P7C3-A20 prevents whole brain radiotherapy-induced chronic hippocampal redox imbalance and neuropsychiatric impairment in mice

TL;DR

A compound called P7C3-A20 protects the brain from long-term damage caused by whole brain radiotherapy in mice, preserving cognitive and emotional health.

Contribution

P7C3-A20 prevents chronic oxidative stress and neuropsychiatric effects of WBRT without reducing its anti-tumor effectiveness.

Findings

P7C3-A20 reduces oxidative stress and hippocampal injury lasting up to one year after WBRT in mice.

The compound prevents cognitive decline and depressive-like behavior caused by WBRT.

P7C3-A20 preserves brain health without compromising the efficacy of radiation therapy.

Abstract

Whole brain radiotherapy (WBRT) prolongs survival for patients with brain metastases but produces persistent oxidative injury to the brain and long-term neuropsychiatric sequelae, for which no approved neuroprotective therapies exist. Here, we show that a single course of WBRT produces chronic oxidative stress in mice that persists for at least one year, equivalent to decades in humans, and selectively injures the hippocampus, causing cognitive decline and depressive-like behavior. Daily administration of P7C3-A20, a neuroprotective compound that stabilizes brain nicotinamide adenine dinucleotide (NAD+) homeostasis, markedly reduced oxidative stress and prevented hippocampal pathology for one year after WBRT. P7C3-A20 treatment suppressed neuroinflammation, axonal injury, loss of hippocampal neural precursor cells, blood-brain barrier breakdown, and microglial lipid droplet accumulation, resulting in long-term preservation of cognition and brain health. Crucially, P7C3-A20 did not impair the antitumor efficacy of radiation. These findings identify NAD+ homeostasis stabilization as a promising strategy to mitigate radiation-induced oxidative damage and to prevent long-term neuropsychiatric complications of WBRT.

Figures

P7C3-A20 treatment does not alter WBRT-mediated tumor cell killing.(A–C) Cell survival post-exposure of breast and lung metastatic cell lines (BRBM-347 and LUMBM-4761, respectively) and primary tumor glioblastoma cell line (U118 MG) to increasing doses of ionizing radiation (IR) in the presence of P7C3-A20 demonstrates no interference with antitumoral efficacy of IR (n = 2–4 biological replicates with n = 6 technical replicates per condition).(D) Time course of glioblastoma (U87 cell line) brain tumor size in male nude mice treated with daily P7C3-A20 (20 mg/kg/day, i.p.) or vehicle beginning

Chronic neuropsychiatric impairment in the year following WBRT is prevented by P7C3-A20.(A) Experimental schematic. Three days after initiating treatment with P7C3-A20 or vehicle, mice received 10Gy WBRT. Daily P7C3-A20 or vehicle were continued for one year, and behavioral analysis was conducted on days 90 (3 months), 210 (7 months) and 365 (12 months). Thereafter, animals were euthanized for biochemical and immunohistochemical analysis.(B) Equivalent swim speed in the Morris water maze test across all groups at all times indicates normal basal motor function throughout all experimental phase

Chronically elevated reactive oxygen species (ROS) damage, neurodegeneration, neural precursor cell loss, blood-brain barrier deterioration, and aberrant microglial deposition in the hippocampus one year after WBRT are prevented by P7C3-A20.(A&B) 8-OHdG staining (red) is chronically elevated in the hippocampus 1 year after WBRT, relative to Sham-Veh mice, and prevented by P7C3-A20. DAPI staining is blue. (Scale bar = 10 μm) (C) 4-HNE-modifed proteins were chronically elevated in the hippocampus one year after WBRT, relative to Sham-Veh mice, and prevented by P7C3-A20.(D&E) Neurodegeneration me

Summary schematic of results. Systemic treatment with P7C3-A20 before and after WBRT prevented long-term hippocampal injuries, including lipid and DNA oxidative damage, neuroinflammation, blood-brain barrier deterioration, axonal demyelination and neurodegeneration, and hippocampal neural stem cell loss. These protective features were accompanied by preservation of cognitive performance and attenuation of depressive-like behaviors.Fig. 4

Full text

Introduction

1

Whole brain radiotherapy (WBRT) is delivered to roughly 200,000 cancer patients annually in the United States to treat both radiographically visible and microscopic brain metastases [1,2]. Although acute WBRT toxicities (nausea, headache, vomiting) are generally transient, as many as half of long-term survivors develop progressive cognitive decline and depressive symptoms within the first year after treatment, substantially reducing quality of life [3].

