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Implementation of ultra-high dose-rate FLASH radiotherapy (FLASH-RT) is rapidly gaining traction as a unique cancer treatment modality able to dramatically minimize normal tissue toxicity while maintaining antitumor efficacy compared with standard-of-care radiotherapy at conventional dose rate (CONV-RT). The resultant improvements in the therapeutic index have sparked intense investigations in pursuit of the underlying mechanisms. As a preamble to clinical translation, we exposed non–tumor-bearing male and female mice to hypofractionated (3 × 10 Gy) whole brain FLASH- and CONV-RT to evaluate differential neurologic responses using a comprehensive panel of functional and molecular outcomes over a 6-month follow-up. In each instance, extensive and rigorous behavioral testing showed FLASH-RT to preserve cognitive indices of learning and memory that corresponded to a similar protection of synaptic plasticity as measured by long-term potentiation (LTP). These beneficial functional outcomes were not found after CONV-RT and were linked to a preservation of synaptic integrity at the molecular (synaptophysin) level and to reductions in neuroinflammation (CD68+ microglia) throughout specific brain regions known to be engaged by our selected cognitive tasks (hippocampus, medial prefrontal cortex). Ultrastructural changes in presynaptic/postsynaptic bouton (Bassoon/Homer-1 puncta) within these same regions of the brain were not found to differ in response to dose rate. With this clinically relevant dosing regimen, we provide a mechanistic blueprint from synapse to cognition detailing how FLASH-RT reduces normal tissue complications in the irradiated brain. Significance: Functional preservation of cognition and LTP after hypofractionated FLASH-RT are linked to a protection of synaptic integrity and a reduction in neuroinflammation over protracted after irradiation times.

Background Ultrahigh dose-rate radiotherapy (FLASH-RT) affords improvements in the therapeutic index by minimizing normal tissue toxicities without compromising antitumor efficacy compared to conventional dose-rate radiotherapy (CONV-RT). To investigate the translational potential of FLASH-RT to a human pediatric medulloblastoma brain tumor, we used a radiosensitive juvenile mouse model to assess adverse long-term neurological outcomes. Methods Cohorts of 3-week-old male and female C57Bl/6 mice exposed to hypofractionated (2 × 10 Gy, FLASH-RT or CONV-RT) whole brain irradiation and unirradiated controls underwent behavioral testing to ascertain cognitive status four months posttreatment. Animals were sacrificed 6 months post-irradiation and tissues were analyzed for neurological and cerebrovascular decrements. Results The neurological impact of FLASH-RT was analyzed over a 6-month follow-up. FLASH-RT ameliorated neurocognitive decrements induced by CONV-RT and preserved synaptic plasticity and integrity at the electrophysiological (long-term potentiation), molecular (synaptophysin), and structural (Bassoon/Homer-1 bouton) levels in multiple brain regions. The benefits of FLASH-RT were also linked to reduced neuroinflammation (activated microglia) and the preservation of the cerebrovascular structure, by maintaining aquaporin-4 levels and minimizing microglia colocalized to vessels. Conclusions Hypofractionated FLASH-RT affords significant and long-term normal tissue protection in the radiosensitive juvenile mouse brain when compared to CONV-RT. The capability of FLASH-RT to preserve critical cognitive outcomes and electrophysiological properties over 6-months is noteworthy and highlights its potential for resolving long-standing complications faced by pediatric brain tumor survivors. While care must be exercised before clinical translation is realized, present findings document the marked benefits of FLASH-RT that extend from synapse to cognition and the microvasculature.

Mice exposed to FLASH-RT performed similar to controls in the NOR, LDB, and FE tests. A, NOR testing. Male mice exposed to FLASH-RT performed similar to controls, while CONV-RT were unable to differentiate between the familiar and novel object. Female mice exposed to FLASH-RT and CONV-RT exhibited no significant difference between controls. B, Measurement of transition between light and dark environments in LDB testing. Male and female mice exposed to CONV-RT performed significantly worse than controls. Male FLASH-RT mice were not significantly different than controls; however, female mice did not transition between arenas as controls did. C and D, Fear extinction training and extinction days. Exposure to CONV-RT caused male mice to exhibit increased freezing during training, while this was not observed in FLASH-RT or females. Exposure to CONV-RT also inhibited mice ability disassociate the tone/shock pairing as well as FLASH and controls in males and females. Group effects (#) were found in training days indicate that CONV irradiated animals exhibited increase freezing behavior. E and F, Fear extinction testing. FLASH and control mice greatly reduced their tone/shock associations while CONV male (left) and female (right) mice did not. Data were analyzed using a one-way or two-way ANOVA followed by Bonferroni multiple comparison test (n = 11–15/sex/treatment). *, P ≤ 0.05; **, P ≤ 0.01; #, P ≤ 0.05; ns, no significance.

FLASH irradiation protects against reductions in LTP after CONV irradiation, 6 months after irradiation. A, Extended novel object recognition testing, 4 months after irradiation. Female mice that exposed to FLASH-RT performed significantly better than those who received CONV-RT (n = 15–16/treatment). B, TBS applied to the Schaffer collaterals produced a robust increase in fEPSP slope (as percent of baseline) in control and FLASH irradiated female mice but reduced in CONV mice 6 months after exposure. C, Levels of potentiation in the fEPSP slope maintained 1 hour after TBS was reduced significantly in the hippocampus of CONV-RT mice, but not in control or FLASH irradiated mice. Data were analyzed using a one-way ANOVA followed by Bonferroni multiple comparison test (n = 10–11/treatment). *, P ≤ 0.05: ***, P ≤ 0.001; ****, P ≤ 0.0001.

FLASH irradiation protected against prolonged inflammation found in CONV mice, 6 months after irradiation. Representative images of reactive microglia CD68 (red) and DAPI (blue) in the male mouse hippocampus (A) and representative images of IBA1 (green), TLR4 (red), and DAPI (blue; Scale bar = 100 μm; B). C, Quantification of CD68 immunofluorescence in the hippocampus and medial prefrontal cortex. Male (left) and female (right) mice exposed to FLASH-RT exhibit no significant change in CD68 expression while CONV mice expressed a neuroinflammatory response. D, Quantification of IBA1 and TLR4 colocalization in the hippocampus and medial prefrontal cortex. Male and female mice exhibited decreased levels of the neuroinflammatory mediator TLR4 when compared with CONV irradiation. E, Inflammatory cytokines measured using ELISA. IL1α exhibited elevated expression after CONV-RT exposure when compared with controls while FLASH induced no changes. No significant changes were observed in TNFα or IL1β. All data were analyzed using a one-way ANOVA followed by Bonferroni multiple comparison test (n = 4/sex/treatment, two sections analyzed/region/animal). *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001.

