Uric acid is a metabolite released by cells to detoxify themselves from nitrogen. Unlike other mammalians, hominids, and also birds, lack uricase, an enzyme that catalyzes the transformation of uric acid to allantoin. Therefore, high levels of uric acid accumulate in the plasma of hominids. Under certain conditions, i.e., the consumption of a fructose-rich diet, which is also often associated with diabetes, the concentrations of uric acid rise. Under such conditions, uric acid may in part exert effects on different cells. Using isolated and cultivated totally differentiated rat cardiomyocytes, we show that uric acid at plasma-relevant concentrations that are similar to those found in patients decreases the calcium-affinity of troponin in cardiomyocytes and thereby reduces cell function. Therefore, the accumulation of uric acid stresses cardiomyocytes, a mechanism that may contribute to heart failure under such conditions.Hyperuricemia is a risk factor for heart disease. Cardiomyocytes produce uric acid via xanthine oxidase. The enzymatic reaction leads to oxidative stress in uric-acid-producing cells. However, extracellular uric acid is the largest scavenger of reactive oxygen species, specifically to nitrosative stress, which can directly affect cells. Here, the effect of plasma-relevant concentrations of uric acid on adult rat ventricular cardiomyocytes is analyzed. A concentration- and time-dependent reduction of load-free cell shortening is found. This is accompanied by an increased protein expression of ornithine decarboxylase, the rate-limiting enzyme of the polyamine metabolism, suggesting a higher arginine turnover. Subsequently, the effect of uric acid was attenuated if other arginine consumers, such as nitric oxide synthase, are blocked or arginine is added. In the presence of uric acid, calcium transients are increased in cardiomyocytes irrespective of the reduced cell shortening, indicating calcium desensitization. Supplementation of extracellular calcium or stimulation of intracellular calcium release by β-adrenergic receptor stimulation attenuates the uric-acid-dependent effect. The effects of uric acid are attenuated in the presence of a protein kinase C inhibitor, suggesting that the PKC-dependent phosphorylation of troponin triggers the desensitizing effect. In conclusion, high levels of uric acid stress cardiomyocytes by accelerating the arginine metabolism via the upregulation of ornithine decarboxylase. Uric acid (UA) is the end-product of the purine nucleotide catabolism and is enzymatically catalyzed by xanthine oxidase in a two-step reaction that converts hypoxanthine into xanthine and xanthine into UA. In both steps, superoxide is a byproduct. Superoxide can generate oxidative stress in cells specifically if the activity of xanthine oxidase is high or the activity of reactive-oxygen-species (ROS)-detoxifying enzymes is low. In most mammalians, UA can be transformed to allantoin by the enzyme uricase. However, hominids lack uricase and instead have a UA transport molecule in the kidney URAT1) that allows the resorption of UA. Therefore, the UA plasma concentration in hominids is high compared to most mammalians. UA itself has antioxidative properties and it has been suggested that high plasma concentrations of UA in hominids contribute to its longevity compared to small rodents  that all,3,4,5. IThe activity of xanthine oxidase is elevated in cardiomyocytes isolated from dogs with pacing-induced heart failure . This isIn this context, it is interesting that UA itself can affect cell function directly. UA can enhance PKC activity in endothelial cells , activatTo challenge this hypothesis, we expose isolated and cultured adult rat ventricular cardiomyocytes to plasma-relevant concentrations of UA. We use the load-free cell shortening of myocytes as an established surrogate parameter of the contractile function. The data of our study show that UA can increase the arginine metabolism in cardiomyocytes and exert effects on cardiomyocytes that are characteristic for arginine depletion. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health . The permission to scarify rats is registered at Justus-Liebig-University.The calcium-sensing receptor protein was detected by the anticalcium sensing receptor antibody produced in rabbit . Ornithine decarboxylase was detected by the anti-ODC antibody produced in goat . The calcium-sensing receptor and ornithine decarboxylase levels were normalized to actin ). Secondary antibodies were directed against rabbit IgG and coupled to alkaline phosphatase . SB202190, chelerythrine chloride, and SP600125 were purchased from Calbiochem . Uric acid, L-nitro arginine methyl ester (L-NAME), Nor-NOHA, and Difluoromethyl ornithine (DFMO) were purchased from Sigma-Aldrich Chemie, Taufkirchen, Germany. w/v) crude collagenase and 25 µM CaCl2 at 5 mL/min. After 25 min, the ventricular tissue was minced and incubated for 20 min in a recirculating medium. The resulting cell suspension was filtered through a 200 µm nylon mesh. The filtered material was washed twice by centrifugation  and resuspended in the collagenase-free perfusate, in which the calcium concentration was step-wise increased to 0.5 mmol/L. After a further centrifugation, the cell pellet was resuspended in serum-free culture medium  and cells were plated on culture dishes . Medium change was performed after 2 h. Cells were analyzed thereafter or after 24 h incubation, as indicated in the Results section. The following inhibitors were used within this study: inhibition of p38 MAPK by SB202190 (10 µmol/L), inhibition of c-jun kinase by SP600125 (10 µmol/L), and inhibition of protein kinase C by chelerythrine chloride . Concentrations were used according to references [Adult rat ventricular cardiomyocytes were isolated from male 4-month-old Wistar rats, as described previously ,18. Brieferences . Arginasferences .Cells were allowed to contract at room temperature and analyzed using a cell-edge-detection system, as previously described . Cells wCalcium transients were measured with the fluorescent dye fura-2 acetoxymethyl ester, as described before . Adult rIsolated cardiomyocytes were incubated with lysis buffer as described before . Samplesp values are given in the legends of the figures. Data are expressed as indicated in the legends. One-way ANOVA and the Student–Newman–Keuls test for post hoc analysis were used to analyze the experiments. Data are presented as box and whisker blots in which the boxes represent the 25, 50, and 75% percentiles, and the whiskers represent the total range of all data. Exact As mentioned in the Introduction, UA may directly affect cell function via the activation of protein kinases in a rapid way or, alternatively, via transcriptional effects that would require long-term regulation. Therefore, we analyzed the acute and long-term effects of UA  on the cell shortening of cardiomyocytes. The long-term incubation of cardiomyocytes with UA decreased cell shortening in a concentration-dependent way, whereas acute exposure had minimal effects A–C. FiguAs UA effects on the load-free cell shortening of cardiomyocytes required long-term exposure, this favored transcriptional modifications as a mechanistic explanation. Arginine metabolism has multiple connections to UA effects in other cells, as mentioned in the Introduction. Arginine can be metabolized via nitric oxide synthase into nitric oxide and citrulline or via arginase feeding the polyamine metabolism. As moderate increases in nitric oxide improve the load-free cell shortening, whereas UA deteriorated cell shortening, we analyzed the protein expression of ornithine decarboxylase (ODC) as the rate-limiting enzyme of the polyamine metabolism and the protein expression of calcium-sensing receptors. Secreted polyamines may activate this receptor. We found that UA increased ODC expression in cardiomyocytes A,B. In cNext, we inhibited several steps of the polyamine metabolism in the cardiomyocytes. Difluoromethyl ornithine (DFMO) was used to inhibit ODC activity, ornithine was added to stimulate ODC activity, Nor-NOHA was used to inhibit arginase activity, and L-NAME was used to inhibit nitric oxide synthase (NOS) in the cardiomyocytes. DFMO reduced the effect of UA on cell shortening, suggesting that the upregulation of the ODC protein expression contributes to the pathway. Ornithine did not affect the UA effects, and the inhibition of arginase had an intermediate effect between the DFMO and ornithine A–C. L-NAThe data of the aforementioned experiments support the idea that UA induces an arginine depletion. This may induce a limitation of intracellular arginine bioavailability required for proper function. This hypothesis was tested by increasing the concentration of l-arginine in the medium in the copresence of UA. Increasing the l-arginine concentration, the natural substrate for NOS, attenuated the effect of UA in a concentration-dependent way A,B. HoweAs mentioned in the Introduction, UA may affect the activity of protein kinases. Therefore, we