The multidisciplinary management of patients with brain metastases consists of surgical resection, radiation treatment and systemic treatment. Tailoring and timing these treatment modalities is challenging. This study presents real-world data from consecutively treated patients and assesses the impact of all treatment strategies and their relation with survival. The aim is to provide new insights to improve multidisciplinary decisions towards individualized treatment strategies in patients with brain metastases.A retrospective consecutive cohort study was performed. Patients with brain metastases were included between June 2018 and May 2020. Brain metastases of small cell lung carcinoma were excluded. Overall survival was analyzed in multivariable models.676 patients were included in the study, 596 (88%) received radiotherapy, 41 (6%) awaited the effect of newly started or switched systemic treatment and 39 (6%) received best supportive care. Overall survival in the stereotactic radiotherapy group was 14 months (IQR 5–32) and 32 months (IQR 11–43) in patients who started or switched systemic treatment and initially did not receive radiotherapy. In patients with brain metastases without options for local or systemic treatment best supportive care was provided, these patients had an overall survival of 0 months (IQR 0–1). Options for systemic treatment, Karnofsky Performance Score ≥ 70 and breast cancer were prognostic for a longer overall survival, while progressive extracranial metastases and whole-brain-radiotherapy were prognostic for shorter overall survival.Assessing prognosis in light of systemic treatment options is crucial after the diagnosis of brain metastasis for the consideration of radiotherapy versus best supportive care. While novel systemic treatments such as targeted molecular treatment and immunotherapy are successfully applied to target extracranial metastases (ECM), the question whether to treat brain metastases (BM) systemically as well is a more debated field –3. IntraIn this study, we analyze real-world data of patients with BM consecutively treated with radiotherapy in our center in the recent era of novel systemic treatments. This study aims to evaluate the role of systemic treatment in the multidisciplinary approach and treatment decisions for patients with BM.A single-center, retrospective cohort study was conducted in a tertiary referral, comprehensive cancer center in the Netherlands. All patients with BM, aged 18 years or older were consecutively included in the study between June 1st 2018 and May 30th 2020. Patients with a primary, non-metastatic intracranial tumor were excluded, as were patients with BM from small cell lung carcinoma who received prophylactic cranial radiotherapy . FurtherThe following baseline characteristics were retrieved from medical charts: sex, age (dichotomized into ≤ 65 years versus > 65 years), primary tumor , breast, melanoma or other), Karnofsky Performance Score (KPS) (< 70 vs. ≥ 70), status of ECM at the time BM were diagnosed  and whether patients had previous radiotherapy for other BM. Furthermore, targeted therapy was defined as therapy against targetable mutations. These included anaplastic lymphoma kinase (ALK), epidermal growth factor receptor (EGFR) and programmed death-ligand 1 (PD-L1) in NSCLC, and BRAF in melanoma. In addition to reviewing patient files, all multidisciplinary neuro-oncology board meetings in the abovementioned period were reviewed in order to collect information about medical decision-making.The following items were assessed for patients undergoing stereotactic radiotherapy: number of irradiated BM, total tumor volume, and the volume of the largest lesion. These data were unavailable for patients receiving WBRT. Furthermore, the type of radiotherapy was evaluated. Patients who received stereotactic radiotherapy (SRT) in a single session were classified as ‘single fraction’, those that received radiotherapy in several consecutive sessions as ‘fractionated’ and patients who underwent radiotherapy in multiple sessions with 2–4 weeks interval as ‘staged’ . PostopeThe status of systemic treatment possibilities at the time of BM diagnosis was classified as follows:‘First ST’: if patients presented synchronously with their primary tumor and BM and started their first line of systemic treatment.‘Continued ST’: if the same systemic treatment was continued after BM diagnosis, in patients with metachronous BM;‘Switched ST’: if the type of systemic treatment was switched after BM diagnosis, in patients with metachronous BM;‘No ST, but options’: if patients had a metachronous BM, without ECM, without the indication for systemic treatment;‘No options for ST’: if no further options for systemic treatment were available.The primary outcome measure was overall survival (OS) which was defined as the time in months between diagnosis of BM and death, assessed using clinical reports. If clinical reports provided insufficient information, general practitioners were contacted to provide the date of death. The last follow-up was on March 22nd 2023.Kaplan-Meier analysis was performed to assess OS. In case patients underwent multiple radiotherapy sessions during the follow-up period, characteristics of the first radiotherapy session were used for analysis.For statistical analysis, patients were divided into three groups, in accordance with treatment decisions made in the multidisciplinary neuro-oncology board meetings:Group 1: patients who received immediate radiotherapy at diagnosis of BM;Group 2: patients awaiting the effects of newly started or switch of systemic treatment , because intracranial response of systemic treatment was expected, which could lead to either a smaller tumor volume at subsequent radiotherapy or no radiotherapy at all;Group 3: patients who received no local treatment, but started best supportive care after diagnosis of BM.The comparison between subgroups was made with the Chi-Square test or Fisher exact test. Univariate analyses were performed with log-rank test for categorical data. The Cox proportional hazards model was used in continuous data. Stepwise forward likelihood ratio multivariate analyses including all statistically significant predictors of the univariate analysis were included to assess the prognostic value of different variables using the Cox proportional hazard. For the group of patients receiving best supportive care, only descriptive statistics were used. Statistical analysis was performed using IBM SPSS Statistics for Windows, version 26 .676 consecutively diagnosed BM patients were included Fig. ; Table 1A total of 596 patients received radiotherapy, of which 501 (84%) patients received SRT and 95 (16%) patients WBRT. Median OS in the group receiving SRT was 14 months (IQR 5–32) versus 2 months (IQR 1–6) in case of WBRT. Median age in the group receiving radiotherapy was 60 years (IQR 51–67), and 331 (55%) patients were female  of NSCLC patients had a targetable mutation, as had 62 (65%) melanoma patients. Of these patients, 95 (65%) with NSCLC received or had an option to start targeted therapy after BM diagnosis, as did 24 (39%) patients with melanoma. Furthermore, 25 of 62 (40%) of patients with melanoma and targetable mutations received immunotherapy.Differences in OS between groups with different systemic treatment possibilities are shown in Fig. . PatientFactors associated with a significant shorter OS in the univariate analysis are shown in Table 3  and > 30 cm3 (HR 2.3 (1.3–4.0), were associated with a shorter OS  patients with BM. 38 patients (93%).started or switched systemic treatment with a potential intracranial response. Three patients (7%) did not receive any systemic treatment, although there were remaining available options Table .Median survival of the total group was 32 months (IQR 11–43). Four patients (10%) died within 3 months, 37 (90%) survived longer than three months. Of the four patients that died within 3 months, 1 patient had rapid cerebral progression leading to coma, 1 patient had cerebral and leptomeningeal progression leading to severe epileptic seizures, 1 patient had rapid progression of ECM and 1 patient opted for best supportive care because of severe pain due to progression ECM.8 (61%) of 13 patients with NSCLC had targetable mutations and all 8 (100%) patients received targeted therapy. 20 (83%) of 24 patients with melanoma had targetable mutations, 12 (60%) received targeted therapy.27 (66%) of the 41 patients did receive radiotherapy in the further course of the disease (4 (10%) within three months, 23 (56%) after three months). The median time between the decision to wait for the effects of systemic treatment and the start of eventual SRT was 4 months (IQR 2–7).39 (6%) patients received best supportive care directly after the BM diagnosis. All patients had a KPS < 70, and 28 patients (72%) had progressive ECM. 11 (28%) patients presented with synchronous BM and therefore had no systemic treatment before, 12 patients (31%) had possibilities for a switch in systemic treatment, 7 (18%) had no systemic options. In 7 (18%) patients clinical condition was too poor for systemic treatment and 2 (5%) patients refused systemic treatment. The median OS was 0 months (IQR 0–1).In this study, we present the OS of a consecutive cohort of patients with BM presented in our center from June 2018 to May 2020 and treated with radiotherapy and/or systemic therapy.OS was 14 months (IQR 5–32) for patients receiving SRT immediately after BM diagnosis and 2 months (IQR 1–6) in patients whom received WBRT. In the group that awaited the effect of systemic treatment on BM, OS was 32 months (IQR 11–43).Our study confirms the earlier stated correlation of clinical predictors such as age, KPS , type ofFor half of patients that awaited the intracranial response of recently started or switched systematic treatment, SRT was administered within 4 months. In NSCLC and melanoma patients that awaited the effect of systemic treatment, 28 of 37 patients (76%) had a targetable mutation. In the group that received radiotherapy directly after BM diagnosis, 208 of 383 (54%) patients with NSCLC and melanoma had a targetable mutation.Also, a larger proportion received, or had options for, targeted therapy. Systemic treatment options for BM is a prognostic factor besides KPS, primary tumor type and status of extracranial metastases, and should be considered in treatment decisions for patients with BM. The expected intracranial response of the systemic treatment and its expected duration determine whether BM are treated with SRT only, a combination of SRT followed by a (switched) systemic treatment, systemic treatment only, or delayed SRT after intracranial response on systemic therapy.The survival of the cohort that received WBRT was poor, half of the patients died within 2 months. Whether WBRT should have a place in treating palliative patients with BM has been debated before , consideOur study stresses the important role of systemic treatment for individualizing treatment strategy in patients with BM. Firstly, systemic treatment with an intracranial response can postpone or prevent local treatment in patients that newly started or recently switched systemic treatment.In our study, the time between the decision to await systemic treatment effect and treat eventually with radiotherapy was close to 4 months for half of the patients. In the group of patients with more than 3 months survival, 41% did not receive radiotherapy at all after the diagnosis of BM. Systemic treatment can be initially preferred over radiotherapy in patients with large tumor volume or high number of BM and stable extracranial disease. A treatment strategy could be to start with systemic treatment  to reduce the volume or number of BM and to give subsequent radiotherapy at the time of progression.Secondly, in case patients are in need for systemic treatment due to extracranial disease and a low likelihood of intracranial response is to be expected, the choice of local treatment modality is important in the overall treatment strategy. Radiotherapy in patients who are not in direct need of surgery  can expedite the start of systemic treatment compared to surgery and this could lead to survival benefits. In our data, this resulted in a relatively low percentage (12%) of patients that underwent surgery as a primary local treatment. This hypothesis stresses that more real world data is needed for optimizing the treatment strategies for individual patients including analysis of survival, tumor response, toxicity and quality of life. More insights have become available to estimate the intracranial responses and ability to defer or prevent local therapy –25, whicThis study presents a large, consecutive cohort of all patients in our center diagnosed with BM, regardless of treatment after the diagnosis and the status of their systemic treatment. To our knowledge, this is the largest cohort of patients with BM in which the systemic treatment status is included.Several limitations should be addressed. Firstly, our series was obtained in a single, tertiary referral, comprehensive cancer center, which could have affected the patient and treatment characteristics and OS. All patients underwent intracranial follow-up in our center, but a subset of patients underwent systemic follow-up in the referring hospital. Although we were able to retrieve dates of death in all patients, it was not always possible to make a robust evaluation whether the cause of death was caused by either progressive intracranial or extracranial disease, or both.Since this study was a retrospective cohort study, we could not accommodate for the differences in assessing clinical features such as KPS between different physicians.In conclusion, this study shows that in a selected group of patients with BM, that started a new line of systemic treatment with an expected significant intracranial response rate, the response on BM could be awaited, and SRT deferred. There is a limited role for radiotherapy in patients with progressive ECM and no systemic treatment options. Furthermore, our results endorse the important role that (new) systemic treatment options have on OS. We recommend that the availability of systemic treatment options should always be considered in determining treatment strategies in patients with BM.