Mice exposed to FLASH-RT performed similar to controls in hippocampal-dependent learning and memory tests OUL while CONV-RT mice did not. A, Objects in updated locations testing experimental design. B, Update session behavior. At 4 months post-irradiation, FLASH and control mice showed preference for the novel toy and location in both males (left) and females (right) while CONV did not. C, Updated information test session. CONV irradiated female mice failed to learn the updated novel (A4) object over its predecessor (A3) when compared with FLASH and control mice. CONV irradiated male mice performed significantly worse than FLASH mice. D, Original information test session. CONV irradiated female mice were unable to differentiate between the updated novel location (A4) and the original location (A1) while FLASH and control performed similarly. No significant changes in male mice were observed. All data were analyzed using a one-way ANOVA followed by Bonferroni multiple comparison test (n = 11–16/sex/treatment). *, P ≤ 0.05; **, P ≤ 0.01; ns, no significance.

Irradiation parameters. Fractionated whole brain irradiation was performed on the Oriatron 6e, 6-MeV electron beam LINAC at Lausanne University Hospital. Mice received three doses of 10 Gy separated by 48 hours using a 17 mm graphite applicator at either CONV (CONV-RT; 0.09 Gy/second) or ultra-high dose-rate FLASH (FLASH-RT, delivered at 5.6 × 106 Gy/second in a single 1.8 μs pulse)

Timeline. Ten-week-old mice received hypofractionated FLASH or CONV irradiation (3 × 10 Gy). A, Male and female mice underwent behavioral testing 4 months after irradiation. At 6 months post-irradiation, mice were sampled, and tissues were prepared for either IHC analysis (n = 4/sex/treatment) or ELISA (n = 5–8/sex/treatment). B, Female mice were used to assess LTP. At 4 months post-irradiation, animals performed in the extended NOR behavioral assay (n = 16/treatment). At 6 months post-irradiation, mice were sacrificed and prepared for electrophysiological analysis of long-term potentiation (n = 10–11/treatment).

FLASH irradiation protects synaptic density and spine morphology, 6 months after irradiation. A, Representative images of synaptophysin (red), DAPI (blue; Scale bar = 100 μm). B, Representative image of Homer1a (red)/Bassoon (green), respectively (Scale bar = 10 μm and 2 μm in the zoomed image). C and D, Quantification of synaptic density using synaptophysin found that FLASH did not induce dendritic disruptions that were observed in mice exposed to CONV-RT in both the Hippocampus and mPFC. E and F, Quantification of Homer1a and Bassoon spots within 120 nm of each other. Male and female mice exhibited no differences between presynaptic and postsynaptic binding after FLASH or CONV irradiation in the hippocampus or mPFC. All data were analyzed using a one-way ANOVA followed by Bonferroni multiple comparison test (n = 4/sex/treatment, two sections analyzed/region/animal). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, no significance.

Supplementary Table from Dose- and Volume-Limiting Late Toxicity of FLASH Radiotherapy in Cats with Squamous Cell Carcinoma of the Nasal Planum and in Mini Pigs

Figure S1B- Higher magnification showing the skin of the mini-pig 15 and 32 weeks post radiotherapy (RT). At 15 weeks, skin area irradiated with 31Gy-FLASH-RT shows hair regrowth but not with 31Gy-Conv-RT. At 32 weeks, skin area irradiated with 34Gy-FLASH-RT is healthy whereas contractile skin fibrosis is observed with 34 Gy-Conv-RT.

Supplementary Figure from Dose- and Volume-Limiting Late Toxicity of FLASH Radiotherapy in Cats with Squamous Cell Carcinoma of the Nasal Planum and in Mini Pigs

Supplementary Data Table S1: WHO staging system (TNM Classification of Tumors in Domestic Animals. Geneva: World Health Organization; 1980) Table S2: Cat patient and treatment characteristics

Purpose:Previous studies using FLASH radiotherapy (RT) in mice showed a marked increase of the differential effect between normal tissue and tumors. To stimulate clinical transfer, we evaluated whether this effect could also occur in higher mammals.Experimental Design:Pig skin was used to investigate a potential difference in toxicity between irradiation delivered at an ultrahigh dose rate called “FLASH-RT” and irradiation delivered at a conventional dose rate called “Conv-RT.” A clinical, phase I, single-dose escalation trial (25–41 Gy) was performed in 6 cat patients with locally advanced T2/T3N0M0 squamous cell carcinoma of the nasal planum to determine the maximal tolerated dose and progression-free survival (PFS) of single-dose FLASH-RT.Results:Using, respectively, depilation and fibronecrosis as acute and late endpoints, a protective effect of FLASH-RT was observed (≥20% dose-equivalent difference vs. Conv-RT). Three cats experienced no acute toxicity, whereas 3 exhibited moderate/mild transient mucositis, and all cats had depilation. With a median follow-up of 13.5 months, the PFS at 16 months was 84%.Conclusions:Our results confirmed the potential advantage of FLASH-RT and provide a strong rationale for further evaluating FLASH-RT in human patients.See related commentary by Harrington, p. 3

Supplementary Tables S1-S5.

Purpose: Medullary thyroid carcinoma (MTC), an aggressive rare tumor due to activating mutations in the proto-oncogene RET, requires new therapeutic strategies. Sunitinib, a potent inhibitor of RET, VEGF receptor (VEGFR)-1, VEGFR-2, VEGFR-3, and platelet-derived growth factor receptor (PDGFR)α/β, has been reported as clinically effective in some patients with advanced MTC. In this study, we examine molecular mechanisms of action of sunitinib and identify candidate soluble biomarkers of response.Experimental Design: Both in vitro and in vivo assays, using the human TT RETC634W MTC cell line, were done to assess the activity of sunitinib. Kinetic microarray studies were used to analyze molecular pathways modified by sunitinib and to identify candidate biomarkers that were subsequently investigated in the serum of patients.Results: Sunitinib displayed antiproliferative and antiangiogenic activities and inhibited RET autophosphorylation and activation of downstream signaling pathways. We showed that sunitinib treatment induced major changes in the expression of genes involved in tissue invasion and metastasis including vimentin (VIM), urokinase plasminogen (PLAU), tenascin-C (TN-C), SPARC, and CD44. Analyzing downregulated genes, we identified those encoding secreted proteins and, among them, interleukin (IL)-8 was found to be modulated in the serum of xenografted mice under sunitinib treatment. Furthermore, we demonstrated that metastatic MTC patients presented increased serum levels of IL-8 and TGF-β2.Conclusions: Experimental models confirm the clinical efficacy of sunitinib observed in a few studies. Molecular pathways revealed by genomic signatures underline the impact of sunitinib on tissue invasion. Selected soluble candidate biomarkers could be of value for monitoring sunitinib response in metastatic MTC patients. Clin Cancer Res; 17(7); 2044–54. ©2011 AACR.

