PAK3 downregulation induces cognitive impairment following cranial irradiation

Reviewer #1 (Public Review):

Summary:

Exposure to cranial irradiation (IR) leads to cognitive deficits in the survivors of brain cancer. IR upregulates miR-206-3p, which in turn reduces the PAK3-LIMK1 axis leading to the loss of F and G-actin ratio and, thereby, mature dendritic spine loss. Silencing miR-206-3p reverses these degenerative consequences.

Strengths:
The authors show compelling data indicating a clear correlation between PAK3 knockdown and the loss of mature dendritic spine density. In contrast, overexpression of PAK3 in the irradiated neurons restored mature spine types and recovered the F/G ratio. These in vitro results support the authors' hypotheses that PAK3 and LIMK1-mediated downstream signaling impact neuronal structure and reorganization in vitro. These data were supported by similar experiments using differentiated human neurons. Importantly, silencing miR-206-30 using antagonist miR also reverses IR-induced downregulation of the PAK3-LIMK1 axis, preventing spine loss and cognitive deficits.

Weaknesses:

All the miR-206-3p data are presented from in vitro cortical neurons or human stem cell-derived neuron cultures. This data (IR-induced elevation of miR-206-3p) should also be confirmed in vivo using an irradiated mouse brain to correlate the cognitive dysfunction timepoint.

Antago-miR-206-3p reversed Ir-induced upregulation of miR-206 (in vitro), and prevent reductions in PAK3 and downstream markers. Importantly, it reversed cognitive deficits induced by IR. This data should be supported by in vivo staining for important dendritic markers, including cofillin, p-cofilin, PSD-95, F- and G-actin within the hippocampal and PFC regions.

Other neuronal and non-neuronal targets of miR-206-3p should be discussed and looked into as a downstream impact of IR-induced functional and physiological impairments in the brain.

Reviewer #2 (Public Review):

Summary:
The paper entitled "PAK3 downregulation induces cognitive 1 impairment following cranial irradiation" by Lee et al. aimed at investigating the functional impact of cranial irradiation in mouse and propose PAK3 as molecular element involved in radiation-induced cognitive decrement. The results provided in this paper are problematic as both the irradiation paradigm (5X2 Gy) as well as the timing of investigation (3 to 8 days post-IR) are completely irrelevant to investigate radiation induced neurocognitive impairment. This testifies to the team's lack of knowledge in radiobiology/radiotherapy and the methodology to explore radiation induced neurocognitive damages. It precludes any further relevance of the molecular results.

Weaknesses:

First and according to the BED equation a single dose of 10 Gy cannot not be approximated by 5 fractions of 2 Gy, as fractionation is known to decrease normal tissue toxicity. Note that in radiobiology/radio-oncology, the BED stands for "Biologically Effective Dose." This equation is used to compare the effects of different radiation treatments on biological tissues, taking into account the dose, fractionation, and the overall biological response of the tissue to radiation.
The BED equation is commonly used to calculate the equivalent dose of a fractionated radiation treatment, which is the dose that would produce the same biological effect as a single, higher dose delivered in a single fraction.
The general formula for BED is:BED = D * (1 + d / α/β)
D is the total physical dose of radiation delivered in Grays (Gy)
d is the dose per fraction in Gy
α/β is the tissue-specific ratio of the linear (α) and quadratic (β) components of the radiation response. It is measured in Gy and describes how the tissue responds to different fractionation schedules (usually equal to 3 for the normal brain).
Please refer to radiobiology/radiotherapy textbooks by Hall or Joiner.

Second, the brain is a late responding organ. GBM patients treated with 60 Gy exhibit progressive and debilitating impairments in memory, attention and executive function several month post-irradiation. In mice, neurocognitive decrements after a single dose of 10 Gy delivered to the whole brain does occur at late time point, usually > 2 months post-exposure. Multiple publications such as the one by Limoli C lab, Rossi S lab, Britten R lab or earlier Fike J lab and Robin M lab support this. Next, 5 fractions of 2 Gy will be more protective than a single dose of 10 Gy and neurocognitive decrements will require at least 5-6 months to occur if they ever occur. In Figure 1, the decrement reported is marginal, the number of animals included (4 to 5 at most?) The number of animals is not specified) is too low to draw any significant conclusions. In addition to the timing issue, the strategy described for NOR analysis shows methodological issues with the habituation period being too short and exploration level being very low.

Author Response

The following is the authors’ response to the original reviews.