Polycystic ovary syndrome (PCOS) seems to be associated with increased ovarian sympathetic nerve activity and in rodent models of PCOS reducing the sympathetic drive to the ovary, through denervation or neuromodulation, improves ovulation rate. We hypothesised that sympathetic nerves work with gonadotropins to promote development and survival of small antral follicles to develop a polycystic ovary phenotype.Using a clinically realistic ovine model we showed a rich sympathetic innervation to the normal ovary and reinnervation after ovarian transplantation. Using needlepoint diathermy to the nerve plexus in the ovarian vascular pedicle we were able to denervate the ovary resulting in reduced intraovarian noradrenaline and tyrosine hydroxylase immunostained sympathetic nerves. We developed an acute polycystic ovary (PCO) model using gonadotrophin releasing hormone (GnRH) agonist followed infusion of follicle stimulating hormone (FSH) with increased pulsatile luteinising hormone (LH). This resulted in increased numbers of smaller antral follicles in the ovary when compared to FSH infusion suggesting a polycystic ovary.Denervation had no effect of the survival or numbers of follicles in the acute PCO model and did not impact on ovulation, follicular and luteal hormone profiles in a normal cycle.Although the ovary is richly inervated we did not find evidence for a role of sympathetic nerves in ovarian function or small follicle growth and survival. Polycystic ovary syndrome (PCOS) is a common endocrine disorder affecting 7-8% of women of reproductive age . AlthougSympathetic nerves are present within the ovary but the role of ovarian sympathetic innervation remains unclear . It has There is evidence of an increased sympathetic tone in women with PCOS . Women wWe hypothesised that the sympathetic nervous system works in parallel with gonadotrophins to facilitate the development of a PCO and that ovarian denervation would improve the polycystic ovarian phenotype. Herein we assessed the effect of ovarian denervation in a large animal model that, unlike rodents, has very similar ovarian function to women. We then developed and validated an acute model of PCO using gonadotrophin manipulation and assessed the effect of denervation on gonadotrophin-driven follicle growth.Ovis aries) weighing 60-75 kg in their third to fifth breeding season. Ewes were housed together in spacious pens with ad libitum hay supplemented with Excel ewe nuts  and Crystalayx extra high energy lick . All experiments were conducted under Project Licence  from the UK Home Office and underwent institutional ethics review. This work was conducted in accordance with Animals (Scientific Procedures) Act 1986, Galvani Policy on the Care, Welfare and Treatment of Animals Policy 040 and approved by the Galvani Bioelectronics Animal Scientific Review Committee and the GSK Policy on the Care, Welfare and Treatment of Animals. Ovarian sections from an earlier study collected 11 months after whole ovarian cryopreservation and transplantation using Scottish Greyface ewes as described in detail previously (We studied adult Scottish Greyface ewes (Ewes were killed using a schedule 1 method and ovaries and ovarian vascular pedicles were collected. The vascular pedicles were fixed in Bouin’s solution for 24 hours and transferred to 70% ethanol for subsequent paraffin wax embedding. The ovaries were either: 1) fixed in Bouin’s solution for 24 hours and embedded in paraffin wax for subsequent immunohistochemistry, 2) halved longitudinally and one half fixed in Bouin’s solution and embedded into paraffin wax and the other half snap frozen and stored at -80 °C for subsequent RNA extraction and measurement of intraovarian noradrenaline or 3) fixed in 4% paraformaldehyde for optical projection tomography, depending on the experiment.2O2 for 10 minutes, blocking with avidin and biotin  and then serum blocking with 20% normal goat serum/5% bovine serum albumin (BSA) in Tris Buffered saline . Slides were washed in TBS between treatments, then in TBS containing 0.025% Triton X-100 (TBS-T) prior to serum block and antibody incubation.Mid-ovarian tissue sections cut to 5μm were mounted on permafrost slides. Sections were dewaxed, rehydrated as described previously . AntigenThe primary antibody diluted in serum block , mouse monoclonal anti-Ki67 1:100  (1:1000) or rabbit polyclonal anti-caspase 3 1:100 ) was applied to sections and incubated overnight at 4°C. Slides were washed in TBS-T and the secondary antibody  diluted 1:500 in serum block) was applied to slides for 1 hour. Slides were washed in TBS-T followed by Vectastain ABC Elite tertiary complex  for 1 hour after which 3,3′-diaminobenzidine  was applied for 3 minutes to visualise binding. Sections were then counterstained with haematoxylin and mounted using Pertex mounting medium . Negative controls were non-specific mouse or rabbit serum of equivalent immunoglobulin concentrations in place of the primary antibody.Dual labelled tissue sections were prepared for confocal microscopy following the immunohistochemistry protocol described above, with the following adjustments. The peroxidase wash step was omitted and the slides were permeabilised normally through a series of two five-minute washes in TBS-T. After an incubation time of one hour in 10% normal goat serum, the endothelial antibody  and mouse anti-tyrosine hydroxylase antibody were diluted together at a concentration of 1:100 and 1:200 respectively in TBS, before being added to the slides and incubated overnight at 4°C.® 594  and goat-anti-mouse IgG secondary antibody conjugated to Alexaflour® 488 , each at 1:100 in 10% NGS. Slides were incubated in the dark at room temperature for two hours in a humidity chamber. After washing with TBS-Tween 20 in the dark slides were mounted in an aqueous solution containing 4’6-diamidino-2-phenyllindole (DAPI), and stored for 12 hours at 4°C, prior to visulisation on a Zeiss LSM 880 AxioObserver Z1 confocal fluorescent microscope .After washing in TBS with 0.01% Tween 20  the slides were incubated with biotinylated goat-anti-rabbit secondary antibody diluted in 10% NGS at a concentration of 1:500 for one hour at room temperature. After washing in TBS-Tween 20 (0.01%) the slides were incubated with DylightTwo examiners, blinded to treatment, graded the immunohistological staining (based on area of staining) of tyrosine hydroxylase independently and the scores were averaged for whole ovary sections. Each ovary section was examined and graded out of four, with zero indicating no staining present and four indicating abundant staining throughout the tissue. Spatiotemporal examinations of vessel and nerve relationships were made using a Zeiss LSM880 Confocal Microscope. Images were captured to illustrate this at 20x magnification, and 63x magnification with oil.Two independent examiners, blinded to treatment, also independently counted the number of follicles from a standardised mid-section of the ovary  as well A mini-laparotomy was performed with sterile technique under general anaesthesia, induced using isoflourane . A small paramedian incision exposed the ovaries. To avoid non-specific ovarian damage we specifically targeted the nerves in the ovarian neurovascular pedicle. Needlepoint diathermy using monopolar coagulation current   was used3 sample from the ovarian cortex at the lateral edge of the ovary was used to measure intraovarian noradrenaline (NA) concentrations. It was weighed and homogenised in lysis buffer . Lysate was spun at 5000 rpm at 4 °C for 10 min and the supernatant analysed. Quantification was carried out using the competitive NA ELISA kit  as described previously . The progesterone assay (Cobas progesterone II) has a sensitivity of 0.48 nmol/l. The cross reactivity with related steroids is <1%. The estradiol assay (Cobas Estradiol III) has a sensitivity of 11 pmol/l. Apart from 6α-OH estradiol the cross reactivity of related steroids is <1%. Both assays have CVs <10%.RNA was extracted from tissue using RNeasy mini spin columns following manufacturer’s protocol and concentration measured using NanoDrop 1000 Spectrophotometer as described previously . ComplimPrimer3 Input version 0.4, online software, was used to design forward and reverse primers from DNA sequences obtained from Ensembl Genome Browser. Sequences were checked for specificity using Basic Local Alignment Search Tool and validity confirmed as previously described . The priIntravaginal progestogen-impregnated sponges  were inserted into ewes (n=6) and then 3 days later gonadotrophin releasing hormone implants  were inserted, through large bore needles, for pituitary suppression. Eleven days later the sponge was removed and the sheep were given an injection of prostaglandin F2α  to ensure luteolysis of any residual corpora lutea and prepare for an artificial follicular phase. Three days later the jugular vein of the sheep was canulated and infusions started. FSH  given at 1mg/hour via jugular catheter using Graseby MS 16A syringe drivers; Luteinizing Hormone  given as 4 hourly pulses via the jugular catheter (18 µg/pulse) using Zyklomat pulse infusion pumps, for 6 days. Sheep were given either physiological FSH concentrations only with baseline endogenous LH (n=3) or physiological FSH + additional exogenous LH infusions (n=3) (Progesterone sponges were inserted in ewes (n=6) and 7 days later GnRH implants were inserted as described above. Five days later mini-laparotomy was performed as described above followed by unilateral diathermy needle denervation to the left ovary was performed, allowing the right ovary to serve as an internal control. Six days later sponges were removed and PG injections were given as described above. Sheep were cannulated 3 days later and infusions started as described above with FSH (1 mg/hour) (n=3) or FSH+LH  (n=3) for 6 days , then 30 minutes each in 30%, 70%, 90%, 100% Ethanol. They were transferred to Methanol for 2 hours, then into fresh Methanol and stored at 4°C until processed for scanning. Ovaries were attached to mounting blocks, and then immersed in BABB  until cleared sufficiently for scanning. Cleared ovaries were scanned in a calibrated Bioptonics 3001 tomograph . Dataviewer  was used to combine the scans into 2D and 3D models to then quantify the size and number of each follicle throughout each ovary. After scanning ovaries were returned to methanol to remove BABB, then washed 30 min each in 100%, 90% and 70% Ethanol. They were stored in 70% Ethanol prior to embedding in paraffin wax for sectioning.Statistical analysis was conducted using GraphPad Prism 8.0  with P<0.05 considered statistically significant. Proportional