Purpose:The FLASH effect is characterized by normal tissue sparing without compromising tumor control. Although demonstrated in various preclinical models, safe translation of FLASH-radiotherapy stands to benefit from larger vertebrate animal models. Based on prior results, we designed a randomized phase III trial to investigate the FLASH effect in cat patients with spontaneous tumors. In parallel, the sparing capacity of FLASH-radiotherapy was studied on mini pigs by using large field irradiation.Experimental Design:Cats with T1-T2, N0 carcinomas of the nasal planum were randomly assigned to two arms of electron irradiation: arm 1 was the standard of care (SoC) and used 10 × 4.8 Gy (90% isodose); arm 2 used 1 × 30 Gy (90% isodose) FLASH. Mini pigs were irradiated using applicators of increasing size and a single surface dose of 31 Gy FLASH.Results:In cats, acute side effects were mild and similar in both arms. The trial was prematurely interrupted due to maxillary bone necrosis, which occurred 9 to 15 months after radiotherapy in 3 of 7 cats treated with FLASH-radiotherapy (43%), as compared with 0 of 9 cats treated with SoC. All cats were tumor-free at 1 year in both arms, with one cat progressing later in each arm. In pigs, no acute toxicity was recorded, but severe late skin necrosis occurred in a volume-dependent manner (7–9 months), which later resolved.Conclusions:The reported outcomes point to the caveats of translating single-high-dose FLASH-radiotherapy and emphasizes the need for caution and further investigations.See related commentary by Maity and Koumenis, p. 3636

Tumor hypoxia is a major cause of resistance to cancer treatments and especially to radiotherapy (RT) and we wanted to assess whether ultra-high dose rate FLASH RT could overcome this resistance. We engrafted tumor cells of various origins subcutaneously in mice to provide a reliable and rigorous way to modulate oxygen supply via vascular clamping or carbogen breathing. We irradiated tumors using a single 20 Gy fraction at either conventional (CONV) or FLASH dose-rate. Using multiple different subcutaneous tumor models, and in contrast CONV-RT, FLASH-RT retained anti-tumor efficacy under extreme hypoxia. These findings demonstrate that in addition to normal tissue sparing, FLASH-RT overcomes hypoxia-mediated tumor resistance. Follow-up molecular analysis using RNAseq profiling uncovered FLASH-specific inhibition of cell proliferation and translation as well as metabolic shifts that discriminated FLASH-RT from CONV-RT. These data provide new and specific insights into the mechanism of action and identify novel targets for intervention.

The physico-chemical and biological response to conventional and UHDR electron and proton beams was investigated, along with conventional photons. The temporal structure and nature of the beam affected both, with electron beam at ≥1400 Gy/s and proton beam at 0.1 and 1260 Gy/s found to be isoefficient at sparing zebrafish embryos.

The physico-chemical and biological response to conventional and UHDR electron and proton beams was investigated, along with conventional photons. The temporal structure and nature of the beam affected both, with electron beam at ≥1400 Gy/s and proton beam at 0.1 and 1260 Gy/s found to be isoefficient at sparing zebrafish embryos.

In France, the human papilloma (HPV) vaccine is recommended to all female adolescents, and since 2016 to men having sex with men (MSM) under the age of 27. Here, we aimed to explore HPV vaccine coverage in adult MSM living in France.We elaborated an anonymous online questionnaire targeting MSM and disseminated it between October 7, 2018 and January 15, 2019 by various means, including a Facebook® discussion forum for pre-exposure prophylaxis (PrEP) users.Overall, 2094 participants completed the questionnaire (mean age 35.4 ± 11 years); 25.8% were in the age class targeted by the HPV vaccine recommendation; 16.1% were in a PrEP program. On a 1-10 scale, they evaluated being "in favor of vaccination" at a median of 10, and general vaccine usefulness and harmfulness at medians of 10 and 2, respectively; 62.4% considered that the HPV vaccine was rather not or not at all dangerous. Those using PrEP had better perceptions of vaccination in general and of the HPV vaccine in particular. For the 1728 participants with a family physician, he/she had proposed the HPV vaccine in 9.9% of cases (9.1% of those in the targeted age class [27 years]). Overall, 1994 knew their HPV vaccine status; 8.0% had received at least 1 dose of the vaccine, including 17.9% of those in the targeted age class (and 52.2% among the 40 participants in this age group who received PrEP). When the 1935 participants who declared to be unvaccinated against HPV were asked whether they would accept to be vaccinated, 34.4% answered "rather yes" and 45.5% "definitely yes".HPV vaccine coverage is low among French MSM. Our results suggest that this trend has more to do with the infrequent proposals made by clinicians than with negative vaccine perceptions.