investigated effects of p38 MAPK kinase inhibition (by SB202190), PKC inhibition , and Jun-Kinase inhibition (by SP600125) on the UA-dependent reduction of load-free cell shortening A–C. AmonReduction in contractility may depend on two different mechanisms that have been reported before. First, the reduced mobilization of calcium from intracellular stores  or, second, the impairment of the calcium sensitivity of troponin subunits. In the first case, calcium transients are decreased, whereas, in the second case, they are normal or even enhanced. To clarify which mechanism may be involved in the UA-dependent effect, we analyzed calcium amplitudes during contraction. As shown in The aforementioned data show that cells exposed to UA can be negatively affected by UA with respect to load-free cell shortening. However, it remained unclear whether this effect requires the presence of UA or whether the effect is reversible by removing UA. Therefore, we exposed cultured adult rat ventricular myocytes for 24 h to UA and subsequently replaced the UA-containing medium with normal medium immediately before measuring the cell function. This washing-out period was sufficient to reverse the effect of UA on the load-free cell shortening .Finally, we addressed the question of whether UA synergistically acts with hyperglycemia on load-freed cell shortening because the high plasma levels of UA are often associated with diabetes and hyperglycemia also negatively affects cell shortening. On the cellular basis, hyperglycemia may induce oxidative stress, whereas UA is an ROS scavenger molecule. In this experiment, we confirmed the deteriorating effect of hyperglycemia and UA on cardiomyocytes’ cell shortening. In both cases (glucose and UA concentration), we used a submaximal concentration. Importantly, the effects of both stressors on load-free cell shortening were additive .The general association between the high plasma levels of UA and heart disease is well established. However, this observation does not answer questions about the mechanism. Is UA a direct stressor for cardiomyocytes, an indicator for oxidative stress in cardiomyocytes, or are the high levels of extracellular UA without intrinsic activity but properly beneficial due to its ROS scavenging character? This study aimed at addressing these questions. The main finding of our study is that UA can directly affect the contractile function of cardiomyocytes. Enzymatic formation of UA by xanthine oxidase can be attenuated by pharmaceuticals, such as allopurinol, and the inhibition of xanthine oxidase activity has beneficial effects in the context of heart disease ,28. NeveThe first and novel observation of our study is that the exposure of cardiomyocytes to UA increases the protein expression of ODC. High ODC activity is linked to heart failure as well as an increased polyamine metabolism . PolyamiWe also observed that the effect of UA depends on the activity of the PKC. Interestingly, it was described in other cells that PKC can inhibit the main arginase transport protein in myocytes, CAT-1 . In contAlthough arginine depletion may contribute to the UA-dependent effect on cardiomyocytes, the question remains how this translates into the loss of contractility. An earlier study published on myofilaments isolated from diabetic cardiomyocytes showed that PKC-dependent phosphorylation of troponin I is associated with decreased calcium sensitivity ,40. FurtOur interest to understand the interaction between UA and cardiomyocytes comes also from the diabetes field, as conditions favoring the onset of diabetes, i.e., dietary uptake of large amounts of fructose-rich drinks, increase the plasma concentration of UA. Therefore, high plasma levels of glucose and UA go hand-in-hand. Chickens have naturally high plasma levels of glucose and UA, and it has been shown that this is a double-edged sword . On the As with all studies, this study has some limitations. First, the UA concentration used here leans on plasma concentrations typically found in humans, but rat cardiomyocytes are normally exposed to lower concentrations of UA. Second, the cellular arginine pool and arginine turnover were not biochemically quantified here, and the argument that arginine depletion triggers the UA-dependent effect comes from experiments showing the dependency of the effects from extracellular arginine. Third, as mentioned already, the observed stress by high UA levels may lead to compensatory mechanisms in vivo, such as the activation of the sympathetic