contingency table analysis was measured by Fisher’s exact test for 2x2 tables and Chi squared for larger tables. After checking for normality and similar variances two column comparisons were examined using unpaired two-tailed t-tests if the data was parametric and Mann Whitney U tests if not parametric or normally distributed. Correlation was assessed using Spearman co-efficient of correlation.The ovaries and ovarian pedicle of Scottish Greyface sheep (n=6) were examined macroscopically  underwent laparotomy and unilateral denervation in the mid-follicular phase using monopolar needle micro-diathermy of the putative ovarian nerves within the neurovascular bundle leaving the vasculature intact. After 21 days the sheep were killed and the ovaries examined. There was a loss of sympathetic nerve fibre immunostaining within the treated ovary compared to the contralateral control ovary  or a sham procedure without diathermy (n=4). The hormonal profiles of the ewes were then examined daily over an ovarian cycle and ovaries were collected 21 days later and at that stage we examined intraovarian noradrenaline concentrations to confirm ongoing denervation during the experiment. In the follicular phase there was no difference in estradiol concentrations . Sheep were given an infusion of FSH with baseline endogenous LH (Control) or with additional high dose pulsatile LH (PCOS-like). After seven days the ovaries were collected and analysed by optical tomography, which allows the whole ovary to be viewed in real-time digital sections to accurately count and measure all the antral follicles (After bilateral denervation we then assessed the effects of gonadotrophin infusion to create the acute PCOS-like model. There was a difference in the pattern of follicles in the control high dose pulsatile LH PCOS-like sheep (We have shown that there is dense sympathetic innervation in the ovine ovary that is not only associated with blood vessels but also seen around the avascular small follicles. Sympathetic denervation, confirmed by intra-ovarian sympathetic nerve immunostaining and NA measurement, has no effect on antral follicle growth, ovulation or luteolysis. In addition, it had no effect on gonadotrophin action in follicular development. We recreated a polycystic ovarian morphology using gonadotrophin manipulation to test the effect of sympathetic denervation. There was no effect on the acute antral follicle response. This has narrowed down the potential roles of the sympathetic nervous system in the ovary.Sympathetic innervation of the ovary is seen in multiple species including rodents , ruminanWe surmised that total severance of the sympathetic nerves entering the ovary would occur during oophorectomy . If ovarWe were able to denervate the ovary and examine what happened during the follicular and luteal phase of a cycle. Follicular growth and ovulation occurred normally. Importantly luteolysis also occurred normally. As luteolysis involves a vascular counter-current between the uterus and the ovary  this wouIf the sympathetic nervous system can augment gonadotrophin action in the ovary, and promote follicular survival, this might facilitate the development of a polycystic ovary in PCOS. Women with PCOS have increased LH action within the ovary . ManipulThis acute model of PCO allowed us to determine the effects of sympathetic denervation on LH action in the development of a polycystic ovary. We hypothesised that after denervation the polycystic ovary would have less and larger follicles. Elegant rodent studies involving reducing sympathetic innervation improved ovarian function in an induced polycystic ovary phenotype . There wIn summary we have developed an acute model of PCO ovarian morphology in an ovine model by manipulating gonadotrophins, which may have future utility in terms of separation of metabolic aspects from ovarian aspects of this syndrome. One benefit of this model was that it allowed us to investigate the role of the sympathetic nervous system in the regulating gonadotrophin action in the follicle. Gonadotrophins are the master regulator of the gonadotrophin dependent follicle and there does not seem to be any significant neural contribution. However, it remains likely that sympathetic action is involved in ovarian function in concert with gonadotrophins. The involvement of sympathetic nerves in the polycystic ovary, and a physiological effect of testosterone secretion, remains possible but that gonadotrophin action is the fundamental driver of ovarian structure and function. There may be effects before gonadotrophins take over follicular growth and development and possible effects on local androgen production that are not able to be ascertained in this model. This suggests that longer term experiments, perhaps using a clinically realistic prenatally programmed ovine model of PCOS , 24 woulThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was approved by The UK Home Office, The University of Edinburgh Institutional review and Galvani Bioelectronics Animal Scientific Review Committee. The study was conducted in accordance with the local legislation and institutional requirements.WCD: Conceptualization, Formal Analysis, Funding acquisition, Methodology, Project administration, Writing – original draft, Writing – review & editing. LN: Data curation, Formal Analysis, Writing – review & editing. RO’H: Formal Analysis, Investigation, Writing – review & editing. JW: Funding acquisition, Resources, Writing – review & editing. JM: Funding acquisition, Resources, Writing – review & editing. BC: Resources, Writing – review & editing. JT: Formal Analysis, Writing – review & editing. MR: Funding acquisition, Methodology, Supervision, Writing – review & editing.


Background: Complex organ formation requires the coordinated morphogenesis of adjacent tissue layers. Here,we report a role for the planar cell polarity (PCP) proteins Fz6 and Celsr1 in generating squamous basal cells in the later stage embryonic epidermis of the mouse is reported, whichmay impact upon the shape of overlying suprabasal cells.Methods: The depth of the epidermis and basal layer as well as cell proliferation index was scored from immunostained wax sections taken from different mouse embryos mutant in planar cell polarity signalling and their wild-type littermates. Orientation of epidermal cell division inCelsr1Crash/Crash mutants was determined from thick frozen immunostained sections. Immunostained wax sections of wild-type skin explants cultured using the Lumox method enabled any changes in epidermal and basal layer depth to be measured following the release of surface tension upon dissection of skin away from the whole embryo.  Results: Increased numbers of columnar and cuboidal basal epidermal cells were observed infz6-/- mutant andCelsr1 mouse mutant Crash/Crash which correlated with visibly more rounded suprabasal cells and a thicker epidermis.Conclusions: Altogether these data support tissue intrinsic roles for PCP proteins in ‘outside-in’  skin architecture. Complex organs comprise multiple tissue layers. During organ formation, morphogenesis of the different tissue layers is coordinated to achieve the correct architecture for healthy organ function. A useful model for this is the mammalian embryonic epidermis which is a stratified epithelium comprising of a progenitor basal cell layer, a suprabasal layer and a surface periderm layer  leads to randomisation of hair follicle orientation in adult back skin  null mutants exhibit an abnormal thickening of the later stage epidermis (E17-E17.5), which correlates with a loss of squamous basal cells. Increased numbers of taller basal cells in each mutant correlate with more rounded suprabasal cells. Altogether the data presented is consistent with a model where PCP mouse mutants exhibit a late stage, tissue intrinsic failure to locally generate or maintain squamous basal cell shape which may directly impact on suprabasal cell shape. This is exciting because PCP proteins are expressed within the basal epidermal layer, raising interesting questions about how basal layer PCP signalling impacts upon adjacent, overlying suprabasal layers. These data also provide additional support for core-PCP protein function(s) in radial skin architecture. In addition to epidermal stratification, embryonic skin becomes globally and locally patterned along the tissue plane. The core planar cell polarity (PCP) pathway plays a major role in this process which manifests in the orientation and alignment of hair follicle down-growth along the head-to-tail axis of mouse back skin  at King’s College London before Home Office review, the Establishment Licence number for King's College London is X24D82DFF. Prior to individual studies taking place, a study plan was also reviewed within King’s College London. Mice were held in individually ventilated cages at an average 21°C within a 12h light/dark cycle with food and water provided ad-lib. Mating trios  were set up and monitored morning and afternoon until all females had plugged. At desired gestational age, pregnant females were killed via schedule 1  with all embryos killed by hypothermia and exsanguination.fz6 andCelsr1 mouse mutants have been described previously (Crsh/Crsh:fz6-/-:Celsr1-/-). Genotypes are:Crsh/Crsh, inbred BALB/c strain;fz6-/-, C57BL6 × 129 background;Celsr1-/-, floxed mice 129 x 1/Sv 129S1/Sv background  were crossed with the germline deleter PGK-Cre and the heterozygous progenies (Celsr1+/-) were inter-crossed to generate homozygous mutants (Celsr1-/-) and controls (Celsr1+/+). All mice were between 8 weeks and 1 year of age. Mice were genotyped by polymerase chain reaction (PCR). Wild-type mice used for explant cultures were the outbred CD-1 strain (in-house). Timed mating was performed and 9am of the first day of plugging was taken as E0.5. Wild-type and homozygous mutant mice from the same litters were compared. No inclusion or exclusion criteria were set, no embryos were excluded from any of the analyses performed. Mutant embryos were not randomised but due to variability inCelsr1-/- embryo phenotypes data from 3 representative embryos are shown of n=8Celsr1-/- embryos analysed. One wild-type embryo for each condition was randomly chosen for data analysis. Analyses were carried out as outlined in ARRIVE checklist (Theecklist .Risk assessments were generated prior to experimentation.Paraffin sections were generated and immunostained according toH&E) staining was as described inSuspension explants were embedded into wax as described inAxiovision rel.4.7 software (Zeiss). Images were exported intoAdobe Photoshop CS5.1 and manipulated using thecrop function.The