Reviewer #1

1. IR reduced mature spines (mushroom) but not immature spines (filopodia) in vitro at 14 days post-2 Gy IR. Please check previous reports by C. Limoli and J. Fike groups (in vivo dendritic spine characterization following proton or photon irradiation).

We appreciate the reviewer's comments. Although IR did not reduce filopodia in the previous study, there are no prior studies using the same time points as ours, 4 days post-2 Gy IR. Additionally, according to other previous studies, PAK3 inhibition led to an increase in filopodia (J Neurosci. 2004 Dec 1;24(48):10816-25), and IR increased thin-type spines and decreased mushroom-type spines at the 7 days after 2 Gy IR (PLoS One. 2012;7(7):e40844). Considering these findings, we believe that the increase in filopodia observed in our study is due to the short-term effects of IR and the consequent PAK3 downregulation. We added the description regarding time point in “Materials and Methods”.

Page 20, line 439-440; "In the analysis of molecular alterations, cultured neurons were sampled 4 days after irradiation."

1. Does IR (2 Gy or 5 x 2 Gy) affect the viability in vitro? This could be linked with reduced dendritic structure and F/G-actin ratios.

As the reviewer mentioned, we evaluated neuronal viability following 2 Gy IR exposure. Consequently, approximately 80% of the cells survived after the IR exposure (Fig. 4A). Although we agree that cognitive abilities may decrease due to the neuron death after IR, we identified that PAK3 overexpression restores the F/G-actin ratio in surviving neurons after IR, suggesting the IR-induced alterations at least in neuronal plasticity are mainly regulated by PAK3 rather than IR itself. Additionally, neurons that survive after IR maintain similar levels of NeuN, a mature neuron marker (Fig. S5A). We added the description regarding additional experiments in “Results”.

Page 10, line 206-209; "IR decreased neuronal viability in human differentiated neurons, with approximately 80% survival (Fig 4A). However, IR did not alter the mature neuronal marker, NeuN (Fig S5A). These results indicate that IR-induced disruption of PAK3 signaling occurs in surviving neurons following irradiation. Consistent with previous murine neuron data, IR reduced the F/G-actin ratio (Fig. 4B)."

1. The authors state, "Overall, these results indicated that IR could induce cognitive impairment by disrupting dendritic spine maturation." Dendritic spine damage may not be the only factor contributing to cognitive dysfunction (neural circuit function, neuroinflammation, astrogliosis, etc., needs to be discussed).

We agree with the reviewer's comment that dendritic spine damage may not be the only factor contributing to cognitive impairment. Since our study has only confirmed the effects on dendritic spines as part of the complex impact of radiation, we added the description of the necessity for further research on various factors related to IR-induced cognitive dysfunction in “Discussion”.

Page 15, line 317-324; >The dendritic spine is one of the major factors influencing cognitive function. In our study, we observed changes in dendritic spines due to radiation exposure, followed by subsequent cognitive impairment. Additionally, we established that regulating PAK3, which affects dendritic spine maturation, can modulate radiation-induced cognitive dysfunction. However, considering that radiation can impact the entire nervous system and that neural circuit function, neuroinflammation, and astrogliosis can also influence cognitive function (Makale et al., 2017), future studies is needed to investigate the mechanisms of factors beyond dendritic spine changes caused by radiation.>

1. Fig 2 and Suppl Fig S2. The in vivo results should be placed in the manuscript Fig 2 as this would provide relevant physiological information on PAK3 downregulation and reduced dendritic spines and cognition.

We appreciated the reviewer's comment. As the reviewer mentioned, we rearranged Fig S2C to Fig 2H.

Page 33, line 825-827; "(H) Left: the protein levels of phosphorylated LIMK1, LIMK1, phosphorylated cofilin, and cofilin after IR in frontal cortex and hippocampus. Right: each western blot bands are quantified by ImageJ."

1. miR-206-3p expression was found to be elevated post-IR in the human and mouse neurons in vitro. This was correlated with IR-induced downregulation of PAK3 using an antagonist miR experiment, wherein PAK3, LIMK1, and downstream makers were restored in the irradiated neurons. MiR-206-3p upregulation data should also be confirmed in vivo using an irradiated mouse brain to correlate the cognitive dysfunction timepoint.

We observed IR-induced miR-206-3p upregulation (Fig 6D) and consequent PAK3 downregulation (Fig 6G) in vivo at 4 days after IR. Considering that the antagomiR significantly restores cognitive dysfunction (Fig 6E) at 1-3 days after IR, we suppose the expression of miR-206-3p would be consistently increased by IR, suppressing the PAK3 signaling pathway and leading to cognitive dysfunction.