nervous system shifting the disease progression to other receptor systems. Finally, load-free cell shortening is used as a well-established surrogate parameter of cardiomyocytes’ function, but in vivo UA may also affect perfusion and imbalance the energy supplementation of myocytes. Therefore, the mechanistic insights outworked in this study require proper in vivo controls. Nevertheless, these limitations are opposed to the advantages of the mechanistic analysis with terminally differentiated cardiomyocytes and the impressive high reproducibility of the effects.Our study used the advantage of an isolated cell model to show that UA has direct effects on cardiomyocytes. This effect adds stress to the heart. On the molecular basis, UA effects are linked to the arginine metabolism  and calcium desensitization most likely triggered by this process. The conclusions drawn from our cell-based experimental study are summarized in


In an independent cohort of 400 patients, we verify that HTLV-1 prevalence correlates with aGVHD incidence, while none of the control viruses shows significant associations. Our findings thus provide new insights into the aetio-pathology of liver-transplantation-associated aGVHD and raise the possibility of preventing aGVHD prior to transplantation.Acute graft versus host disease (aGVHD) is a rare, but severe complication of liver transplantation (LT). It is caused by the activation of donor immune cells in the graft against the host shortly after transplantation, but the contributing pathogenic factors remain unclear. Here we show that human T cell lymphotropic virus type I (HTLV-1) infection of donor T cells is highly associated with aGVHD following LT. The presence of HTLV-1 in peripheral blood and tissue samples from a discovery cohort of 7 aGVHD patients and 17 control patients is assessed with hybridization probes (TargetSeq), mass cytometry (CyTOF), and multiplex immunohistology (IMC). All 7 of our aGVHD patients display detectable HTLV-1 Tax signals by IMC. We identify donor-derived cells based on a Y chromosome-specific genetic marker, EIF1AY. Thus, we confirm the presence of CD4 Acute graft versus host disease is a rare but deadly complication following liver transplantation. Author show here, upon screening a large cohort of liver transplanted patients and detailed immune phenotyping of samples from the 7 affected individuals and appropriate controls, that human T cell lymphotropic virus type I infection of donor immune cells appear to correlate with the occurrence of acute graft versus host disease. It is a type of rejection that is stronger than typical rejections, destroying the skin and mucosal barriers of the host and leading to severe infections and ultimately lethal septic shock. The disease progression can be less than two weeks from onset to mortality3. Generally, GVHD can be categorized into two types, namely acute (aGVHD) and chronic (cGVHD), depending on whether the disease manifests before or after the 100th day since transplantation4. In contrast to the high incidence (20–80%) of aGVHD in patients receiving hematopoietic stem cell transplantation (HSCT)6, the incidence is much lower for solid organ transplantations, such as liver transplantation (LT) (0.1–2%)7. However, the mortality of LT patients with aGVHD is as high as 80–100%8, whereas the overall lethality of transplant recipients is 9.4% within one year9. The low incidence of GVHD with high mortality is presented in all kinds of solid organ transplantations. However, there is still a lack of theory to explain this issue10.Graft-versus-host disease (GVHD) is defined as immunological damage to host tissues and organs due to the activation of donor immune cells in graft by the host’s major histocompatibility complex (MHC)11. The introduction of massive donor-derived T cells contributes to the high incidence of GVHD in HSCT patients6, whereas LT patients receive a tiny proportion of donor immune cells. When a liver graft is implanted, 109−1010 donor-derived immune cells are transferred into the recipient’s body, including T cells, B cells, natural killer (NK) cells, and monocytes12. In some cases, these cells can be detected in the recipient’s peripheral blood and bone marrow between 3 and 100 days after surgery which matches the onset time of aGVHD13. Any transplant that transfers viable allogeneic lymphocytes into the recipient carries the potential risk of GVHD, such as transfusion-associated GVHD15.More than 20 years ago, donor-derived T cells were identified as