primary antibodies used were cytokeratin1 (K1) antibody , Ki67  and PhosphoHistoneH3 . Secondary antibodies (Life Technologies) were fluorescently labelled and used at 1:1000.measure tool of Axiovision rel.4.7 software (Zeiss). Measurements for total height were excluded if the epidermis in section appeared distorted. For each basal cell measured, total height of the overlying epidermis including the height of the basal cell was also scored. At least 75 measurements were made from three independent sections from each of n=3 embryos (from two litters) for each condition. Total adult animals used was n=6. Analysis was blinded, embryos embedded in wax for each gene knockout condition and wild-type were assigned a number by the histologist. One wild-type was randomly chosen for analysis from each mutant cohort. Mutant embryos (n=8) from more than two litters were analysed forCelsr1-/- to confirm the variability in phenotype observed in original litters for one embryo. Embryos exhibiting representative phenotypes are shown forCelsr1-/-. Immunostained sections were analysed using Axiovision rel 4.7 software, measurements were taken and then the genotype linked to each number was revealed. Histograms were generated usingGraphpad Prism 7, butMicrosoft Excel could also be used. Statistical analysis was one-way ANOVA with Bonferetti post-hoc test, using Prism software. Frequency graphs, correlation plots and their R-squared values were generated by Prism software.For whole embryos, epidermal thickness and basal cell height measurements were from longitudinal wax sections, every 10 basal cells across inter-follicular epidermis from mid-trunk back skin. Immunostained or H&E-stained wax sections of epidermis were viewed under 40X magnification, bright field illumination and measurements were taken using the. Analysis of explants was not blinded.For suspension explants, five measurements of epidermal thickness and basal cell height were taken from a wax section derived from the central portion of each explant, n=3 explants, total of 15 measurements, using the measure tool of Axiovision rel 4.7 software. For each basal cell measured, total height of the overlying epidermis including the height of the basal cell was again scored. A total or two measurements were taken from each mid-flank of n=3 transverse wax sections taken from each of n=3 mouse embryos, total of 18 measurements. All embryos were taken from the same two wild-type litters. Total adult animals used was n=2. Histograms were generated using Graphpad Prism 7. Statistical analysis was unpaired two-tailed Student’s t-test, using Prism softwareKi67 staining was used to assess any unusual patterns of cell proliferation. Mitotic index was scored manually using immunostaining for pHH3 and H&E staining of telophase divisions from three images analysed from three embryos (from two litters) for each condition, basal and suprabasal divisions were scored for each 1000 basal cells counted, n> 1000 basal cells for each condition. Total adult animals used was n=6, the same embryos were used to score for cell proliferation as were used to measure epidermal thickness. Histograms were generated using Graphpad Prism 7 and statistical analysis was unpaired two-tailed Student’s t-test, using Prism software. Analysis was blinded, embryos embedded in wax for each condition were assigned a number by the histologist. Immunostained sections were analysed using Axiovision rel 4.7 software, measurements were taken and then the genotype linked to each number was revealed.Volocity software were generated  was calculated. A total of n=37 wild-type divisions and n=36Crsh/Crsh divisions were analysed from skin sections taken from n=3 embryos (from four litters) for each condition. Total adult animals used was n=4. Statistical analysis using unpaired two-tailed Student’s t-test compared wild-type andCrash/Crash divisions in bins shown, significant differences were not found. Histograms were generated using Graphpad Prism 7 and statistical analysis was unpaired two-tailed Student’s t-test, using Prism software. Analysis was not blinded.Thick longitudinal frozen sections (100μm) were immunostained with fibronectin to label the basal lamina, E-cadherin to label cell outlines and DAPI to label nuclei. Sequential images along a stretch of mid-trunk back skin were taken on a confocal microscope (Nikon A1R). Z-stack images were generated using 0.3µm steps. For each image, 3-D reconstructions of each Z-stack usingated see. All telCrsh/Crsh embryonic skin is that the suprabasal layer was thickened compared to wild-type, with visibly more rounded, cytokeratin-1 expressing suprabasal cells (nuclear shape was taken as a proxy for cell shape) overlying the basal layer in mutant skin compared to wild-type littermates  mouse embryos, which exhibit closed neural tubes, revealed that the epidermis offz6-/- embryos was significantly thickened compared to wild-type embryos  were generated for individual embryos revealing strong similarities betweenld-type . Individidermis . Indeed,idermis  for all fz6-/- embryos were observed that might account for the epidermal thickening phenotype  in wild-type contained a mixture of tall (columnar), square  and flattened (squamous) epithelial cells with a bias towards squamous-type: most frequent height was found for bin centre 6μm  Converse height .fz6-/- andCrsh/Crsh correlated with suprabasal cells which were more visibly rounded in shape  supporting a positive correlation between basal cell height and total epidermal height  and 0.17  respectively. Mean r-squared value for representativeCelsr1-/- embryos was 0.33 . One way ANOVA (Bonferonni post-hoc test) across all PCP mutants and wild-type controls gave aP value of 0.024 with multiple comparisons revealingP values of 0.0266 (fz6KO), 0.0223 (Crsh/Crsh) and 0.22 (Celsr1-/-) for individual mutants compared to wild-type controls. We found therefore a generally lower correlation between basal cell height and total epidermal height for all PCP mutants analysed.Notably, the bias towards taller basal cells inn shape . In wildn shape . Cuboidan shape . Correlan shape  revealed height . Mean r-fz6-/- andCrsh/Crsh mutants show visibly more rounded suprabasal cells, we were not able to robustly quantify suprabasal cell shape however in wax sections. One simple explanation for the presence of more rounded suprabasal cells directly above the more columnar height of basal cells observed infz6-/- andCrsh/Crsh skin is direct mechanical contact. We have recently used suspension explant (ex-vivo) culture to investigate skin morphogenesis and found that explants of wild-type surface ectoderm deform when excised away from the intact embryo, demonstrating that the embryonic skin is under extrinsic mechanical tension  epidermal basal cells. A similar later stage thickening of the epidermis has also been reported in theVangl2 Lp/Lp mutant, which was linked to the presence of taller basal cells and an increased frequency of oblique/perpendicular basal cell divisions at the expense of planar orientations much earlier in skin development i.e., at around E14.5  basal cell shape in later stage skin impacts on suprabasal cell shape. Suspension explants of E15.5-E16 wild-type skin provides evidence for a mechanical coordination in cell height between different epidermal layers. Significantly lower r-squared values forCrash andLp homozygote hair placode/follicle phenotypes  organisation of the developing mammalian skin. Future work will focus on a more detailed analysis of the correlation between basal and suprabasal cell shape in wholemounted skin at earlier embryonic stages which will enable packing and cell shape in both layers to be fully investigated. The authors have taken on board our suggestions and we have no further comments.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Cell and developmental biology of planar polarityWe confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. No further comments.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. A diagram of the epidermal layers would be useful to help orient less expert readers. An alternative to a cartoon might be clear ‘brackets’ beside one of the figure panels indicating the cell layers. The white arrows in panel C partly address this point, but are rather thin and hard to see. Could the authors zoom in on one of the panels B-D and point out the basal cell layer, and also examples of squamous, cuboidal and columnar basal cells?Panel E, F: Total epidermal depth and basal cell height are measured, n = 75 measurements from 3 embryos, measurements taken every 10 basal cells. Given the very low p-values, we wonder if the ‘n’ value used for the statistics was in fact n=75 rather than n=3. In our view, the true n is 3 (number of embryos), not 75. The 25 measurements within an embryo seem to fall into the category of technical replicates, not biological replicates. We would suggest taking an average per embryo and doing stats between embryos (if this wasn’t what was done). Most likely the result will still be significant (or close to significant), but in either case reporting the actual p-values would provide useful information. Regarding the statistical test used, the legend indicates ‘Students t-test’, however multiple comparisons are shown and the Methods mention both t-tests and ANOVA. We think the appropriate test for multiple comparisons is ANOVA with post-hoc correction for multiple samples (e.g. Bonferroni – we think ‘Bonferretti’ in the Methods is a typo?). Could the authors clarify the test used? Panel H legend also states t-test, but as there are 3 genotypes, ANOVA looks more appropriate. Although the authors state in the text that differences are not significant, giving the actual p-value would be more informative . Panel M: We have similar questions regarding technical vs biological replicates and the appropriate n number.It would be nice  to have a sample image to accompany panel I, showing examples of different orientations of cell division.Panel J: Celsr, Fz6 and Crsh mutants had more columnar and cuboidal cells relative to wild-type. The total epidermal depth for each of these classes was then plotted, with the conclusion that having columnar/cuboidal cells rather than flat/squamous cells correlates with overall epidermal thickness. As there will be a range of cell heights within each class, we wonder whether it might be also useful to show scatter plots of individual basal cell height versus total epidermal thickness? This might firm up the connection between basal cell height and epidermal thickness. We were again uncertain about the stats. Is the p-value given the overall ANOVA p-value for comparisons of epidermal depth for all classes, or for comparison of epidermal depth in columnar vs squamous (as the bars above the graphs might imply)? Clarifying this in the figure legend would be good. If the former, why not also show the pair-wise values on the graph? There is also the question of the correct n number for the ANOVA. Averaging within animals and then comparing between animals would again seem most appropriate? However, there may not be enough measurements in each animal for some classes  to do this. This is why scatter plots of individual cell height vs epidermal depth might be more valid, using either pooled data or plotting each animal individually, and looking at the r-squared values for these plots to show a correlation.The authors say the bias towards taller basal cells in fz6-/-, Celsr1-/- and Crsh/Crsh correlated with suprabasal cells which were more rounded in shape. While not essential, can this be quantitated in some way?This short manuscript follows up on previous observations of epidermal thickening in Vangl2 mutant mice. The authors report a similar thickening of the epidermis in Celsr1 and Fz6 mutant animals, and describe cell shape changes in both the basal cell layer and the suprabasal cell layer.