Aims/hypothesisBeta-site amyloid precursor protein-cleaving enzyme 1 (BACE1) is required for the production of toxic amyloid peptides and is highly expressed in the brain, but also to a lesser extent in major peripheral organs such as muscle and liver. In contrast, BACE2 is mainly expressed in peripheral tissues and is enriched in pancreatic beta cells, where it regulates beta- cell function and mass. Previous reports demonstrated that loss of BACE1 function decreases body weight, protects against diet-induced obesity and enhances insulin sensitivity in mice, whereas mice lacking Bace2 exhibit reduced blood glucose levels, improved intraperitoneal glucose tolerance and increased beta-cell mass. Impaired glucose homeostasis and insulin resistance are hallmarks of type 2 diabetes and have been implicated in Alzheimer’s disease. Therefore, we tested the contribution of the individual BACE isoforms to those metabolic phenotypes by placing Bace1 knockout (KO), Bace2 KO, Bace1/2 double knockout (dKO) and wild-type (WT) mice on a high-fat high-cholesterol diet (HFD) for 16 weeks.MethodsBace1 KO (n = 18), Bace2 KO (n = 18), Bace1/2 dKO (n = 18) and WT C57BL/6N mice (n = 54) were fed a HFD for 16 weeks (age 9–25 weeks). Body composition was measured before initiation of the HFD and after 11 weeks of HFD. Oral glucose tolerance and insulin sensitivity tests were performed after 12 and 13 weeks of HFD, respectively, and full blood chemistry was analyzed after 16 weeks of HFD. The effects of subchronic BACE1/2 inhibition were assessed by administration of 10 mg/kg/day of the dual BACE1/2 inhibitor MBi-3 in a HFD fed to C57BL/6N mice for 3 weeks.ResultsBace1 KO and Bace1/2 dKO mice showed decreased body weight and improved glucose tolerance and insulin resistance vs. WT mice. Conversely, Bace2 KO mice did not show any significant differences in body weight, glucose tolerance or insulin resistance under our experimental conditions. Finally, subchronic MBi-3–mediated BACE1/2 inhibition in mice in conjunction with a HFD resulted in a modest improvement of glucose tolerance.Conclusions/interpretationOur data indicate that lack of BACE1 – but not BACE2 – function contributes mainly to the metabolic phenotypic changes observed in Bace1/2 dKO mice, suggesting that inhibition of BACE1 has the greater role (vs. BACE2) in any potential improvements in metabolic homeostasis.HIGHLIGHTSInsulin resistance may develop in the brains of patients with Alzheimer’s disease (83/85 characters)BACE1 and BACE2 may play a role in glucose homeostasis and insulin sensitivity (80/85 characters)Body weight in mice decreased with Bace1 KO and Bace1/2 KO but not Bace2 KO alone (83/85 characters)Bace1 and Bace1/2, but not Bace2, KO improved glucose tolerance/insulin resistance (84/85 characters)Improved metabolic homeostasis may follow loss of BACE1 rather than BACE 2 activity (85/85 characters)

Simple Summary The objective of the present study was to investigate the impact of sex in the development of long-term toxicities affecting quality of life in cancer survivors after systemic paclitaxel treatment and cardiac irradiation. Sex-specific differences may affect tumor biology, drug pharmacokinetics and dynamics, and response to local treatment such as radiation therapy (RT). However, sex is rarely taken into consideration when administering cancer therapies. Interestingly, female mice are protected from paclitaxel-induced neurotoxicity as well as from radiotherapy-induced cardiotoxicity, and deficiency in the small GTPase RhoB reversed the protection in females but not in males. In conclusion, our results are the first to identify sex- and organ-specific responses to systemic paclitaxel administration and localized RT. These results may have important implications for the management of cancer patients and implementation of personalized medicine in oncology. Abstract The impact of sex in the development of long-term toxicities affecting the quality of life of cancer survivors has not been investigated experimentally. To address this issue, a series of neurologic and cardiologic endpoints were used to investigate sex-based differences triggered by paclitaxel treatment and radiotherapy exposure. Male and female wild-type (WT) mice were treated with paclitaxel (150 and 300 mg/kg) administered weekly over 6 weeks or exposed to 19 Gy cardiac irradiation. Cohorts were analyzed for behavioral and neurobiologic endpoints to assess systemic toxicity of paclitaxel or cardiovascular endpoints to assess radiotherapy toxicity. Interestingly, female WT mice exhibited enhanced tolerance compared to male WT mice regardless of the treatment regimen. To provide insight into the possible sex-specific protective mechanisms, rhoB-deficient animals and elderly mice (22 months) were used with a focus on the possible contribution of sex hormones, including estrogen. In females, RhoB deficiency and advanced age had no impact on neurocognitive impairment induced by paclitaxel but enhanced cardiac sensitivity to radiotherapy. Conversely, rhoB-deficiency protected males from radiation toxicity. In sum, RhoB was identified as a molecular determinant driving estrogen-dependent cardioprotection in female mice, whereas neuroprotection was not sex hormone dependent. To our knowledge, this study revealed for the first time sex- and organ-specific responses to paclitaxel and radiotherapy.

Background Cancerous cells can recycle metabolic ammonium for their growth. As this ammonium has a low nitrogen isotope ratio (15N/14N), its recycling may cause cancer tissue to have lower 15N/14N than surrounding healthy tissue. We investigated whether, within a given tissue type in individual mice, tumoral and healthy tissues could be distinguished based on their 15N/14N. Methods Micro-biopsies of murine tumors and adjacent tissues were analyzed for 15N/14N using novel high-sensitivity methods. Isotopic analysis was pursued in Nude and C57BL/6 mice models with mature orthotopic brain and head&neck tumors generated by implantation of H454 and MEERL95 murine cells, respectively. Results In the 7 mice analyzed, the brain tumors had distinctly lower 15N/14N than healthy neural tissue. In the 5 mice with head&neck tumors, the difference was smaller and more variable. This was at least partly due to infiltration of healthy head&neck tissue by tumor cells. However, it may also indicate that the 15N/14N difference between tumoral and healthy tissue depends on the nitrogen metabolism of the healthy organ in question. Conclusions The findings, coupled with the high sensitivity of the 15N/14N measurement method used here, suggest a new approach for micro-biopsy-based diagnosis of malignancy as well as an avenue for investigation of cancer metabolism.

Microglia play a critical role in maintaining neural function. While microglial activity follows a circadian rhythm, it is not clear how this intrinsic clock relates to their function, especially in stimulated conditions such as in the control of systemic energy homeostasis or memory formation. In this study, we found that microglia-specific knock-down of the core clock gene, Bmal1, resulted in increased microglial phagocytosis in mice subjected to high-fat diet (HFD)-induced metabolic stress and likewise among mice engaged in critical cognitive processes. Enhanced microglial phagocytosis was associated with significant retention of pro-opiomelanocortin (POMC)-immunoreactivity in the mediobasal hypothalamus in mice on a HFD as well as the formation of mature spines in the hippocampus during the learning process. This response ultimately protected mice from HFD-induced obesity and resulted in improved performance on memory tests. We conclude that loss of the rigorous control implemented by the intrinsic clock machinery increases the extent to which microglial phagocytosis can be triggered by neighboring neurons under metabolic stress or during memory formation. Taken together, microglial responses associated with loss of Bmal1 serve to ensure a healthier microenvironment for neighboring neurons in the setting of an adaptive response. Thus, microglial Bmal1 may be an important therapeutic target for metabolic and cognitive disorders with relevance to psychiatric disease.