WBRT triggers persistent oxidative stress that engages multiple interrelated neurotoxic pathways, including impaired hippocampal neuronal stem cell survival, neuroinflammation, blood-brain barrier (BBB) disruption, and neurodegeneration. The hippocampus is particularly susceptible to these injuries and is a major contributor to radiation-associated cognitive dysfunction and depression. Current clinical approaches, such as hippocampal-sparing radiation techniques and agents like memantine, provide only modest protection [4,5]. Thus, there remains a critical need for neuroprotective therapies that preserve hippocampal integrity and neuropsychiatric function without reducing oncologic efficacy.

Physiological maintenance of nicotinamide adenine dinucleotide (NAD^+^) homeostasis attenuates oxidative stress through multiple interlinked pathways [6]. NAD^+^ is a key modulator of redox homeostasis and crucial for numerous cellular process, including supporting sirtuin-dependent antioxidant programs that participate in DNA repair, as well as mitochondrial function [7]. Deficient NAD^+^ homeostasis in the brain weakens redox buffering capacity and increases susceptibility to neurodegeneration in conditions such as Alzheimer's disease and traumatic brain injury, and stabilizing NAD homeostasis is protective [[8], [9], [10], [11], [12]]. We therefore tested whether systemic administration of P7C3-A20, a blood-brain barrier permeant neuroprotective small molecule that stabilizes NAD^+^ homeostasis without driving supraphysiologic NAD^+^ elevation [11,[13], [14], [15], [16], [17]], could prevent WBRT-induced hippocampal pathology and the resulting cognitive dysfunction and depression-like behavior. P7C3-A20 has shown neuroprotective efficacy across diverse preclinical models of neurodegeneration, including mitigation of oxidative injury and rescue of cognitive function in advanced Alzheimer's disease and chronic traumatic brain injury models, and chronic oral dosing in nonhuman primates has been reported to increase survival of proliferating hippocampal neural precursor cells without safety issues [[8], [9], [10],[18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42]].

Methods

2

Animals

2.1

All procedures were approved by The University of Iowa Institutional Animal Care and Use Committee and complied with NIH guidelines. Seven-week-old male C57BL/6J (stock no. 000664) and Nu/J (stock no. 002019) mice were obtained from The Jackson Laboratory. Food and water were provided ad libitum, and environmental conditions (temperature, humidity, light cycle) were controlled. Animals were randomized to experimental groups and researchers were blinded to exposure and treatment throughout testing.

Cell lines

2.2

U-87 MG-Luc2 (ATCC, HTB-14-LUC2) cells (source: male) and U118 MG (ATCC, HTB-15) cells (source: male) were grown in DMEM F12 medium supplemented with 15 % FBS, penicillin (100 U/mL) and streptomycin (0.1 mg/mL). Tissues used to generate the LUBM-4761 and BRBM-347 lines were collected in accordance with a Cleveland Clinic Institutional Review Board-approved protocol. LUBM-4761 is a metastatic poorly differentiated lung adenocarcinoma derived from a left frontal lobe brain metastasis resected from a 56-year-old female. BRBM-347 is a metastatic breast adenocarcinoma (ER-, PgR-, HER2+) derived from a right posterior temporal lobe brain metastasis in a 52-year-old female. Brain metastasis tissues were disaggregated using the Papain Dissociation System (Worthington Biochemical) as per manufacturer's instructions. Isolated cells were grown in DMEM/F-12 supplemented with B27, EGF (20 ng/mL), FGF (20 ng/mL), 1 mM l-Glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin. All cells were cultured at 37^o^C, 5 % CO_2_, and in a humidity-controlled environment.

Additional experimental details (WBRT, drug treatment, behavioral assays, tissue collection, immunohistochemistry, hippocampal neurogenesis assay, BODIPY and 4-hydroxy-2-nonenal (4-HNE) analysis, image acquisition and analysis, western blotting, electron microscopy of the BBB and lipid droplets, cytokine analysis, clonogenic survival assay, brain tumor xenografts, and statistical methods) are provided in Supplementary Materials.