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Cell and developmental biology of planar polarityWe confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above. The manuscript by Hobbs and Formstone, “Planar cell polarity protein-dependent basal cell height in the later stage of embryonic mouse epidermis impacts on the shape of overlying suprabasal cells” reports a brief and targeted analyses of epidermal dynamics in the planar cell polarity (PCP) constitutive mutants for Fzd6 and Celsr1. The authors focus mainly on embryonic day (E) 17.5 of skin development and the data correlate epidermal thickness with basal cell height as well as suprabasal cell shape.et al, 2019 on Vangl2 mutants to other PCP mutants, namely Celsr1 and Fzd6. The authors conclude that “basal cell height…impacts on the shape of overlying suprabasal cells”; however, a causal relationship is difficult to establish from correlation in fixed samples and at a single timepoint during development. The authors acknowledge the correlation in the abstract and main text but the title asserts a causal link with the word “impacts on”. A more objective conclusion from the data maybe that the thickness of the epidermis correlates with the packing of the cells and their shapes in both the basal and suprabasal layers, which also applies to the data on the ex-vivo skin cultures in K-M, and perhaps the basal cell packing reported in Box et al., 2019. In my opinion, the outstanding questions are: why is the cellular packing different in PCP mutants? If it does not correlate with proliferation rates as shown in Fig. 1H , then what about cellular densities and cell sizes in both the basal and suprabasal layers from E13.5 – E17.5? A more thorough analysis of PCP mutants is required to address these questions. The data in the manuscript extend previous findings by Box The authors raise an interesting point on a potential non-cell autonomous role for PCP proteins that are expressed in the basal layer and affect the suprabasal layers, which is an area for further investigation into the relationships between the different layers of the epidermis.Fig. 1A: The examples of squamous suprabasal cells shown by the black arrows coincide with the troughs of epidermal ridges, which are hallmarks of the morphogenetic processes that occur in the skin after E15.5. The suprabasal cell shape was not quantified to support the author’s conclusion regarding the correlation with the basal cell height.Fig. 1D: The orientation of the section shown appears slanted and may impact on the thickness of the epidermis and cell shapes.et al. 2021 is not correctly cited in reference to the roles of asymmetric cell division in the later stages of epidermal development. The author’s conclusion in this article is more focused on the roles of delamination than asymmetric cell division per se.Damenet al. 2019 analyzing Fzd6 mutants, when the authors likely mean Vangl2 mutants instead.Perhaps a typo in reference to Box Below are more specific comments:Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.


LOX, OPR3) and JA signalling genes (JAZ and JAR) were found in higher transcription levels in plants treated with HA. In addition, ethylene (ETR4) and auxin (ARF6) signalling components were positively modulated by HA, revealing a hormonal cross-talk. Our results prove that the plant defence system linked to JA can be emulated by HA application without growth inhibition.Intensive agriculture maintains high crop yields through chemical inputs, which are well known for their adverse effects on environmental quality and human health. Innovative technologies are required to reduce the risk generated by the extensive and harmful use of pesticides. The plant biostimulants made from humic substances isolated from recyclable biomass offer an alternative approach to address the need for replacing conventional agrochemicals without compromising the crop yield. The stimulatory effects of humic substances are commonly associated with plant hormones, particularly auxins. However, jasmonic acid (JA) is crucial metabolite in mediating the defence responses and governing plant growth and development. This work aimed to evaluate the changes in the biosynthesis and signalling pathway of JA in tomato seedlings treated with humic acids (HA) isolated from vermicompost. We use the tomato model system cultivar Micro-Tom (MT) harbouring a reporter gene fused to a synthetic promoter that responds to jasmonic acid (JERE::GUS). The transcript levels of genes involved in JA generation and activity were also determined using qRT-PCR. The application of HA promoted plant growth and altered the JA status, as revealed by both GUS and qRT-PCR assays. Both JA enzymatic synthesis ( Conventional agricultural strategies are characterized by fast growing production trends in a short-term period, and weak flexibility of cropping practices limited to the unplanned use of available resources and intensive distribution of synthetic agrochemicals. However, the maintenance of this productive model is currently hampered by both negative externalities related to the impact on environmental quality and human health, to the progressive degradation of soil fertility, and to the long-term loss of crop productivity . The lacAmong the different products, soluble humic extracts are one of the main PB categories applied either as specific application, as mixed formulation or as bio-composite carriers . CurrentThe valorisation of recycled agro-industrial by-products represent a reliable alternative source of bioactive molecules. Organic solid wastes can be converted in to soil amendments, biofertilizers and biostimulants, creating a win–win approach toward more sustainable, profitable and safe agriculture . VermicoThe stimulatory action of HS is generally attributed to the presence of hormone-like components acting preferentially by the emulation of auxin pathways, due to their remarkable effect on the architecture of radicular systems . HoweverArabidopsis /Σ[(45–60)/2 + (60–110) + (160–190)].the alkyl ratio determines the relative contribution of apolar versus polar alyl molecules.(2) A/OA = (0–45)/(60–110).The aromaticity index is the total area assigned to aromatic compounds.(3) Ar = Σ[110–160).The lignin ratio relates the area of methoxyl-C+N-alkyl groups to that of O-aryl-C.(4) LigR = (45–60)/(145–160).S. lycopersicum L.) cv. Micro-Tom (MT) harbouring the JA response synthetic element (AGACCGCC) fused to the gusA gene was used [Tomato , and the RNA was extracted using the mini-plant RNeasy Qiagen ® kit . Reverse transcription (RT) followed by polymerase chain reaction (PCR) 1 μg of total RNA was used to produce cDNAs. The synthesis was performed using the high-capacity cDNA reverse transcription kit Applied Biosystems, USA). A PCR with a gradient temperature  was performed to confirm the specificity of the primers and the actual melting temperature. Electrophoresis in 2.0% agarose gel with TAE buffer was also performed to confirm PCR products with the specific primers. Primers for the genes SlPDLa3 ; SlLOX2S (Lipoxygenase 2); SlOPR3 (12-oxiphytodienoate reductase 3); SlAOS2 (Allene oxide synthase 2); SlMYC2 (Transcription factor); SlARF6a (Auxin response factors); SlARF8a (Auxin response factors); SlJAR1 (Jasmonoyl-isoleucine synthetase); SlJAZ (Jasmonate ZIM Domain-containing protein); SlETR4 (Etileno response sensor); SlCEVI57 (Proteinase inhibitor II); SlACT4  were designed with the Primer3 program and their characteristics were evaluated in the Oligothech program, and after a rigorous analysis, they were synthesised by IDT technology  was dissected along the longitudinal axis into 15 × 3 mm strips covering the entire leaf blade (avoiding the primary veins) and fixed on microscope slides with transparent enamel. 10 individuals per treatment were sampled, and four different ranges were analysed per plant (n = 40) for each side . Photographs were taken using a Leica S8AP0 loupe  set to 80×, coupled to a Leica DFC295 camera . Trichome counts were performed on the images, and side views of the images were used to allow for the correct classification of trichome types. Trichome densities were then calculated as trichome counts per unit leaf area.We proved using a gene-reporter staining assay that HA from vermicompost modified the JA content in tomato plants. The qRT-PCR analysis showed that both JA synthesis and signalling were positively affected by the application of HA solutions resulting in larger transcription than in control plants. However, phenotypes traits, such as root growth and trichome density, in leaves were divergent from JA activity, suggesting a complex integration with other plant regulators also induced by HA, as observed in this study with auxin response factor (ARF6). These findings are ground-breaking contributions to enforce the knowledge and use of HA-based biostimulant from local available recycled biomasses as accessible technologies to strengthen the plant adaptation and responses to biotic and abiotic stress conditions. The comprehensive understanding of structural activity relationship is an unavoidable requirement to conceive the tailored exploitation of humic materials. The update of scientific insights on the metabolic regulation of soil–plant resilience mechanisms represents a valuable repository support for the reliable adoption of sustainable, affordable and safety farming practices.