Despite recent advances in pharmacological research and microsurgery, lymphoedema remains an incurable disease that deeply affects quality of life. There is an urgent need for innovative approaches to restore continuous lymph flow in affected tissues. To this end, the efficacy of a subcutaneously implanted draining device in reducing lymphoedema volume in a rat hindlimb lymphoedema model was tested. A rat model of chronic lymphoedema was developed by surgical removal of popliteal and inguinal lymph nodes, followed by irradiation. The model was characterised by monitoring limb volume via tape measure, skin water content via dielectric constant measurement, and lymphatic drainage via lymphofluoroscopy. After lymphoedema establishment in 16 Wistar rats, a device made of fenestrated tubing equipped with a miniaturised pumping system, was implanted subcutaneously in the affected limb to restore continuous recirculation of interstitial fluid. Lymphofluoroscopy imaging showed impaired lymphatic drainage following lymphadenectomy and irradiation. Affected limb volume and skin water content increased significantly compared with the untreated limb, with a median (interquartile range) of 3.85 (0.38) cm 3 versus 3.03 (0.43) cm 3 for volume (n = 16, p = .001) and 26.6 (9.1) versus 16.6 (3.7) cm 3 for skin dielectric constant (n = 16, p = .001). Treatment of lymphoedema with the implanted drainage device showed that 5 weeks post-implant excess volume was significantly reduced by 51 ± 18% compared with the pre-implant situation (n = 9 sham group, n = 7 pump group). Lymphoedema volume in the rat model was significantly reduced by restoring continuous drainage of excess fluid using a novel subcutaneously implanted device, opening the way to the development of an artificial lymphatic vessel.

This study is the first proof of concept that the FLASH effect can be triggered by X-rays. Our results show that a 10 Gy whole-brain irradiation delivered at ultra-high dose-rate with synchrotron generated X-rays does not induce memory deficit; it reduces hippocampal cell-division impairment and induces less reactive astrogliosis.

Human stem cell-derived extracellular vesicles (EV) provide many advantages over cell-based therapies for the treatment of functionally compromised tissue beds and organ sites. Here we sought to determine whether human embryonic stem cell (hESC)-derived EV could resolve in part, the adverse late normal tissue complications associated with exposure of the lung to ionizing radiation. The hESC-derived EV were systemically administered to the mice via the retro-orbital sinus to explore the potential therapeutic benefits following exposure to high thoracic doses of radiation (14 Gy). Data demonstrated that hESC-derived EV treatment significantly improved overall survival of the irradiated cohorts (P < 0.001). Increased survival was also associated with significant reductions in lung fibrosis as quantified by CBCT imaging (P < 0.01, 2 weeks post-irradiation). Qualitative histological analyses revealed reduced indications of radiation induced pulmonary injury in animals treated with EV. EV were then subjected to a rigorous proteomic analysis to ascertain the potential bioactive cargo that may prove beneficial in ameliorating radiation-induced normal tissue toxicities in the lung. Proteomics validated several consensus exosome markers (e.g., CD68) and identified major classes of proteins involved in nuclear pore complexes, epigenetics, cell cycle, growth and proliferation, DNA repair, antioxidant function, and cellular metabolism (TCA cycle and oxidative phosphorylation, OXYPHOS). Interestingly, EV were also found to contain mitochondrial components (mtDNA, OXYPHOS protein subunits), which may contribute to the metabolic reprograming and recovery of radiation-injured pulmonary tissue. To evaluate the safety of EV treatments in the context of the radiotherapeutic management of tumors, mice harboring TC1 tumor xenografts were subjected to the same EV treatments shown to forestall lung fibrosis. Data indicated that over the course of one month, no change in the growth of flank tumors between treated and control cohorts was observed. In conclusion, present findings demonstrate that systemic delivery of hESC-derived EV could ameliorate radiation-induced normal tissue complications in the lung, through a variety of potential mechanisms based on EV cargo analysis.

Purpose: Recent data have shown that single-fraction irradiation delivered to the whole brain in less than tenths of a second using FLASH radiotherapy (FLASH-RT), does not elicit neurocognitive deficits in mice. This observation has important clinical implications for the management of invasive and treatment-resistant brain tumors that involves relatively large irradiation volumes with high cytotoxic doses. Experimental Design: Therefore, we aimed at simultaneously investigating the antitumor efficacy and neuroprotective benefits of FLASH-RT 1-month after exposure, using a well-characterized murine orthotopic glioblastoma model. As fractionated regimens of radiotherapy are the standard of care for glioblastoma treatment, we incorporated dose fractionation to simultaneously validate the neuroprotective effects and optimized tumor treatments with FLASH-RT. Results: The capability of FLASH-RT to minimize the induction of radiation-induced brain toxicities has been attributed to the reduction of reactive oxygen species, casting some concern that this might translate to a possible loss of antitumor efficacy. Our study shows that FLASH and CONV-RT are isoefficient in delaying glioblastoma growth for all tested regimens. Furthermore, only FLASH-RT was found to significantly spare radiation-induced cognitive deficits in learning and memory in tumor-bearing animals after the delivery of large neurotoxic single dose or hypofractionated regimens. Conclusions: The present results show that FLASH-RT delivered with hypofractionated regimens is able to spare the normal brain from radiation-induced toxicities without compromising tumor cure. This exciting capability provides an initial framework for future clinical applications of FLASH-RT. See related commentary by Huang and Mendonca, p. 662

Encephalic radiation therapy delivered at a conventional dose rate (CONV, 0.1–2.0 Gy/min) elicits a variety of temporally distinct damage signatures that invariably involve persistent indications of neuroinflammation. Past work has shown an involvement of both the innate and adaptive immune systems in modulating the central nervous system (CNS) radiation injury response, where elevations in astrogliosis, microgliosis and cytokine signaling define a complex pattern of normal tissue toxicities that never completely resolve. These side effects constitute a major limitation in the management of CNS malignancies in both adult and pediatric patients. The advent of a novel ultra-high dose-rate irradiation modality termed FLASH radiotherapy (FLASH-RT, instantaneous dose rates ≥ 10(6) Gy/s; 10 Gy delivered in 1–10 pulses of 1.8 μs) has been reported to minimize a range of normal tissue toxicities typically concurrent with CONV exposures, an effect that has been coined the “FLASH effect.” Since the FLASH effect has now been found to significantly limit persistent inflammatory signatures in the brain, we sought to further elucidate whether changes in astrogliosis might account for the differential dose-rate response of the irradiated brain. Here we report that markers selected for activated astrogliosis and immune signaling in the brain (glial fibrillary acidic protein, GFAP; toll-like receptor 4, TLR4) are expressed at reduced levels after FLASH irradiation compared to CONV-irradiated animals. Interestingly, while FLASH-RT did not induce astrogliosis and TLR4, the expression level of complement C1q and C3 were found to be elevated in both FLASH and CONV irradiation modalities compared to the control. Although functional outcomes in the CNS remain to be cross-validated in response to the specific changes in protein expression reported, the data provide compelling evidence that distinguishes the dose-rate response of normal tissue injury in the irradiated brain.

