We identified previously undefined subcellular-specific functions of tau in acute neuronal injury in multiple model systems that otherwise simulate normal physiological conditions. The effects of radiotherapy used for cancer treatment have generally focused on radiation-induced DNA damage, particularly double strand breaks [61]. Such damage activates a diverse array of repair machinery that prevents cell cycle progression at specific checkpoints thus allowing for DNA repair [62]. Some investigators have proposed that tau in the nucleus has a non-canonical neuroprotective function by maintaining and regulating heterochromatin, localizing with acrocentric chromosomes, binding with gene-coding and intergenic regions, and being active at pre-RNA processing sites [63]. Tau also participates in protecting neuronal DNA from damage through heat and oxidative stress [34]. In this study, we discovered a unique role for tau in radiation-induced neuronal DDR via direct interactions (evaluated via IP-MS) with key DDR proteins such as H2Ax.
In traumatic brain injury or neurodegenerative diseases, H2AX has been shown to be a sensitive marker of DNA damage through phosphorylation of its serine residue at Ser139 (γH2Ax) [64]. We found that γH2Ax foci were diminished in the absence of tau and in the presence of radiation-induced injury, and our ChIP-Seq findings show ptau enrichment in gene regions involved in DDR. Curiously, we detected basal levels of ptau in the nucleus before irradiation, which may reflect a homeostatic role of tau in normal neurons. In the event of an injury, ptau rapidly localizes and increases in the nucleus, setting in motion an injury response by interacting with proteins involved with repairing damaged DNA. In line with established functions of DDR markers in disease, our results suggest that tau acts as a regulator of the damage response in the injured brain.
In addition to its functions in the neuronal nucleus for damage repair, tau has critical functions in other neuronal compartments as well. Tau has a sirtuin-dependent role in protein synthesis [65]. In neurons, homeostatic translation requires the coordinated transportation of ribosomes, tRNAs, mRNAs, and other important machinery at proximal and distal sites of neuronal dendrites with pristine fidelity to avoid mistranslation and ribosomal frameshifting [66–72]. Even the subtlest changes in this finely tuned machinery can lead to misfunction [73] and conditions such as epileptic seizures and frontotemporal dementia [74–76]. We found that tau has a unique interaction with a crucial translation marker, eEF2. In cells with loss of tau and exposed to injury, active status of eEF2 is restored, indicating a shift in translation balance. This shift can have downstream repercussions, as shown in studies showing localized eEF2/eEF2K activity in dendrites, which affects glutamate signaling, downstream NMDAR activation, subsequent increase in calcium levels, and long-term depression [72, 74, 77–79]. Our results suggests that tau has a critical role in maintaining translation homeostasis via its interaction with eEF2. Indeed, regulation of eEF2 by tau could prevent superfluous protein synthesis and ensure translation of proteins essential for neuronal maintenance post-injury, especially because translation is an energy-demanding process. Moreover, the synthesis of synaptic proteins amplified in the absence of tau and injury, perhaps because of unchecked eEF2 active status. Taken together, these results suggest that tau has a regulatory role in the translation machinery of the injured brain.
The cumulative effects of radiation-induced insult have detrimental effects on neuronal and dendritic architecture [80–84]. Our lab has shown that cranial irradiation induces axon initial segment alternations and dysfunction in neurons [17]. Structural alterations such as these are detrimental for axonal transport and neuronal communication. We also observe reductions in tau levels (relative to the 0 Gy control) after irradiation in cortical (Supplementary Fig. 5A,D) and hippocampal (Supplementary Fig. 5B,E) neurons and hiPSC-derived cerebral organoids (Supplementary Fig. 5C,F). Others have shown that tau knockdown affects neuronal damage via growth cone impairment, delayed maturation, reduced microtubule density, and synaptic changes [85]. Our results show that radiation-induced injury results in decreases in pre- and post-synaptic proteins relative to controls. However, we also report that both loss of tau and neuronal injury led to increases in pre- and post-synaptic proteins GRIN2A, PSD95, and BDNF, and increases in glutamate and eEF2 levels. These findings lead us to hypothesize a complex “feed-forward” adaptive mechanism of higher synaptic translation, elevated glutamate content and hence increased post-synaptic protein expression in the absence of tau and the presence of neuronal injury, resulting in excitotoxicity and aberrant neuronal firing. Although the molecular basis of this relationship needs to be investigated further, our findings on cognitive performance and EEG spectral power in TKO mice exposed to neuronal injury suggest a role for tau as a neuroprotector. TKO mice performed worse in novel object recognition test after irradiation compared to wild-type control. Besides, persistent firing of neurons in the PFC during working memory tasks has been well documented to orchestrate cognitive dynamics [58]. In TKO mice, neuronal firing was aberrant for up to 1 month after injury, suggesting that tau has a sustained role in maintaining optimal firing in the injured brain long-term.
A limitation of our experimental approach is that our model relies on neuronal injury induced by a specific treatment modality used in clinic for cancer patients. Injury models often recapitulate a ‘pathology state’, which was not the aim here since our model lacked classical disease-specific properties observed in traumatic brain injury or stroke models. The strength of our study is using normal physiologically relevant systems across multiple species, namely rat cortical and hippocampal neurons, mouse whole brains, as well as human iPSCs to generate neurons in both 2D cultures and 3D cerebral organoid systems. All systems revealed the same tau response to radiation.
In this study, we present evidence that tau has an important function to play in vertebrate central nervous system in an acute response to ionizing radiation-induced injury. We provide evidence that tau directly interacts with proteins involved in brain homeostasis and participates in preventing cognitive decline. These findings provide a baseline to study the chronic aspects of tau in radiation-induced injury, as well as identify pathways for potential therapeutic targets.