The impact of high-intensity interval training (HIIT) on the central nervous system (CNS) in autoimmune neuroinflammation is not known. The aim of this study was to determine the direct effects of HIIT on the CNS and development of experimental autoimmune encephalomyelitis (EAE). Healthy mice were subjected to HIIT by treadmill running and the proteolipid protein (PLP) transfer EAE model was utilized. To examine neuroprotection, PLP-reactive lymph-node cells (LNCs) were transferred to HIIT and sedentary (SED) mice. To examine immunomodulation, PLP-reactive LNCs from HIIT and SED donor mice were transferred to naïve recipients and analyzed in vitro. HIIT in recipient mice did not affect the development of EAE following exposure to PLP-reactive LNCs. HIIT mice exhibited enhanced migration of systemic autoimmune cells into the CNS and increased demyelination. In contrast, EAE severity in recipient mice injected with PLP-reactive LNCs from HIIT donor mice was significantly diminished. The latter positive effect was associated with decreased migration of autoimmune cells into the CNS and inhibition of very late antigen (VLA)-4 expression in LNCs. Thus, the beneficial effect of HIIT on EAE development is attributed solely to systemic immunomodulatory effects, likely because of systemic inhibition of autoreactive cell migration and reduced VLA-4 integrin expression. Various mechanisms have been suggested to underlie the beneficial effects of ET in experimental autoimmune encephalomyelitis (EAE), an animal model of MS3. Accordingly, in a series of studies, we utilized the passive transfer EAE model, that enabled us to differentiate between systemic immunomodulatory and direct neuroprotective effects of ET, to examine the impact of various training paradigms on the progression of EAE8.Beneficial outcomes of exercise training (ET) in multiple sclerosis (MS) patients are well documented8. We demonstrated that both direct effects of HICT on the CNS and indirect effects via systemic immunomodulation mitigate disease progression8. Specifically, we found that HICT inhibits several key molecules involved in encephalitogenic lymph node cell (LNC) migration into the CNS7, and modulates the properties of the blood–brain-barrier (BBB) to maintain the integrity of the BBB following EAE induction7. Moreover, the results indicated that ~ 70% of the beneficial effects of HICT on disease development derive from systemic immunomodulation and ~ 30% from direct protection of the CNS7.We found that high intensity continuous training (HICT) was superior to moderate intensity continuous training in yielding favorable effects on disease progression9. Its popularity derives from studies that show similar or superior physiological and metabolic adaptations achieved by HIIT in a shorter time duration than HICT12. The beneficial effects of HIIT have also been documented in autoimmune diseases such as rheumatoid arthritis, as well as diabetes15. Several studies also indicate that HIIT improves fitness in people with MS16. However, the direct effects of HIIT on the CNS in autoimmune neuroinflammation have not been studied. Considering that HICT results in direct neuroprotective effects and attenuates development of EAE7 and that HIIT enhances exercise performance more than HICT6, we suspected that HIIT can also impact neuroprotection, possibly even more positively than HICT.HIIT has become a popular and efficient mode of ETTherefore, in this study we investigated the direct effects of HIIT on the CNS, as well as the development of EAE. Additionally, we examined potential mechanisms whereby HIIT exerts its effects on autoimmune neuroinflammation.Female SJL/JCrHsd mice (6–7 weeks of age) were purchased from Envigo Inc, Israel. Animal experimentation was approved by the Institutional Animal Care and Use Committee. The studies were conducted in accordance with the United States Public Health Service's Policy on Humane Care and Use of Laboratory Animals.139–151 transfer EAE model and the HIIT treadmill running program were utilized as previously described6.The PLPThe direct neuroprotective effects of HIIT were assessed in HIIT and SED recipient mice following autoimmune LNC transfer Fig. A. HealthThe modulatory effects of HIIT on systemic autoimmunity and on the migratory potential and encephalitogenicity of proteolipid protein (PLP)- reactive LNCs were examined in vivo following HIIT and sedentary (SED) donor mice- derived autoimmune cell transfer in recipient groups and in vitro Fig. B. Health6.Healthy mice underwent 6-weeks of HIIT treadmill running, including pre- and post- training performance tests on a 5-lane treadmill designed for mice  as previously described139–151 transfer EAE model was utilized as previously described8. EAE was induced either in recipient HIIT  and SED  mice by transfer of encephalitogenic cells obtained from PLP-immunized donor mice , SED-EAE (n = 12), HIIT-transfer-EAE (n = 10) and SED-transfer-EAE (n = 10) recipient mice developed EAE and were scored daily for neurological symptoms up to 30 days after EAE induction as follows: 0—asymptomatic; 1—partial loss of tail tonicity; 2—atonic tail; 3 -hind leg weakness, difficulty to roll over, or both; 4—hind leg paralysis; 5—four leg paralysis; 6—death due to EAE. To assess disease severity, day of onset, maximal clinical score, and cumulative score (“area under curve”) were calculated for each mouse at the end of the follow-up. The cumulative score was calculated in individual mice by summation of the daily clinical scores over the entire follow-up period.17. Mice were anesthetized with a lethal dose of sodium pentobarbital and subjected to perfusion via the ascending aorta with ice cold phosphate-buffered saline followed by 4% paraformaldehyde. The tissues were dissected and post-fixated in 4% paraformaldehyde for 24 h. Serial 6 μm paraffin embedded transverse sections were obtained from mid-cervical, mid-thoracic and mid-lumbar levels of the spinal cord. Sections underwent routine histology staining. Myelin maintenance was assessed using Luxol fast blue  counterstained with nuclear fast red . Axon maintenance was evaluated using Bielschowsky silver impregnation using silver nitrate (Chem-lab CL00.2614.0250) and ammonia solution . For each stain, the whole white matter of three sections per mouse was quantified in a blind fashion, one section per spinal cord level. Demyelination was assessed by calculating the area of LFB loss. For chronic axonal damage, the area of reduced axonal density in Bielschowsky silver staining was assessed and scored as follows: 0 = normal/even silver stain throughout the white matter; 1 = small sporadic areas in the white matter that lack silver stain; 2 = small but frequent areas in the white matter that lack silver stain; 3 = extensive loss of silver stain throughout the white matter. All pathology measurements were performed by using the Image J software analysis .At the end of the clinical follow-up, a group of SED-EAE and HIIT-EAE mice . At the day of clinical onset, recipient mice were sacrificed by intracardial perfusion with 4% paraformaldehyde. Thereafter, spinal cords were removed and serial 10 μm longitudinal frozen sections were prepared, and counterstained with DAPI. For each mouse, a total of 100 microscopic images (X40 magnification) from six sections at 60 μm apart were captured and quantified in a blind fashion. PKH+ cells were counted in the spinal cord parenchyma and reported as total average number ± SEM per square millimeter. Measurements were performed by using the Image J software analysis .Lymph nodes were excised from naïve Fig. A or HIITThe BBB properties and permeability were evaluated in spinal cords of HIIT and SED recipient mice after transfer of encephalitogenic PLP-reactive cells from donor mice Fig. A by: (1)7  extravasation method as previously described7 Figs. A–D. At 57  from freshly isolated LNCs that were excised from HIIT and SED mice 10 days after PLP immunization  was used. If a significant F ratio was found, then the Newman-Keuls multiple comparison test was used to identify the location of significance. Data were analyzed in GraphPad Prism software v.5. Differences were considered statistically significant at p < 0.05. All data are presented as mean ± standard error of mean (SEM).Normality of distribution of variables was tested by the Shapiro–Wilk test followed by the appropriate statistical test for comparison. Two experimental groups were compared using the unpaired Student’s Animal experimentation was approved by the institutional ethics committee, approval number IL-185-11-19. The study is reported in accordance with ARRIVE guidelines.First, we evaluated if HIIT affected the clinical severity and pathological process of EAE Fig. . TransfeSince HIIT did not affect the clinical severity in EAE recipient mice, we studied the direct effects of HIIT on the BBB. To that end, encephalitogenic LNCs were transferred to HIIT and SED recipient mice Fig. A. Since To examine the direct effect of HIIT on the profile of endothelial cells of the blood vessels, CD31+ endothelial cells in spinal cords of naïve healthy mice, SED- and HIIT- pre EAE mice were double stained with VCAM-1 34, as well as higher catecholamine levels35. The extent to which excessive levels of such factors (or other exerkines) could affect on the CNS is not clear.The finding that the HIIT-mediated improvement in BBB integrity was associated with increased migration of LNCs into the CNS and enhanced demyelination of spinal cord sections was surprising. Even more surprising was that the latter findings were not associated with an even more negative clinical progression versus SED see Fig. . When co40. Accordingly, there is evidence that mitochondria in neurons produce ROS, and that mitochondrial ROS overproduction and changes in mitochondrial redox homeostasis have been shown to be involved in a number of neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease and Amyotrophic Lateral Sclerosis41. Previously we showed that HICT had a large positive effect on the development of EAE both via direct neuroprotection and systemic immunomodulation8. These positive effects were associated with a decreased production of ROS by isolated neuroglia following exposure to PLP8. Similarly, it was demonstrated that administration of a mitochondria-targeted antioxidant (Mito-Q) to EAE-SED mice diminished the clinical progression of the disease42. Thus, the negative effects of HIIT on the CNS and lack of positive effect on disease progression may be related to excessive ROS production. Therefore, the links between ROS, HIIT and CNS inflammation warrant future investigation.In this context, the ROS component is of interest, as it has been shown to contribute to microglial neurotoxicity, demyelination, and neuronal deathThe next question is why HIIT did not result in even a greater deterioration in terms of disease progression versus SED. To address this question, we examined the effect of HIIT on systemic immunomodulation. We observed a number of changes that could counter the negative effects observed in the CNS and thereby mitigate the clinical deterioration. The observation that HIIT positively affected the systemic immune system in donor mice to attenuate disease progression in recipient mice is consistent with this explanation.43. Accordingly, we previously showed that the HICT paradigm inhibits several key molecules involved in encephalitogenic LNC migration7. We therefore hypothesized that HIIT may also interfere with induction of integrins on LNCs in the peripheral lymphoid system upon immunization with the PLP peptide. However, while HICT induced a reduction in both VLA-4 and LFA-1 expression on PLP-reactive LNCs in vitro7, the HIIT paradigm induced an inhibition only on the VLA-4 integrin expression in LNCs and T cells.Specifically, we found that transfer of PLP- reactive encephalitogenic LNCs obtained from HIIT mice resulted in reduction of PKH26+ autoimmune cell