Results

3

Tumoricidal effects of radiation are not blocked by P7C3-A20

3.1

To ensure neuroprotection without compromising oncologic efficacy, we tested whether P7C3-A20 affects radiotherapy-induced tumor cell killing. In vitro, patient-derived brain metastasis cell lines from breast and lung cancer (BRBM-347, LUBM-4761) and a primary glioblastoma cell line (U-118 MG) were exposed to graded ionizing radiation (IR) with or without P7C3-A20. P7C30-A20 treatment did not reduce radiation-induced cytotoxicity across the dose range tested (Fig. 1A–C). In vivo, daily P7C3-A20 administration likewise did not attenuate the tumoricidal effect of 10 Gy WBRT in nude mice bearing U87 glioblastoma xenografts (Fig. 1D). Together, these results indicate that P7C3-A20 preserves radiotherapy efficacy, warranting further evaluation of its neuroprotective potential in this context.Fig. 1P7C3-A20 treatment does not alter WBRT-mediated tumor cell killing.(A–C) Cell survival post-exposure of breast and lung metastatic cell lines (BRBM-347 and LUMBM-4761, respectively) and primary tumor glioblastoma cell line (U118 MG) to increasing doses of ionizing radiation (IR) in the presence of P7C3-A20 demonstrates no interference with antitumoral efficacy of IR (n = 2–4 biological replicates with n = 6 technical replicates per condition).(D) Time course of glioblastoma (U87 cell line) brain tumor size in male nude mice treated with daily P7C3-A20 (20 mg/kg/day, i.p.) or vehicle beginning at the same time as a single dose of 10 Gy WBRT. There were no detected effects of P7C3-A20, consistent with no interference with the antitumoral efficacy of WBRT (n = 6 per condition). Flank glioblastoma tumors not exposed to ionizing radiation from the same animals were used as control.All values are mean ± SEM. In Panel D, significance determined by mixed-effects linear regression with a random intercept for individual animals and an assessed 3-way interaction of day, WBRT, and P7C3-A20.Fig. 1

Animal experimental design

3.2

To evaluate behavioral and histopathologic outcomes following a single whole brain exposure of ionizing radiation (10 Gy) in mice out to 12 months (equivalent to decades in humans), eight-week-old C57BL/6J male mice were randomized to four groups (Sham-Vehicle, Sham-P7C3-A20, WBRT-Vehicle, WBRT-P7C3-A20; n = 15 per group). P7C3-A20 (20 mg/kg/d, intraperitoneal) or vehicle was administered once daily beginning three days before a single 10 Gy WBRT or sham irradiation exposure (Fig. 2A). The three-day lead-in was chosen based on prior pharmacokinetic data showing attainment of steady-state brain levels of P7C3-A20 [8,20]. Mice were singly housed for the 12 months follow-up to minimize social influences on hippocampal neurogenesis and cognition. No adverse effects of chronic P7C3-A20 dosing were observed.Fig. 2. Chronic neuropsychiatric impairment in the year following WBRT is prevented by P7C3-A20.(A) Experimental schematic. Three days after initiating treatment with P7C3-A20 or vehicle, mice received 10Gy WBRT. Daily P7C3-A20 or vehicle were continued for one year, and behavioral analysis was conducted on days 90 (3 months), 210 (7 months) and 365 (12 months). Thereafter, animals were euthanized for biochemical and immunohistochemical analysis.(B) Equivalent swim speed in the Morris water maze test across all groups at all times indicates normal basal motor function throughout all experimental phases.(C) Learning curve of the Morris water maze test shows no effect of P7C3-A20 or WBRT until 12 months, at which point WBRT is associated with impaired learning.(D) Assessment of memory through recording the number of platform crossings in the probe test of the Morris water maze showed no differences between groups 3 months after WBRT. At 7- and 12-month time points, however, WBRT-Veh mice exhibited memory deficit compared to Sham-Veh mice. WBRT-P7C3-A20 mice were protected from memory impairment at both time points, with performance equivalent to Sham-Veh mice.(E) WBRT-Veh mice showed impaired memory in the novel object recognition test at 3,7 and 12 months, relative to Sham-Veh mice, which was prevented by treatment with P7C3-A20.(F) Elevated depression-like behavior measured by immobility time in the tail suspension test 3 months after WBRT in Veh mice, compared to Sham-Veh mice, was not displayed in WBRT-P7C3-A20 mice.(G) Elevated depression-like behavior measured by immobility time in the forced swim test 7 months after WBRT in Veh mice, compared to Sham-Veh mice, was prevented by P7C3-A20.All values are mean ± SEM. Individual data points represent individual animals. In Panel C, significance was determined by mixed-effects linear regression estimated separately with 3-month, 7-month, and 12-month data with a random intercept for individual animals and an assessed 3-way interaction of day, WBRT, and P7C3-A20. In all other panels, significance was determined by ANOVA or, in cases of imbalance, ordinary least squares (OLS) regression was estimated separately with 3-month, 7-month, or 12-month data and assessing the 2-way interaction of WBRT and P7C3-A20.Fig. 2