the primary cause of GVHD16. Donor CD8+ T cells alone can induce GVHD by identifying disparities of MHC class I molecules, while CD4+ T cells induce GVHD by recognizing MHC class II molecules18. T cells are the main effectors causing cell death in target tissues, mediated by various cytotoxic effects10. As a pathogenic and T-cell-tropic human retrovirus, human T-cell lymphotropic virus type I (HTLV-1) can infect and immortalize CD4+ T cells20. In different animal models of HTLV-1 infection, established in macaques, rabbits, and humanized mice, the virus can cause lethal CD4+ T-cell expansion23. Of note, the bone marrow-liver-thymus (BLT) mouse model was prone to aGVHD, related to elevated levels of HTLV-1 RNA in blood21. HLA matching is performed before HSCT to avoid massive donor-derived T cells attacking the hosts, while HLA matching is not mandatory in liver transplantations. Generally, the proportion of donor lymphocytes in LT is too small to induce aGVHD even though HLA is mismatching between the donors and the recipients. Additionally, HLA miss-matching did not correlate with LT outcome or rejection rate25. In HSCT, tissue damage and GVHD seem to be the reciprocal causation28. However, in LTs, without tissue damage caused by conventional chemotherapy, the risk factors that trigger and induce the expansion of donor-derived T cells for aGVHD development remain unknown, which is our focus.The critical process of the development of aGVHD is the activation of T-cell receptors (TCR) on donor-derived T cells by the antigen-presenting cells (APC) with corresponding host MHC or minor histocompatibility antigen (miH) peptides+ T cells are increased by HTLV-1 infection. Additionally, joint analysis of mass cytometry (CyTOF) and IMC results suggest the aGVHD-specific Tax+CD68+ APCs take up HTLV-1 and transmit the virus and immune signals. Thus, our study indicates that HTLV-1 is involved in the entire process of aGVHD.In this study, we hypothesize that viral infection plays a role in expanding donor-derived T cells in LT, which might have been masked by the severe fungi and bacteria infections in the course of aGVHD. From 2015 to 2020, seven aGVHD patients that were DCD (donor after circulatory death) recipients had been identified among 3763 liver transplant recipients in our center. Therefore, the incidence of aGVHD is 0.18% . To reveal the cryptogenic infections associated with developing aGVHD, we design a cocktail of probes targeting eight non-hepatotropic viruses. As a result, HTLV-1 infections are explicitly found in all the seven aGVHD patients. Furthermore, the multiplex immunohistology (IMC) results confirm that donor-derived CD429. Latent infection, where viral DNA had been integrated into the host genome, is a way for viruses to achieve persistent and lifelong infection in humans and participate in the pathogenesis of various complications30. In both the aGVHD and control groups, HTLV-2 and HTLV-4 were not detected and therefore excluded from subsequent analysis   in the aGVHD group and 17 recipients in the control group Fig. . Detailesis Fig. . HHV-6A sis Fig. . Viral hsis Fig. . The presis Fig. . Compare06) Fig. . Parvovi06) Fig. . Althoug06) Fig. . Therefo06) Fig. .Fig. 1A 4)31. The other HTLV-1-positive patient with co-infection with hepatitis C died on the second day after surgery for acute liver failure. None of the 400 patients in this cohort was diagnosed with aGVHD, and there was no HTLV-1 positive recipient. Therefore, the prevalence of HTLV-1 in our center in patients without aGVHD was 0.5%, with a 95% confidence interval of , which matched the reported prevalence of 0.1% to 1%32. Given that, the possibility that the co-occurrences of the two rare diseases were a coincidence was less than 10−4 using a binomial probability calculation, suggesting that HTLV-1 infection should be closely related to aGHVD development after LT.To further investigate the prevalence of HTLV-1 in our center and verify our hypothesis of HTLV-1 infection and aGVHD process, an independent cohort of 400 patients, including liver surgery patients, recipients, and donors of liver transplants Table , were reN = 3) and control (N = 10) patients  were selected for subsequent data analysis  was adopted for systematic and deep phenotyping of the PBMCs from the aGVHD . Dendritic cells (DC), constituting the mononuclear phagocyte system (MPS), are considered to be the most potent APCs36. Because CD68 is a selective marker for human monocytes and macrophages, Clusters 22#, 23#, and 