Persistent vasculature abnormalities contribute to an altered CNS microenvironment that further compromises the integrity of the blood-brain barrier and exposes the brain to a host of neurotoxic conditions. Standard radiation therapy at conventional (CONV) dose rate elicits short-term damage to the blood-brain barrier by disrupting supportive cells, vasculature volume and tight junction proteins. While current clinical applications of cranial radiotherapy use dose fractionation to reduce normal tissue damage, these treatments still cause significant complications. While dose escalation enhances treatment of radiation-resistant tumors, methods to subvert normal tissue damage are clearly needed. In this regard, we have recently developed a new modality of irradiation based on the use of ultra-high-dose-rate FLASH that does not induce the classical pathogenic patterns caused by CONV irradiation. In previous work, we optimized the physical parameters required to minimize normal brain toxicity (i.e., FLASH, instantaneous intra-pulse dose rate, 6.9 · 10(6) Gy/s, at a mean dose rate of 2,500 Gy/s), which we then used in the current study to determine the effect of FLASH on the integrity of the vasculature and the blood-brain barrier. Both early (24 h, one week) and late (one month) timepoints postirradiation were investigated using C57Bl/6J female mice exposed to whole-brain irradiation delivered in single doses of 25 Gy and 10 Gy, respectively, using CONV (0.09 Gy/s) or FLASH (>10(6) Gy/s). While the majority of changes found one day postirradiation were minimal, FLASH was found to reduce levels of apoptosis in the neurogenic regions of the brain at this time. At one week and one month postirradiation, CONV was found to induce vascular dilation, a well described sign of vascular alteration, while FLASH minimized these effects. These results were positively correlated with and temporally coincident to changes in the immunostaining of the vasodilator eNOS colocalized to the vasculature, suggestive of possible dysregulation in blood flow at these latter times. Overall expression of the tight junction proteins, occludin and claudin-5, which was significantly reduced after CONV irradiation, remained unchanged in the FLASH-irradiated brains at one and four weeks postirradiation. Our data further confirm that, compared to isodoses of CONV irradiation known to elicit detrimental effects, FLASH does not damage the normal vasculature. These data now provide the first evidence that FLASH preserves microvasculature integrity in the brain, which may prove beneficial to cognition while allowing for better tumor control in the clinic.

Purpose Ultra-high-dose-rate FLASH radiation therapy has been shown to minimize side effects of irradiation in various organs while keeping antitumor efficacy. This property, called the FLASH effect, has caused enthusiasm in the radiation oncology community because it opens opportunities for safe dose escalation and improved radiation therapy outcome. Here, we investigated the impact of ultra-high-dose-rate FLASH versus conventional-dose-rate (CONV) total body irradiation (TBI) on humanized models of T-cell acute lymphoblastic leukemia (T-ALL) and normal human hematopoiesis. Methods and Materials We optimized the geometry of irradiation to ensure reproducible and homogeneous procedures using eRT6/Oriatron. Three T-ALL patient–derived xenografts and hematopoietic stem/progenitor cells (HSPCs) and CD34+ cells isolated from umbilical cord blood were transplanted into immunocompromised mice, together or separately. After reconstitution, mice received 4 Gy FLASH and CONV-TBI, and tumor growth and normal hematopoiesis were studied. A retrospective study of clinical and gene-profiling data previously obtained on the 3 T-ALL patient-derived xenografts was performed. Results FLASH-TBI was more efficient than CONV-TBI in controlling the propagation of 2 cases of T-ALL, whereas the third case of T-ALL was more responsive to CONV-TBI. The 2 FLASH-sensitive cases of T-ALL had similar genetic abnormalities, and a putative susceptibility imprint to FLASH-RT was found. In addition, FLASH-TBI was able to preserve some HSPC/CD34+ cell potential. Interestingly, when HSPC and T-ALL were present in the same animals, FLASH-TBI could control tumor development in most (3 of 4) of the secondary grafted animals, whereas among the mice receiving CONV-TBI, treated cells died with high leukemia infiltration. Conclusions Compared with CONV-TBI, FLASH-TBI reduced functional damage to human blood stem cells and had a therapeutic effect on human T-ALL with a common genetic and genomic profile. The validity of the defined susceptibility imprint needs to be investigated further; however, to our knowledge, the present findings are the first to show benefits of FLASH-TBI on human hematopoiesis and leukemia treatment.

Major advances in high precision treatment delivery and imaging have greatly improved the tolerance of radiotherapy (RT), however, the selective sparing of normal tissue and the reduction of neurocognitive side effects from radiation-induced toxicities remain significant problems for pediatric patients with brain tumors. While the overall survival of pediatric patients afflicted with medulloblastoma (MB), the most common type primary brain cancer in children, remains high (&ge, 80%), lifelong neurotoxic side-effects are commonplace and adversely impact patients&rsquo, quality of life. To circumvent these clinical complications, we have investigated the capability of ultra-high dose rate FLASH-radiotherapy (FLASH-RT) to protect the radiosensitive juvenile mouse brain from normal tissue toxicities. Compared to conventional dose rate (CONV)irradiation, FLASH-RT was found to ameliorate radiation-induced cognitive dysfunction in multiple independent behavioral paradigms, preserve developing and mature neurons, minimize microgliosis and limit the reduction of the plasmatic level of growth hormone. The protective &ldquo, FLASH effect&rdquo, was pronounced, especially since a similar whole brain dose of 8 Gy delivered with CONV-RT caused marked reductions in multiple indices of behavioral performance (objects in updated location, novel object recognition, fear extinction, light-dark box, social interaction), reductions in the number of immature (doublecortin+) and mature (NeuN+) neurons and increased neuroinflammation, adverse effects that were not found with FLASH-RT. Our data point to a potentially innovative treatment modality that is able to spare, if not prevent, many of the side effects associated with long-term treatment that disrupt the long-term cognitive and emotional well-being of medulloblastoma survivors.