Behavioral assessments were conducted by investigators blinded to treatment and exposure at 3, 7, and 12 months post-WBRT (90, 210, and 365 days) using a standardized testing sequence to reduce confounding from repeated measures.

Chronic cognitive impairment after WBRT is prevented by P7C3-A20

3.3

Swim speeds in the Morris water maze (MWM) were equivalent across groups at all time points (Fig. 2B), indicating comparable motor function and motivation. Although WBRT produced a learning deficit in the MWM at 12 months that was not prevented by P7C3-A20 (Fig. 2C), treatment robustly preserved memory, with WBRT-P7C3-A20 mice showing intact probe trial performance at 7 and 12 months, whereas WBRT-Vehicle mice showed progressive memory loss (Fig. 2D).

Consistent with these findings, WBRT-Vehicle mice exhibited persistent recognition deficits in the novel object recognition (NOR) test at all time points, which were prevented by treatment with P7C3-A20 (Fig. 2E). Together, these results indicate that P7C3-A20 preserves hippocampal-dependent memory after WBRT.

Chronic depression-like behavior after WBRT is prevented by P7C3-A20

3.4

Depression-like behavior was assessed using two complementary assays to limit the habituation that can occur with repeated testing: forced swim test (FST) at 3 months and tail suspension test (TST) at 7 months. WBRT-Vehicle mice showed significantly increased immobility in both tests, consistent with depressive-like behavior. Daily P7C3-A20 treatment prevented these increases in immobility, with WBRT-P7C3-A20 mice performed comparably to Sham-Vehicle controls in both TST and FST (Fig. 2F and G). These data indicate that P7C3-A20 blocks WBRT-induced depression-like outcomes.

Chronic hippocampal oxidative stress after WBRT is prevented by P7C3-A20

3.5

To link the behavioral effects to underlying pathology, we performed molecular, cellular, and ultrastructural analyses of hippocampus and cortex one year following WBRT or sham exposure. WBRT produced a selective, sustained increase in 8-hydroxy-2-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage, in the hippocampus, which was prevented by P7C3-A20 (Fig. 3A and B). In contrast, cortical 8-OHdG levels were unchanged by WBRT one year after exposure (Fig. S1A and B). P7C3-A20 also reduced WBRT-induced accumulation of 4-HNE modified proteins (Fig. 3C and Fig. S1C), a marker of lipid peroxidation-related protein damage linked to mitochondrial dysfunction and neurodegeneration [43]. Together with prior evidence connecting NAD^+^ decline to oxidative stress and 8-OHdG accumulation [44], these results indicate that WBRT produces a chronic, region-specific redox imbalance in the hippocampus that is preventable by P7C3-A20.Fig. 3. Chronically elevated reactive oxygen species (ROS) damage, neurodegeneration, neural precursor cell loss, blood-brain barrier deterioration, and aberrant microglial deposition in the hippocampus one year after WBRT are prevented by P7C3-A20.(A&B) 8-OHdG staining (red) is chronically elevated in the hippocampus 1 year after WBRT, relative to Sham-Veh mice, and prevented by P7C3-A20. DAPI staining is blue. (Scale bar = 10 μm) (C) 4-HNE-modifed proteins were chronically elevated in the hippocampus one year after WBRT, relative to Sham-Veh mice, and prevented by P7C3-A20.(D&E) Neurodegeneration measured by silver staining (black) 1 year after WBRT is significantly elevated in the hippocampus of WBRT-Veh mice, relative to Sham-Veh, and prevented by P7C3-A20. (Scale bar = 20 μm) (F&G) Axonal demyelination measured by loss of immunostaining of myelin oligodendrocyte glycoprotein (MOG) (red) is observed in the hippocampus 1 year after WBRT in WBRT-Veh mice, relative to Sham-Veh mice, and prevented by P7C3-A20. DAPI staining is blue. (Scale bar = 20 μm).(H–J) Quantification of hippocampal neurogenesis measured by BrdU staining (brown) demonstrates lower proliferation of neural stem cells and lower survival of young hippocampal neurons in WBRT-Veh mice, relative to Sham-Veh mice. Both measures are protected in WBRT-P7C3-A20 mice, with levels equivalent to Sham-Veh mice. (Scale bar = 50 μm) (K&L) Transmission electron microscopy of hippocampus 1 year after WBRT shows capillary endothelium breaks (red arrow) in WBRT-Veh mice, relative Sham-Veh mice, prevented by P7C3-A20. (Scale bar = 1–2 μm) (M&N) TEM of hippocampus shows increased number of microglia with lipofuscin containing LD (red arrows) in WBRT-Veh mice, relative to Sham-Veh mice, and prevented by P7C3-A20.All values are mean ± SEM. Individual data points represent individual animals. For panels a–c, significance determined by one-way ANOVA and Dunnett's post hoc analysis. For panels e–h, significance was determined by mixed-effects linear regression estimated separately with hippocampus and cortex data with a random intercept for individual animals and an assessed 2-way interaction of WBRT and P7C3-A20. For panels i and j, two-way ANOVA assessing the interaction of WBRT and P7C3-A20 was estimated. For panels l and m, significance was determined using Kruskal-Wallis tests with post-hoc Dunn Test comparing paired groups and using a Holm p-value adjustment. Significance for panels n and o was determined using exact logistic regression (elrm) predicting the LD + Microglia out of the total microglia with a 3-level group variable.Fig. 3

