. The notion about the relative resistance of the CRB against the pathological accumulation of pTau and β-amyloid lesions has been attributed to some genetic or biological protective factor, which remains mostly unknown. If there are direct interactions between APP and GAP43 in the CRB and why this particular region of the CNS appears to be more susceptible to a low-dose of brain radiation compared to other brain regions such as H or FC remains elusive, and it represents a fascinating question deserving of future research efforts.
In general, the reduction of soluble levels of pTau, APP, and GAP43 across different mammalian brain regions could also be due to the terminal effects of more basic mechanisms associated with DNA (or RNA) damage or, alternatively, repair activities, especially in those regions of the mammalian brain more susceptible to radiation. In support of an early DNA radiation-induced activation, we did find an increased level of DNA-polymerase- in the FC region. This is not so surprising since the FC is one of the latest neuroanatomical regions to ontogenetically and phylogenetically fully develop very late during the CNS maturation process due to its high level of circuital complexity and biological instability51. Importantly, though, DNA-polymerase- is one of the enzymes involved in reparative processes and in various duplicative and reparative DNA mechanisms that could have been indeed triggered by low-dose brain radiation.
In contrast to the lower levels of pTau, APP, and GAP43 in FC, H and CRB regions, but consistent with previous clinical and experimental observations, the level of GFAP in the H was higher in RAD- vs. SH-animals. Furthermore, the increased level of GFAP in the H (as measured by WB quantifications) was confirmed by an initial by obvious process of astroglial cell reaction as detected at immunohistochemistry. More specifically, based on the immunohistochemistry-microscopic inspection, the astrogliosis seems to be localized at level of the peri-dental gyrus (peri-DG) area of the H (Figure 6) - one of the hippocampal subregions more often activated during various physiological and pathological processes affecting the H. WB and neuropathological findings suggest then the co-presence of an early neuroinflammatory response in the H associated with a reduction of other proteins levels (e.g. pTau) in the H itself and FC region. Furthermore, levels of IBA–1 (a marker of microglia) and MBP (a marker of myelin), did not differ in RAD- vs. SH-animals in any of the considered brain regions and no changes were observed at histological level or immunohistochemistry at high-magnification light microscopy inspection. These last findingssuggest that microglial cells and oligodendrocytes may possibly react at a later stage or at a much slower reacting rate compared to other possible more “radio-sensitive” cell types.
In general, the WB results obtained from low-dose radiation did not parallel any obvious brain lesion observable by histology stains (HE, LFB, CV) or immunohistochemistry, except for the increase of GFAP as a signal of astrogliosis in the H (Figure 6).. We would like to emphasize that increased levels of GFAP are not necessarily linked to detrimental effects since astroglial cells are also involved in various regenerative and neurogenesis phenomena52. Moreover, the absence of immunohistochemistry-based histopathological brain lesions in RAD-vs. SH-animals indirectly confirmed the reduced levels of pTau, APP, and GAP43 across all the examined regions. In fact, those reduced levels of soluble proteins could have prevented the generation of the biochemical conditions necessary to determine molecular changes (e.g. increase levels of Tau, pTau/Tau ratio abnormalities) that are expected to induce the pathological accumulation of insoluble hyperphosphorylated-Tau in long-distance cortical neurons for example.
One of the major points of strength of this study is that we have used a large mammalian brain under normal anatomical and physiological conditions in order to observe possible molecular and neurohistopathological changes due to low-dose brain radiation after 4 weeks and that a compound like captopril is involved in those specific changes. These results in swine are relevant since the swine brain represents a neural system that is much closer to humans than rodents in terms of neuroanatomy, neurocircuitry complexity, cerebro-vascular physiology, liver metabolism, pharmacology, etc. This makes swine brain an excellent model for brain radiation research while searching for its potential beneficial effects at molecular and behavioral level. These species-related considerations are especially important when considering that recent studies have shown that the gene responses to radiation (e.g. in the blood) vary greatly across different species53.
To the best of our knowledge, these molecular and neuropathological findings observed 4 weeks following low-dose total-body radiation in a large mammalian brain are unique and of special relevance for possible future therapeutic applications to conditions affecting the CNS, especially those conditions associated with different types and mechanisms of misfolded protein accumulation. Future larger studies are necessary to precisely define other possible important brain-radiation effects and possible interacting or modifying factors such as minimally necessary effective total dose to administer, best fractionated scheme for each specific brain region and condition, definition of possible stereotactic approaches, genetic background-based response, and other factors not determined yet. Establishing all these parameters could greatly improve the beneficial and therapeutic applicability of low-dose brain radiation during the early phases of various neurodegenerative conditions in humans.