This study shows for the first time that normal brain tissue toxicities after WBI can be reduced with increased dose rate. Spatial memory is preserved after WBI with mean dose rates above 100Gy/s, whereas 10Gy WBI at a conventional radiotherapy dose rate (0.1Gy/s) totally impairs spatial memory.

International audience; PURPOSE:The "PRAVACUR" phase 2 trial (NCT01268202) assessed the efficacy of pravastatin as an antifibrotic agent in patients with established cutaneous and subcutaneous radiation-induced fibrosis (RIF) after head and neck squamous cell carcinoma (HNSCC) radiation therapy and/or radiochemotherapy.METHODS AND MATERIALS:The main inclusion criteria were: NSCC in remission, grade ≥2 cutaneous and subcutaneous neck RIF (National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.0), and no current treatment with statins or fibrates. Patients received pravastatin 40 mg/d for 12 months. The primary endpoint was reduction of RIF thickness by more than 30% at 12 months, as measured by cutaneous high-frequency ultrasonography. Secondary endpoints included RIF severity reduction, pravastatin tolerance, and quality of life.RESULTS:Sixty patients with grade 2 (n = 37), grade 3 (n = 22), or grade 4 (n = 1) RIF were enrolled from February 2011 to April 2016. The mean interval between RIF diagnosis and pravastatin initiation was 17.1 months. Pravastatin was stopped before 11 months of treatment in 18 patients (because of grade ≥2 adverse events related to pravastatin in 8 patients [13%]). In the 40 patients in whom pravastatin efficacy was assessed by high-frequency ultrasonography at baseline and at 12 months of treatment, a reduction of RIF thickness ≥30% was observed in 15 of 42 patients (35.7%; 95% confidence interval, 21.6%-52.0%). At the 12-month clinical evaluation, RIF severity was decreased in 50% of patients (n = 21; 95% confidence interval, 34.2%-65.8%), and the patients' self-perception, mood state, and social functioning were significantly improved. Pravastatin was well tolerated, with a very low occurrence of grade 3 toxicities (myalgia, n = 1) and grade 2 toxicities (myalgia/arthralgia or esophagitis, n = 3).CONCLUSIONS:This phase 2 prospective study supports the notion of radioinduced fibrosis reversibility. It showed that pravastatin (40 mg/d for 12 months) is an efficient antifibrotic agent in patients with grade ≥2 cutaneous and subcutaneous fibrosis after HNSCC radiation therapy.

Here, we highlight the potential translational benefits of delivering FLASH radiotherapy using ultra-high dose rates (>100 Gy⋅s-1). Compared with conventional dose-rate (CONV; 0.07-0.1 Gy⋅s-1) modalities, we showed that FLASH did not cause radiation-induced deficits in learning and memory in mice. Moreover, 6 months after exposure, CONV caused permanent alterations in neurocognitive end points, whereas FLASH did not induce behaviors characteristic of anxiety and depression and did not impair extinction memory. Mechanistic investigations showed that increasing the oxygen tension in the brain through carbogen breathing reversed the neuroprotective effects of FLASH, while radiochemical studies confirmed that FLASH produced lower levels of the toxic reactive oxygen species hydrogen peroxide. In addition, FLASH did not induce neuroinflammation, a process described as oxidative stress-dependent, and was also associated with a marked preservation of neuronal morphology and dendritic spine density. The remarkable normal tissue sparing afforded by FLASH may someday provide heretofore unrealized opportunities for dose escalation to the tumor bed, capabilities that promise to hasten the translation of this groundbreaking irradiation modality into clinical practice.

Over the past decades, technological advances have transformed radiation therapy (RT) into a precise and powerful treatment for cancer patients. Nevertheless, the treatment of radiation-resistant tumors is still restricted by the dose-limiting normal tissue complications. In this context, FLASH-RT is emerging in the field. Consisting of delivering doses within an extremely short irradiation time, FLASH-RT has been identified as a promising new tool to enhance the differential effect between tumors and normal tissues. Indeed, preclinical studies on various animal models and a veterinarian clinical trial have recently shown that compared to conventional dose-rate RT, FLASH-RT could control tumors while minimizing normal tissue toxicity. In the present review, we summarize the main data supporting the clinical translation of FLASH-RT and explore its feasibility, the key irradiation parameters and the potential technologies needed for a successful clinical translation.

OBJECTIVE Systemic sclerosis (SSc) is a rare intractable disease with unmet medical need and fibrosis-related mortality. Absence of efficient treatments has prompted the development of novel therapeutic strategies, among which mesenchymal stem cells/stromal cells (MSCs) or progenitor stromal cells appear to be one of the most attractive options. The purpose of this study was to use the murine model of hypochlorite-induced SSc to investigate the systemic effects of MSCs on the main features of the diffuse form of the disease: skin and lung fibrosis, autoimmunity, and oxidative status. METHODS We compared the effects of different doses of MSCs (2.5 × 10(5) , 5 × 10(5) , and 10(6) ) infused at different time points. Skin thickness was assessed during the experiment. At the time of euthanasia, biologic parameters were quantified in blood and tissues (by enzyme-linked immunosorbent assay, quantitative reverse transcription-polymerase chain reaction, assessment of collagen content). Assessments of histology and immunostaining were also performed. RESULTS A lower expression of markers of fibrosis (Col1, Col3, Tgfb1, and aSma) was observed in both skin and lung following MSC infusion, which was consistent with histologic improvement and was inversely proportional to the injected dose. Importantly, sera from treated mice exhibited lower levels of anti-Scl-70 autoantibodies and enhanced antioxidant capacity, confirming the systemic effect of MSCs. Of interest, MSC administration was efficient in both the preventive and the curative approach. We further provide evidence that MSCs exerted an antifibrotic role by normalizing extracellular matrix remodeling parameters as well as reducing proinflammatory cytokine levels and increasing antioxidant defenses. CONCLUSION The results of this study demonstrate the beneficial and systemic effects of MSC administration in the HOCl murine model of diffuse SSc, which is a promising finding from a clinical perspective.