Chronic hippocampal neurodegeneration after WBRT is prevented by P7C3-A20

3.6

Although NeuN labeling showed no overt neuronal loss in hippocampus or cortex across groups (Fig. S2A–C), silver staining revealed widespread neurodegeneration in the hippocampus of WBRT-Vehicle mice, an effect that was prevented by P7C3-A20 and absent in cortex (Fig. 3D and E and Fig. S2D&E). Consistent with axonal injury, myelin oligodendrocyte glycoprotein (MOG) expression was significantly reduced in the hippocampus after WBRT but preserved by P7C3-A20 and unchanged in cortex (Fig. 3F and G and Fig. S2F&G). WBRT also reduced survival of both quiescent and newly proliferating hippocampal neural precursor cells, cell populations critical for cognitive function, and both deficits were prevented by P7C3-A20 (Fig. 3H–J and Fig.S2H&I). Together, these findings show that P7C3-A20 preserves hippocampal structural integrity and precursor cell survival one year after WBRT.

Chronic hippocampal blood-brain barrier breakdown after WBRT is prevented by P7C3-A20

3.7

One year after WBRT, the hippocampus (but not the cortex) showed persistent vascular injury, with reduced capillary endothelial length and fewer pericytes (Fig. S3A–F), accumulation of ultrastructural endothelial breaches assessed by transmission electron microscopy (TEM) (Fig. 3K and L andFig. S3K&L), and pathological extravasation of immunoglobulin into hippocampal parenchyma (Fig. S3M–O). P7C3-A20 prevented these changes and augmented hippocampal expression of the tight junction protein zonula occludens 1 (ZO-1) (Fig. S3G–J). Together, the structural and functional data indicate that P7C3-A20 robustly protects the hippocampal microvasculature and preserves BBB function after WBRT, consistent with its reported BBB-stabilizing effects in rodent models of traumatic brain injury and Alzheimer's disease [8,10].

Chronic hippocampal neuroinflammation after WBRT is prevented by P7C3-A20

3.8

One year after WBRT, the hippocampus (but not the cortex) showed a pro-inflammatory shift that was prevented by P7C3-A20. Hippocampal interleukin-13 (IL-13), a cytokine important for cognitive function [45], was reduced by WBRT and preserved with P7C3-A20 (Fig. S4A). RANTES, a chemokine involved in axogenesis and neuronal repair after brain injury [46], was decreased by WBRT in both hippocampus and cortex, which was blocked by P7C3-A20 (Fig. S4B). Microglial activation, indexed by increased ionized calcium-binding adaptor molecule 1 (IBA1), a marker of neuroinflammatory microglial activation [47], was elevated in hippocampus (but not cortex) after WBRT, which was also blocked by P7C3-A20 (Fig. S4C–E). Glial fibrillary acidic protein (GFAP), a marker of astroglial reactivity, showed no group differences in hippocampus or cortex (Fig. S4F and G). These results indicate that P7C3-A20 prevents persistent, region-specific neuroinflammatory changes after WBRT. A comprehensive cytokine profile for hippocampus and cortex across all groups is provided in Fig. S5.