infiltration into the CNS recipient mice. Blocking the integrin-adhesion molecule interactions is an effective strategy to prevent CNS inflammation in MS and EAE44. The VLA-4 integrin was shown to mediate initial recruitment of encephalitogenic T cells, as well as the secondary infiltration of inflammatory cells, thereby contributing to and sustaining CNS inflammation45. Thus, our findings suggest that the inhibitory effect of HIIT on the expression of VLA-4 in PLP-reactive encephalitogenic LNCs contributes to the decrease in inflammatory infiltration into the CNS and attenuation of the clinical severity of EAE. Interestingly, VLA-4 expression in F4/80+ macrophages was not affected by HIIT, suggesting that the inhibitory effect of HIIT may be targeted to the T cell populations.Extensive research has been conducted on the role of VLA-4 integrin in leukocyte recruitment to the inflamed CNS during EAEAlthough the number of injected encephalitogenic LNCs from donors to recipient groups was equal, we cannot rule out that the reduction in cell infiltration in the CNS occurred due to differences in post-injection proliferation between groups. Similarly, we cannot exclude other post-injection phenomena, such as differences in apoptosis to account for differences in T cell infiltration between groups. Other possibilities worth investigating in the future are changes in the differentiation and profile of the transferred cells in the CNS between the groups.6. Again, this may be related to excessive ROS production during HIIT. The partial effects of HIIT on integrin expression may also, partially, explain the milder inhibitory effects of HIIT on EAE clinical course compared to the HICT program. Moreover, we demonstrated that while HICT causes a marked inhibition of PLP-induced T-cell proliferation without affecting the T-cell profile, HIIT does not alter T-cell proliferation, but rather inhibits polarization of T cells into T-helper 1 and T-helper 17 autoreactive populations6. We concluded that HICT and HIIT attenuate systemic autoimmunity and T cell encephalitogenicity by distinct immunomodulatory mechanisms. Thus, the current work corroborates the beneficial systemic immunomodulatory effects of HIIT in attenuation of EAE and further highlights the variations between HICT and HIIT paradigms.Of particular interest in this respect is the observation that although HIIT in donor mice impacts positively on disease progression in recipient mice, the effect is not as large as seen with HICT7, the current study demonstrates that the beneficial effect of HIIT stems solely from systemic immunomodulation. Thus, the clinical implication of the findings in the present study is that HICT is superior to HIIT in terms of impacting positively on disease progression, possibly because of differences in ROS production or other metabolic factors. This highlights the need for a systematic comparisons of various training paradigms for designing effective clinical treatments in MS patients and other patients with autoimmune diseases.In conclusion, HIIT does not induce direct neuroprotective effects in an experimental model of autoimmune neuroinflammation but induces systemic immunomodulation to attenuate EAE Fig. . Our res


Preterm infants risk deficits of long-chain polyunsaturated fatty acids (LCPUFAs) that may contribute to morbidities and hamper neurodevelopment. We aimed to determine longitudinal serum fatty acid profiles in preterm infants and how the profiles are affected by enteral and parenteral lipid sources.−1) units.Cohort study analyzing fatty acid data from the Mega Donna Mega study, a randomized control trial with infants born <28 weeks of gestation (n = 204) receiving standard nutrition or daily enteral lipid supplementation with arachidonic acid (AA):docosahexaenoic acid (DHA) (100:50 mg/kg/day). Infants received an intravenous lipid emulsion containing olive oil:soybean oil (4:1). Infants were followed from birth to postmenstrual age 40 weeks. Levels of 31 different fatty acids from serum phospholipids were determined by GCeMS and reported in relative (mol%) and absolute concentration . For AA, absolute postnatal levels were lower compared to cord blood from week 4 throughout the study period (p < 0.05).Higher parenteral lipid administration resulted in lower serum proportion of AA and DHA relative to other fatty acids during the first 13 weeks of life (p < 0.001 for the 25th vs the 75th percentile). The enteral AA:DHA supplement increased the target fatty acids with little impact on other fatty acids. The absolute concentration of total phospholipid fatty acids changed rapidly in the first weeks of life, peaking at day 3, median (Q1-Q3) 4452 (3645–5466) μmol lin utero. Further research is needed to establish optimal postnatal fatty acid supplementation and profiles in extremely preterm infants to promote development and long-term health.Our data show that parenteral lipids aggravate the postnatal loss of LCPUFAs seen in preterm infants and that serum AA available for accretion is below that  PreteFortification of intravenous lipid emulsions and enteral supplementation have been assessed as ways to improve LCPUFA status in preterm infants –19. SuppThe Mega Donna Mega (MDM) trial recently investigated the effect of enteral supplementation with AA:DHA (2:1 ratio) on the outcome of severe retinopathy of prematurity (ROP) in extremely preterm infants. Daily supplementation from birth to term equivalent age significantly reduced the risk for severe ROP by 50% and increased the molar fractions of AA and DHA in serum phospholipids .In this secondary analysis of the MDM trial, we aimed to investigate infants’ serum complete fatty acid profiles over the first months of life, expressed in relative and absolute units, and how the profiles were modulated by the administration of parenteral lipids and enteral AA:DHA supplementation.2.2.1.ClinicalTrials.gov (Identifier: NCT03201588).The current study was performed within the randomized, open-labeled Mega Donna Mega trial . Postnat2.2.™ , a triglyceride oil made from extracts of the filamentous fungus Mortierella alpina, and the dinoflagellate Crypthecodinium cohnii, containing AA and DHA in a two-to-one ratio. Formulaid™ was administrated enterally starting within 72 h after birth and lasted until 40 weeks postmenstrual age (PMA). The dose was 0.39 mL kg−1 daily, equal to 100 mg AA and 50 mg DHA per kg body weight and day. Infants assigned to control received standard care.The Regional Ethical Review Board at the University of Gothenburg approved the study . The study was performed following theethical standards of the Helsinki Declaration of 1975, as revised in 1983. Informed signed consent for all participants was obtained from parents or legal guardians. Infants were recruited between December 2016 and August 2019 at three centers in Sweden: Queen Silvia Children’s Hospital in Göteborg, Skåne University Hospital in Lund, and Karolinska University Hospital in Stockholm . The ran2.3.™ Software . Standardized birth weight was calculated based on growth charts by Fenton and Kim  μmol l−1 at day 3, p < 0.0001). The peak in serum phospholipid fatty acids around day 3 coincided with the maximum administration of parenteral lipids  μmol l−1 (p = 0.022). No specific trends could be observed for absolute levels of DHA  showed significantly lower relative serum levels of AA and DHA compared to infants with the lowest intake (≤25th percentile)  and B. Bs of DHA .3.4.−1]). In both treated and control groups, oleic (18:1 n–9) and linoleic acid were the fatty acids that showed the greatest change over time, both in relative and absolute units. The change in oleic acid was transient over the first week, whereas linoleic acid levels increased rapidly after birth and then were maintained at high levels throughout the study period. As no placebo was given to the control group, the additional fat provided to the intervention group through the supplement could have affected the total lipid intake. However, there was no difference between groups in lipid intake, nor in parenteral fluid or human milk intake over the first four weeks of life  to reduce the dimensionality of the data and compare individuals’ complete fatty acid profiles . This anCord blood and day 1 samples clustered together while samples collected from postnatal day 3 to postnatal week 4 displayed higher heterogeneity; the latter was especially true for when the fatty acid content was expressed as absolute serum concentration. Samples from week 6 to week 12 formed a relatively cohesive cluster in both data sets (relative and absolute).4.This study provides a comprehensive overview of the extremely preterm infants’ fatty acid status during early postnatal life and how fatty acid levels are affected by external lipid sources. The largest changes in serum fatty acid levels occurred in the first few weeks after birth. This period was also defined by a sizeable inter-individual variability, which could partly be explained by the intake of parenteral lipids.4.1.−l) fatty acid concentrations were related to a significant increase in total serum phospholipids starting soon after birth. The magnitude of this phospholipid expansion appears to be strongly related to parenteral lipid administration: at one week postnatal age, infants who received the most parenteral lipids (the ≥75th percentile category) showed around 40% higher total serum phospholipid fatty acids compared to infants who received the lowest amount of parenteral lipids (the ≤25th percentile category). Nevertheless, infants who primarily received enteral nutrition also showed an increase in total serum phospholipid fatty acids in the first week of life. Thus, the increase in total serum phospholipids appears to represent a physiological response to extrauterine life and the transition from glucose to fat as the main energy source. Previous studies have shown a similar increase in the concentration of serum/plasma phospholipids after birth in preterm infants [The strikingly different patterns observed for relative (mol%) and absolute  DHA, but not AA, decreased more in extremely low birth weight infants with prolonged exposure (>28 days) to a lipid emulsion based on soybean oil than in infants with shorter exposure (≤28 days) . However4.2.in utero [In preterm infant blood, AA and DHA are mainly transported as esters of phospholipids in lipoproteins that supply tissues with the material needed for metabolism and cell expansion . We founin utero . We specAs we reported previously , the AA:Collins et al. investigated dose–response of emulsified DHA via tube feeding on circulating RBC levels in infants born less than 30 weeks’ GA . Adminis4.3.We grouped fatty acids according to postnatal level profiles and found several distinct clusters. Some fatty acids clustered according to class; for example, the proportion of the n–3 fatty acids, except DHA, were found in one cluster. The proportion of DHA, on the other hand, shared profile with the long-chain saturates stearic (18:0), behenic (22:0), and lignoceric acid (24:0). The n–6 LCPUFAs AA, adrenic (22:4 n–6), and docosapentaenoic acid (22:5 n–6) clustered together in data based on both relative and absolute concentration, albeit with very different profiles. This analysis highlights the complexity of lipid metabolism during early postnatal life and calls for further studies to determine the origin and fate of circulatory fatty acids.4.4.There are limitations to our study. First, infants in the control group did not receive a placebo treatment. However, there was no difference in total lipid intake between the active and the control group. Second, when investigating the impact of parenteral lipids on infant LCPUFA levels we did not account for other potential confounders such as blood transfusions, LCPUFAs in human milk, LCPUFAs in the human milk fortifiers, and lipids present in other fortifiers, which may impact infant plasma levels ,38. Thir4.5.in utero. The long-term consequences of this LCPUFA deprivation during a sensitive time of development are not yet fully understood. No current nutritional management program restores preterm infant fatty acid profiles to match the intrauterine situation, and whether this is achievable or even desirable, has not been established. This and other studies have provided in-depth knowledge on circulating fatty acids in the preterm infant in the first months of life. However, there is limited consensus on the ideal fatty acid supplementation and profile to aim for when feeding the preterm infant to promote optimal growth and long-term health, warranting further studies.Following preterm birth, infant LCPUFA levels fall below those of the fetus supplementary material