Purpose: Previous studies using FLASH radiotherapy (RT) in mice showed a marked increase of the differential effect between normal tissue and tumors. To stimulate clinical transfer, we evaluated whether this effect could also occur in higher mammals. Experimental Design: Pig skin was used to investigate a potential difference in toxicity between irradiation delivered at an ultrahigh dose rate called “FLASH-RT” and irradiation delivered at a conventional dose rate called “Conv-RT.” A clinical, phase I, single-dose escalation trial (25–41 Gy) was performed in 6 cat patients with locally advanced T2/T3N0M0 squamous cell carcinoma of the nasal planum to determine the maximal tolerated dose and progression-free survival (PFS) of single-dose FLASH-RT. Results: Using, respectively, depilation and fibronecrosis as acute and late endpoints, a protective effect of FLASH-RT was observed (≥20% dose-equivalent difference vs. Conv-RT). Three cats experienced no acute toxicity, whereas 3 exhibited moderate/mild transient mucositis, and all cats had depilation. With a median follow-up of 13.5 months, the PFS at 16 months was 84%. Conclusions: Our results confirmed the potential advantage of FLASH-RT and provide a strong rationale for further evaluating FLASH-RT in human patients. See related commentary by Harrington, p. 3

Summary Background Metabolic syndrome is a combination of symptoms including obesity, dyslipidaemia, glucose intolerance and hypertension. Oxidative stress appears to be a pathophysiological factor that links these signs and encourages progression towards heart failure and diabetes. Nox4 is a hydrogen peroxide nicotinamide adenine dinucleotide phosphate (NADPH) oxidase isoform – found in various cardiovascular cells and tissues, but also in tissues such as the liver – which is involved in glucose and lipid homeostasis. Aims To test whether inhibition of the Nox4 enzyme could improve blood pressure and metabolic parameters in mice receiving either angiotensin II or a high-fat diet. Methods Systolic and diastolic arterial pressures, pulse rate and heart rate were obtained in 24 male mice (12 wild-type [WT] and 12 Nox4–/–) before and during 14 days of angiotensin II infusion. After angiotensin II infusion, cardiac histological remodeling was assessed. Weight and biochemical parameters were measured in 18 male and 18 female mice (nine WT and nine Nox4–/– per gender) after 10 weeks on a standard chow diet, then 15 weeks on a high-fat diet. Glucose tolerance and insulin sensitivity were tested at age 25 weeks. Results Knock-out animals did not demonstrate a baseline blood pressure phenotype, but blocking Nox4 protected against angiotensin II-mediated arterial and pulse pressure increases. No protection against angiotensin II-induced cardiac fibrosis was observed. From a metabolic point of view, Nox4 inhibition reduced plasma triglycerides in male and female mice under a chow diet. However, Nox4 deletion did not affect the metabolic profile under a high-fat diet in males or females, but increased glucose intolerance in females. Conclusion Our data identify Nox4 as a key source of radical oxygen species involved in hypertension and some metabolic problems.

Although opiates represent the most effective analgesics, their use in chronic treatments is associated with numerous side effects including the development of pain hypersensitivity and analgesic tolerance. We recently identified a novel orally active neuropeptide FF (NPFF) receptor antagonist, RF313, which efficiently prevents the development of fentanyl-induced hyperalgesia in rats. In this study, we investigated the properties of this compound into more details. We show that RF313 exhibited a pronounced selectivity for NPFF receptors, antagonist activity at NPFF1 receptor (NPFF1R) subtype both in vitro and in vivo and no major side effects when administered in mice up to 30 mg/kg. When co-administered with opiates in rats and mice, it improved their analgesic efficacy and prevented the development of long lasting opioid-induced hyperalgesia. Moreover, and in marked contrast with the dipeptidic NPFF receptor antagonist RF9, RF313 displayed negligible affinity and no agonist activity (up to 100 muM) toward the kisspeptin receptor. Finally, in male hamster, RF313 had no effect when administered alone but fully blocked the increase in LH induced by RFRP-3, while RF9 per se induced a significant increase in LH levels which is consistent with its ability to activate kisspeptin receptors. Altogether, our data indicate that RF313 represents an interesting compound for the development of therapeutic tools aiming at improving analgesic action of opiates and reducing adverse side effects associated with their chronic administration. Moreover, its lack of agonist activity at the kisspeptin receptor indicates that RF313 might be considered a better pharmacological tool, when compared to RF9, to examine the regulatory roles of RF-amide-related peptides and NPFF1R in reproduction.

International audience; Mutations of the AMP-activated kinase gamma 2 subunit (AMPKγ2), N488I (AMPKγ2NI) and R531G (AMPKγ2RG), are associated with Wolff-Parkinson-White (WPW) syndrome, a cardiac disorder characterized by ventricular pre-excitation in humans. Cardiac-specific transgenic overexpression of human AMPKγ2NI or AMPKγ2RG leads to constitutive AMPK activation and the WPW phenotype in mice. However, overexpression of these mutant proteins also caused profound, non-physiological increase in cardiac glycogen, which might abnormally alter the true phenotype. To investigate whether physiological levels of AMPKγ2NI or AMPKγ2RG mutation cause WPW syndrome and metabolic changes in other organs, we generated two knock-in mouse lines on the C57BL/6N background harboring mutations of human AMPKγ2NI and AMPKγ2RG, respectively. Similar to the reported phenotypes of mice overexpressing AMPKγ2NI or AMPKγ2RG in the heart, both lines developed WPW syndrome and cardiac hypertrophy; however, these effects were independent of cardiac glycogen accumulation. Compared with AMPKγ2WT mice, AMPKγ2NI and AMPKγ2RG mice exhibited reduced body weight, fat mass, and liver steatosis when fed with a high fat diet (HFD). Surprisingly, AMPKγ2RG but not AMPKγ2NI mice fed with an HFD exhibited severe kidney injury characterized by glycogen accumulation, inflammation, apoptosis, cyst formation, and impaired renal function. These results demonstrate that expression of AMPKγ2NI and AMPKγ2RG mutations at physiological levels can induce beneficial metabolic effects but that this is accompanied by WPW syndrome. Our data also reveal an unexpected effect of AMPKγ2RG in the kidney, linking lifelong constitutive activation of AMPK to a potential risk for kidney dysfunction in the context of an HFD.