Chronic accumulation of lipid droplets in microglia after WBRT is prevented by P7C3-A20

3.9

We next examined microglial lipid handling. Lipid droplet (LD) accumulation in microglia impairs phagocytosis [48] and is linked to neurodegeneration [49]. BODIPY labeling of neutral lipids revealed WBRT-induced increases in microglial LD content (BODIPY + IBA1+ cells) in both hippocampus and cortex, which was blocked by P7C3-A20 (Fig. S6A–C). TEM confirmed a higher frequency of microglia containing lipofuscin-associated LDs in hippocampus (Fig. 3M and N) and cortex (Fig. S6D and E) after WBRT, which was blocked by P7C3-A20. These results show that P7C3-A20 preserves long-term microglial lipid homeostasis after WBRT, and identify a common locus of chronic pathology across these regions.

Conclusions and clinical implications

4

Systemic P7C3-A20 administered before and after WBRT robustly prevented a range of long-term hippocampal injuries in our preclinical model, including oxidative damage, axonal and myelin deterioration, loss of hippocampal neural precursor cells, BBB disruption, neuroinflammation, and maladaptive microglial lipid droplet accumulation. These structural and cellular protections were accompanied by preservation of cognitive performance and attenuation of depressive-like behaviors (Fig. 4). Importantly, P7C3-A20 produced these neuroprotective effects without detectable interference with the tumoricidal efficacy of radiation in vitro or in vivo.Fig. 4. Summary schematic of results. Systemic treatment with P7C3-A20 before and after WBRT prevented long-term hippocampal injuries, including lipid and DNA oxidative damage, neuroinflammation, blood-brain barrier deterioration, axonal demyelination and neurodegeneration, and hippocampal neural stem cell loss. These protective features were accompanied by preservation of cognitive performance and attenuation of depressive-like behaviors.Fig. 4

This study used a single 10 Gy WBRT exposure to create a controlled, reproducible module of radiation-induced neurotoxicity, while clinical WBRT typically involves fractionated regimens delivered over multiple sessions (i.e. 30 Gy total delivered over ten fractions). Using the linear–quadratic model with an α/β of 3 Gy for late-responding normal brain tissue, a single 10 Gy fraction corresponds to approximately 26 Gy equivalent dose in 2-Gy fractions (EQD2), while 30 Gy in ten fractions corresponds to approximately 36 Gy EQD2. Thus, the single-fraction regimen used here represents a somewhat lower exposure than conventional fractionated WBRT. The single dose design was used to intentionally minimize the confounding effects of repeated anesthesia, handling, and systemic stress in animals and served to establish a proof of concept for both injury mechanisms and therapeutic rescue.

Translationally, demonstration of neuroprotection at ∼26 Gy EQD2 supports the potential clinical relevance of P7C3-A20 within a clinically meaningful dose range. We hypothesize that clinically used fractionation, by permitting sublethal damage repair and cell redistribution, may modulate and possibly enhance the therapeutic window for neuroprotection. Future studies will test clinically relevant fractionated WBRT regimens (e.g., 30 Gy in 10 fractions or biologically matched fractionated schedules) to determine whether neuroprotection by P7C3-A20 is maintained, augmented, or altered under conditions that more closely mimic clinical practice, and to characterize time-dependent repair and cumulative injury processes.

Additional work will also examine whether protection extends to other brain regions when exposure is more substantial. Although translation from mice to humans requires further validation, prior evidence that P7C3-A20 safely and effectively supports proliferating hippocampal neural precursor cells in nonhuman primates [13], together with the demonstrated absence of interference with radiation-induced tumor killing in our preclinical models, provide a strong rationale for continued evaluation. If these findings are replicated across dosing paradigms and species, P7C3-A20 or related compounds could provide a promising adjunct to WBRT to preserve long-term neurocognitive and neuropsychiatric function without compromising oncologic outcomes.