Page 33, line 825-827; "(H) Left: the protein levels of phosphorylated LIMK1, LIMK1, phosphorylated cofilin, and cofilin after IR in frontal cortex and hippocampus. Right: each western blot bands are quantified by ImageJ."

1. Fig 5 shows that in vivo administration of antago-miR-206 reversed IR-induced upregulation of miR-206, reductions in PAK3 and downstream markers, and, importantly, reversed cognitive deficits induced by IR. This data should be supported by in vivo staining for important dendritic markers, including cofillin/p-cofilin, PSD-95, F- and G-actin within the hippocampal and PFC regions.

We appreciated the reviewer's comment. Based on previous studies on intranasal administration, the substance is delivered to the PFC and hippocampus through the olfactory pathway in both humans and mice (Exp Neurobiol. 2020 Dec 31;29(6):453-469, Stem Cells. 2021 Dec;39(12):1589-1600). Even though we did not show direct evidence that antagomiR-206 is delivered to both regions, we confirmed its actual delivery to the brain using Cy5 fluorescence and examined PAK3 signaling (Fig. 6G) and the F/G-actin ratio (Fig. 6H) in both regions. To show the reliability of the tissue separation, we added a detailed description of the tissue separation method in “Materials and Methods”.

Page 19, line 410-423; "Dissection of prefrontal cortex and hippocampus. The dissection of mouse brain regions was performed following a previous study (Spijker, 2011). First, to obtain the hippocampal region, we gently held the brain and opened the forceps, slowly separating the cortical halves. Once an opening had been created along the midline for approximately 60%, we directed the forceps (in the closed position) counterclockwise by 30–40° to expose the left cortex from the hippocampus, repeatedly opening the forceps as necessary. We then repeated the same procedure for the right cortex by pointing the forceps in a 30–40° clockwise direction until the upper part of the hippocampus became visible. At the most caudal part of the hippocampus/cortex boundary, we moved the small forceps through the cortex and used them to separate the hippocampus from the fornix. After removing the hippocampus, we used the large forceps to fold the cortex back into its original position. Subsequently, we placed the brain with the dorsal side and cut coronal sections to reveal the prefrontal cortex and striatum at different levels. Using a sharp razor blade, we made the first cut to remove the olfactory bulb and cut the section containing the prefrontal cortex."

1. Does this change in the F/G actin ratios, Cofillin, and/or p-Cofillin impact any particular neuronal subtypes, including excitatory, inhibitory or any particular layers of major neurons? This point can't be appreciated from the WB data.

The excitatory and inhibitory neurons do play crucial roles in cognitive function. In terms of response to radiation, excitatory neurons are more likely to be responsive. A previous study showed that spike firing and excitatory synaptic input were reduced by cranial irradiation, while inhibitory input was increased (Neural Regen Res. 2022 Oct;17(10):2253-2259). Additionally, PSD-95 is localized to dense specialized regions within the dendritic spines of excitatory synapses and is associated with synaptic plasticity (Neuron. 2001 Aug 2;31(2):289-303). Indeed, IR decreases the mRNA level of PSD-95 in differentiated human neurons (Fig S5A). Considering the previous research and our data, IR-induced PAK3 downregulation may occur primarily in excitatory neurons.

1. Discussion: "In this study, we investigated the effect of cranial irradiation on cognitive function and the underlying mechanisms in a mouse model." Please change this statement to "....underlying neuronal mechanisms using in vivo and in vitro models."

We appreciate the reviewer’s comment. We replaced ‘mechanisms in a mouse model’ with ‘neuronal mechanisms using in vivo and in vitro models.’ in the manuscript.

Page 14, line 283; "In this study, we investigated the effect of cranial irradiation on cognitive function and the underlying neuronal mechanisms using in vivo and in vitro models."

1. Discussion: "Furthermore, our study identifies a potential mechanism underlying the cognitive impairment associated with cranial irradiation, which downregulates PAK3 expression." This statement should be supported by the in vivo immunofluorescence data for the synaptic markers, including cofilin, p-cofillin, PSD-95, and F/G-actin staining.