Our results indicate that a genetic variant of FNDC5 is associated with low brain glucose metabolism in elderly individuals and suggest that FNDC5 may participate in the regulation of brain metabolism in brain regions vulnerable to Alzheimer’s disease pathophysiology. Understanding the associations between genetic variants in metabolism-linked genes and metabolic brain signatures may contribute to elucidating genetic modulators of brain metabolism in humans.Fibronectin type III domain–containing protein 5 (FNDC5) and its derived hormone, irisin, have been associated with metabolic control in humans, with described FNDC5 single nucleotide polymorphisms being linked to obesity and metabolic syndrome. Decreased brain FNDC5/irisin has been reported in subjects with dementia due to Alzheimer’s disease. Since impaired brain glucose metabolism develops in ageing and is prominent in Alzheimer’s disease, here, we examined associations of a single nucleotide polymorphism in the FNDC5 gene (rs1746661) with brain glucose metabolism and amyloid-β deposition in a cohort of 240 cognitively unimpaired and 485 cognitively impaired elderly individuals from the Alzheimer’s Disease Neuroimaging Initiative. In cognitively unimpaired elderly individuals harbouring the FNDC5 rs1746661(T) allele, we observed a regional reduction in low glucose metabolism in memory-linked brain regions and increased brain amyloid-β PET load. No differences in cognition or levels of cerebrospinal fluid amyloid-β et al. reported that cognitively unimpaired elders carrying the FNDC5 rs1746661(T) allele develop low brain glucose metabolism and increased amyloid deposition. These findings indicate that FNDC5 may contribute to regional glucose metabolism in the human brain.Lima-Filho   Investigation of brain glucose metabolism using [18F]fluorodeoxyglucose positron emission tomography (FDG-PET) is a powerful tool to identify and study brain metabolic dysfunction in ageing and age-related diseases.25AD has been linked to defective brain hormonal signalling and energy metabolism.FNDC5 gene may affect brain function and AD biomarkers in the elderly. We thus analysed data from 240 cognitively unimpaired (CU) and 485 cognitively impaired (CI) participants from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) cohort to investigate if SNPs within the FNDC5 gene are associated with changes in FDG-PET, cognition and AD biomarkers. Allele-specific changes associated with the FNDC5 gene may shed light on the roles of FNDC5/irisin in brain physiology and neurodegeneration.Here, we hypothesized that polymorphisms in the adni.loni.usc.edu). The ADNI was launched in 2003 as a public–private partnership led by Principal Investigator Michael W. Weiner, MD. The primary goal of ADNI has been to test whether serial MRI, PET, other biological markers and clinical and neuropsychological assessment can be combined to measure the progression of mild cognitive impairment and early AD. The ADNI studies obtained appropriate approvals from institutional review boards. Access to data was preapproved by the ADNI Review Board.Data used to prepare this article were obtained from the ADNI database (18F]FDG-PET and genotyping data: 240 CU (n = 82 carriers and 158 non-carriers) and 485 CI individuals (n = 167 carriers and 318 non-carriers). Patients in the CI group were clinically diagnosed with either AD-linked dementia (n = 66) or mild cognitive impairment (n = 419). For more detailed demographics, see The current study comprised 725 elderly individuals with available cross-sectional [http://adni.loni.usc.edu/methods). [18F]FDG-PET images were preprocessed to produce an effective point spread function of full-width at half maximum of 8 mm. Subsequently, linear registration and nonlinear normalization to the MNI152 template were performed with the linear and nonlinear transformations derived from the automatic PET to MRI transformation and the individual’s anatomical MRI coregistration. [18F]FDG-PET standardized uptake value ratio (SUVR) maps were generated using pons as the reference region.27 The global average SUVR was obtained from ADNI. Additional details on our processing pipeline can be found elsewhere29 . Aβ load was estimated using [18F]florbetapir, and the SUVR was calculated using the cerebellar grey matter as the reference region. The global amyloid PET average SUVR was obtained from ADNI, and the cut-off for positivity is 1.11 SUVR.PET acquisitions followed the protocols established by ADNI  and total tau (t-Tau) on an automated Elecsys Cobas e601 instrument, as described by ADNI (https://adni.loni.usc.edu/methods/documents/). Precision and accuracy of runs were assessed and were within the stated limits set by the manufacturer.31 The following thresholds were applied to determine amyloid or tau positivity: Aβ42 < 680 pg/ml [defined as amyloid-positive (A+)]32 and t-Tau > 266 pg/ml [defined as tau-positive (T+)].33CSF was collected from donors by lumbar puncture following ADNI protocols and analysed by the electrochemiluminescence immunoassay Elecsys Aβ−6. Subjects would be excluded if they had a call rate of <95% or if genetic relatedness was detected (PI_HAT > 0.5). Redundant SNPs in high linkage disequilibrium were removed based on pairwise correlation (R2 = 0.8). After the initial processing of the genetic data, SNPs from the FNDC5 gene were extracted within a 10 kb window upstream and downstream of the gene. We followed the STREGA guidelines,34 as noted in the accompanying checklist.PLINK (v1.9) was used to preprocess genetic data and SNP selection. Participants were genotyped with the HumanOmni2.5 BeadChip array . Quality control was performed by excluding SNPs with a genotyping efficiency of <95%, a minor allele (MA) frequency of <5%, or a deviation from the Hardy–Weinberg equilibrium of <1 × 10post hoc test, was performed. P-values are reported in each graph.Statistical analysis was performed using GraphPad Prism 8 . Data were checked for normal distribution using the D’Agostino & Pearson Omnibus normality test. Statistical significance of differences between average values of carriers and non-carriers was assessed using a two-way ANOVA test, followed by Tukey’s posttest, unless otherwise stated in legends. In the supplementary figures, where carriers were subdivided between homozygous and heterozygous, a two-tailed one-way ANOVA test, followed by Dunnett’s 18F]FDG-PET imaging analysis, voxel-wise analyses were performed using Rminc, where linear models tested the association between the FNDC5 SNP carriership and [18F]FDG-PET in all participants, adjusting for age, sex and diagnosis. In addition, the analysis was repeated within CU and CI groups, adjusting for covariates. Adjustment for multiple comparisons was done with random field theory,35 and significant t-values are below or equal to −3.1.For FDG-PET signal was observed in CI patients when compared to CU patients, but we found no differences between genotype groups 32 or t-Tau 33 was similar between carriers and non-carriers  on AD biomarkers and FDG-PET in a cohort of human subjects with or without cognitive impairment.Irisin has been shown to mediate brain benefits of physical exercise,37 and brain9 pathways linked to metabolism.39 Our finding that rs1746661(T) carriers exhibit FDG hypometabolism in brain regions important for cognition advocates for the potential roles of this FNDC5 genomic variant in the control of brain metabolism and function.Irisin has been shown to modulate key peripheral6 rs1746661(T) has been associated with increased blood pressure and imbalanced plasma cholesterol and triglyceride levels40 in type 2 diabetes patients. Since FNDC5 is notably expressed in the brain and in peripheral tissues,2 additional investigation should aim to establish whether impaired brain metabolism in SNP carriers originates from brain or peripheral metabolic alterations.The functional relevance of rs1746661 to brain metabolism needs to be further investigated. While previous reports found little evidence of diabetes-linked peripheral metabolic changes associated with rs1746661 in humans,41‐43 Here, the carriership of rs1746661(T) did not result in changes in the total number of FNDC5 transcripts  in brain metabolism and AD pathological features.Accumulating evidence indicates that intronic SNPs play key roles in regulating transcription.nscripts , suggest45 No significant difference in the prevalence of cognitive impairment or AD was detected between carriers and non-carriers of rs1746661 MA. However, brain Aβ deposition was increased in carriers, even when compared with CI non-carriers, and a trend towards CSF tau build-up was observed in subjects carrying two copies of rs1746661(T), though a larger cohort is necessary to confirm this observation.We focused on potential associations of the rs1746661 SNP with cognitive function, Aβ or tau pathology, as they are hallmarks of AD pathology and disease progression.FNDC5 (rs1746661) with known genetic risk factors for sporadic AD, such as ApoE-ε4 and triggering receptor expressed on myeloid cells 2, to predispose the brain to neurodegenerative changes should also be investigated.Our results also show a more prominent regional low glucose metabolism in CU subjects, while results from the CI cohort denote more subtle changes, which may be explained by the fact that CI subjects already present a larger, global low glucose metabolism in the brain. It is possible that the extent of brain hypometabolism found in SNP carriers may contribute to preclinical memory defects at very early phases of disease progression. This possibility could be explored in longitudinal studies with SNP carriers in the future. The interaction of FNDC5 SNP rs1746661(T), which prevented further stratification in our analysis. Moreover, the use of cross-sectional data limits our exploratory potential to predict disease progression in this cohort, and we feel that longitudinal studies of disease progression in carriers versus non-carriers are warranted.Limitations of the current study include the reduced size of the group of homozygous We acknowledge that altered blood perfusion may impact FDG-PET signals. This issue has not been considered in our work because the ADNI study has strict inclusion/exclusion criteria in which the subject must not have other neurological conditions and no signs of previous cerebrovascular disorders, pacemakers, aneurysm clips or artificial heart valves. The subjects must also have good general health and show no local lesions or infarctions in a baseline MRI scan. Therefore, subjects in this cohort are unlikely to present conditions that can impact their cerebral blood perfusion.FNDC5 SNP with low brain glucose metabolism and AD biomarkers. The finding that an FNDC5 SNP is associated with regional low brain glucose metabolism in humans encourages future studies in larger cohorts to replicate and extend these observations. Further, it may also stimulate investigation of the genomic control and physiological roles of FNDC5/irisin in the brain and how they may interact with the pathophysiology of AD.To the best of our knowledge, this is the first report to associate an fcad216_Supplementary_DataClick here for additional data file.