Funding

This study was funded by the Valour Foundation (AAP), Project 19PABH134580006 AHA/Allen Initiative in Brain Health and Cognitive Impairment (AAP and BDP), T32 CA078586 Free Radical and Radiation Biology, University of Iowa (EV-R), Department of Defense Peer-Reviewed Alzheimer’s Research Program (PRARP) Award AZ210092 (W81XWH-22-1-0129) (EV-R), R01NS124081-01A1 (JSY), VeloSano (JSY), Case CCC (JSY), Cleveland Clinic (JSY), NIA RO1AG066707 (AAP), the Rebecca E. Barchas, M.D., DLFAPA Case Western Reserve University Chair in Translational Psychiatry (AAP), Elizabeth Ring Mather & William Gwinn Mather Fund (AAP), S. Livingston Samuel Mather Trust (AAP), G.R. Lincoln Family Foundation (AAP), Wick Foundation (AAP), Leonard Krieger Fund of the Cleveland Foundation (AAP), NIA/NIH R01AG071512 (BDP and AAP), JHU Catalyst Award (BDP), Louis Stokes VA Medical Center resources and facilities (AAP), NCI/NIH UIHC-CCOM Iowa Aging Initiative P01 CA217797 (BGA and DRS), NCI R25 CA221718 (AAP, KPL), Gateway for Cancer Research G-17-1500 (BGA), DRS was additionally supported by NCP/NIH PO1 CA244091 (DRS), and NCI/NIH P30 CA086862 (BGA, DRS, AAP). SB and EM were supported by the Alzheimer’ s Disease Translational Data Science Training Program NIH T32 AG071474. SB was also supported by Case Western Medical Scientist Training program NIH T32 GM007250. EM was also supported by NIH F31 5F31Ag089912. YK was supported by NIH/NIA F99 Ag083111. M − KS was supported by the New Faculty Startup Fund (370C-20220110), Creative-Pioneering Researchers Program (370C-20230108), and a research grant (370C-20240120) from Seoul National University. M − KS also acknowledges support from the National Research Foundation of Korea (RS-2023-00209597, RS-2024-00352229, RS-2024-00440679, RS-2024-00466703), Seoul R&BD program (BT240041) and donors of Alzheimer's Disease Research, a program of BrightFocus Foundation (A2019551F). Views expressed are those of the authors and do not reflect the position or policy of the Department of Veterans Affairs of the United States government.

CRediT authorship contribution statement

Edwin Vázquez-Rosa: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Writing – original draft, Writing – review & editing. Min-Kyoo Shin: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Writing – review & editing. Kalyani Chaubey: Investigation, Methodology. Sarah Barker: Investigation, Methodology. Sofia G. Corella: Investigation, Methodology. Suwarna Chakraborty: Investigation, Methodology. Sunil Jamuna Tripathi: Investigation, Methodology. Youngmin Yu: Investigation, Methodology. Jiwon Hyung: Investigation, Methodology. Himanshu Dashora: Investigation, Methodology. Jing Hao: Investigation, Methodology. Coral J. Cintrón-Pérez: Project administration, Supervision. Zea Bud: Investigation, Methodology. Matasha Dhar: Investigation, Methodology. Emiko Miller: Investigation, Methodology. Yeojung Koh: Conceptualization, Investigation, Methodology. Kate P. Lindley: Investigation, Methodology. Vidya Indrakumar: Investigation, Methodology. Rocio Aguila Rodriguez: Investigation, Methodology. Kranti A. Mapuskar: Investigation, Methodology. Joshua D. Schoenfeld: Investigation, Methodology. Hisashi Fujioka: Investigation, Methodology. Luke I. Szweda: Investigation, Methodology. Brigid M. Wilson: Data curation, Formal analysis, Funding acquisition, Writing – review & editing. Jennifer S. Yu: Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Writing – review & editing. Bindu D. Paul: Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing – review & editing. Douglas R. Spitz: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Resources, Supervision, Writing – review & editing. Bryan G. Allen: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing – review & editing. Andrew A. Pieper: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing.

Declaration of competing interest

AAP and EV-R hold patents related to P7C3 compounds. AAP is a co-founder of Glengary Brain Health. All other authors declare that they have no financial or personal relationships with any individuals or organizations that could potentially influence the work presented in this manuscript. There are no conflicts of interest related to the research, data analysis, interpretation, or publication of this work.