Even though we did not show the in vivo immunofluorescence data for the synaptic markers, we examined PAK3 signaling (Fig. 6G) and the F/G-actin ratio (Fig. 6H) in the hippocampal and PFC regions. Additionally, according to The Allen Mouse Brain Atlas, PAK3 is mainly expressed in the PFC and hippocampus regions (Fig S2A), suggesting that IR-induced PAK3 downregulation in both regions may have a significant impact on the cognitive impairment. Considering these data, we strongly believe that cranial irradiation downregulates PAK3 levels in the PFC and hippocampus, thus inducing cognitive impairment.

1. miR modulate function by affecting multiple targets. The other potential neuronal and non-neuronal targets for miR-206-3p were not discussed. This possibility should be confirmed using relevant markers.

According to the reviewer’s comment, we performed real-time PCR to examine whether miR-206-3p affects the expressions of neuronal and non-neuronal markers (Fig S5A and S5B). As a result, the post-synaptic marker, PSD-95, was reduced by miR-206-3p treatment. However, a mature neuronal marker (NeuN) and non-neuronal markers (GFAP and IBA-1) did not change upon miR-206-3p treatment. We added the related description in “Results”.

Page 12, line 240-243; "Additionally, the post-synaptic marker, PSD-95, was decreased by miR-206-3p treatment. However, a mature neuronal marker (NeuN) and non-neuronal markers (GFAP and IBA-1) were not alterd upon miR-206-3p treatment (Fig. S5A and S5B)."

1. Irradiation procedure: Please confirm that sham (0 Gy)-irradiated mice were also anesthetized for a similar procedure carried out for the 2 Gy or fractionated irradiation.

According to the reviewer's comment, we added a description of sham (0 Gy)-irradiated mice in “Materials and Methods”.

Page 17, line 359-360; "All mice, including those in the sham (0 Gy) group, were anesthetized with an intraperitoneal (i.p.) injection of zoletil (5 mg/10 g) daily for five days."

1. 24 mL volume (antagomir treatment) via intra-nasal delivery is a rather unusually high volume. Please clarify if such a procedure was approved by the regulatory committee and if 24 mL volume led to any hemodilution.

We appreciate the reviewer's comment. We referred to the protocol of intranasal administration from a previous study (Mol Ther. 2021 Dec 1;29(12):3465-3483), and made an error in specifying the miRNA unit. We corrected it from mL to μL.

Page 19, line 399-402; "According to the manufacturer’s instructions and previous study (Zhou et al., 2021), 40 nmol of antagomiR-206-3p (sequence: 5’-CCACACACUUCCUUACAUUCCA -3’) or antagomiR-NC (the antagomiR negative control, its antisense chain sequence: 5’-UCUACUCUUUCUAGGAGGUUGUGA-3’) was dissolved in 1 mL of RNase-free water."

Page 19, line 402-403; "A total of 24 μL of the solution (1 nmol per one mouse) was instilled with a pipette, alternately into the left and right nostrils (1 μL/time), with an interval of 3–5 min."

Reviewer #2

1. To show the relevance of PAK3 in Radiation-induced neurocognitive decrements, I suggest using 10 Gy WBI, group of 15-16 animals and long-term follow up >2 months post-RT.

We appreciate the reviewer's comment. Biologically Effective Dose (BED) represents the most accurate quantitative prediction of biological effects of radiation. However, our study aimed to analyze the mechanisms underlying cognitive dysfunction induced not by a total dose of 10 Gy but rather by repeating 2 Gy fractions, which is used in clinical practice such as prophylactic cranial irradiation. In this regard, the administration of 2 Gy fractions holds significant relevance in our research.

In statistical analysis, a larger sample size tends to be more accurate. However, we determined the sample size based on ethical considerations in animal research, taking into account the parameter (Effect size: 1.2 / alpha value: 0.05 / Group: 3 groups), resulting in a total sample size of 15, five mice per group (G Power 3.1 software). Despite the relatively small sample size, radiation exposure significantly reduced PAK3 expression with marginal variance, thereby inducing cognitive impairment.

As the reviewer mentioned, the long-term effect (>2 months) of WBI may show more severe cognitive impairment, considering results from the previous studies. Nevertheless, previous research has revealed a correlation between mouse age and human age, suggesting that 2 months in mice is roughly equivalent to 5 years in humans (Life Sci. 2020 Feb 1;242:117242). Due to the substantial difference in biological time between humans and mice, 2 months in mice might be an excessive long-term period. Additionally, our study aims to investigate short-term changes rather than long-term effects. It is clear that IR-induced PAK3 downregulation induces cognitive impairment at least in the short-term period, and we believe that our findings may contribute to preventing serious neuronal dysfunction as the long-term side effects of PCI.