30# (CD3−CD19−CD14−CD16+HLA-DR−CD68+) were considered non-classical monocytes37. Cluster 25# was intermediate monocytes (CD3−CD19−CD14+CD11c+CD16+CD68+). Cluster 24# (CD3−CD19−CD14−CD16−CD11c−CD123−HLA-DR−CD68+) expressed activation markers such as CD3838 and CD6939, indicating these cells were highly activated. Meanwhile, cluster 26# (CD19−CD14−CD3+CD8+Tax+) was characterized as Tax+CD8+T cells . The four methods cross-validated that all and only the seven aGVHD patients were infected with HTLV-1.To fully display the HTLV-1 detection result assessed with multiple methods in the discovery cohort, the positive rate and specific detection information of HTLV-1 are listed in Table + CD68+ cells in peripheral blood and skin sections, the characteristics of APCs in skin lesions of all aGVHD patients were investigated by IMC to further reveal the role of HTLV-1 infection in aGVHD. The primary APCs in the skin are tissue mononuclear phagocyte (MNP) subsets, which are two prominent families, DCs and tissue-resident macrophages40. DCs are further divided into conventional DC (cDC) and plasmacytoid DC (pDC). cDCs, pDCs, monocyte-derived macrophages (MDM), and tissue-resident macrophages respectively express CD11c+HLA-DR+CD68+, CD123+HLA-DR+CD68+, CD14+CD11c−CD68+, and CD14-CD11c−CD68+ cells53. The phenotypes and distributions of the four MNPs found by IMC in the skin lesions were presented by three representative patients  from female patients who received the organ from male donors (ID139), the Tax protein was detected in CD68aft Fig.  and S9. nts Fig. . For botnts Fig.  but negants Fig. , suggestnts Fig. . These r+CD68+ immune cells in peripheral blood, including monocytes and DCs. Integrated analysis of CyTOF and IMC results reproduced the antigen-presenting responses during aGVHD after LT. Some MNPs, mainly DCs, MDMs, and macrophages, in the skin co-expressed Tax and CD68 proteins. It explains the migration of APCs from peripheral blood to skin lesions, during which they took up HTLV-1 and transmitted the viruses and immune signals. Most importantly, we discovered the pathogenic host-targeting T cells by IMC, which were “triple-positive T cells”. Our results confirmed the existence of donor-derived CD4+ T lymphocytes in targeted recipient organs, which were possibly expanded by HTLV-1 infection, as a potential initiator of aGVHD after LT. These findings associate the incidence of aGVHD with a viral infection, providing new perspectives for its prevention and treatment.In this study, we detected HTLV-1 cDNA and HTLV-1 specific Tax proteins from peripheral blood samples, fresh tissue sections, and formalin-fixed and paraffin-embedded (FFPE) tissue sections of all aGVHD-diagnosed patients by pre-synthesized TargetSeq, CyTOF, and IMC. The association between HTLV-1 infection and the occurrence of aGVHD has been determined by statistical analysis. Furthermore, CyTOF uncovered aGVHD-specific circulating Tax20. In contrast to HIV causing T-cell death, HTLV-1 can efficiently infect T cells, sustain their growth, and immortalize them through Tax protein-inducing kappa B-specific proteins20. Expression of the HTLV-1 Tax protein induces enhancement of T-cell promoting factors, IL-2, IL-4, IL-6, T-cell telomerase, and Tax/rex mRNA, and reduction of T-cell apoptotic factors, such as Bim (a pro-apoptotic protein), leading to excessive proliferation of T cells  and unlimited expression of the IL-2R alpha and IL-2 genes20. Unlimited proliferation of T cells, caused by HTLV-1 infection, acts on the human immune system and becomes an enhancer of rejection55. Therefore, HTLV-1 virus infection may expand T cells, possibly for both hosts and donors, resulting in a potential tendency for host-to-graft and graft-to-host rejections.HTLV-1 is a highly pathogenic virus similar to HIV, but due to its geographic distribution and long latency period, HTLV-1 has not been adequately heeded431. Due to the low infection rate and geographical limitations of HTLV-1, many transplantation centers do not routinely screen for HTLV-1, resulting in overlooked viral infection. In our study, expansion of donor T cells occurred regardless of the HTLV-1 infection source . The lethal CD4+ T cells expansion is supposed to underlay aGVHD onset and progression. Therefore, our data suggest that donors, recipients, and blood supply sources should undergo strict testing for HTLV-1 infection before surgery to prevent the disordered proliferation of T cells in organ recipients. In this study, the specificity of HTLV-1 virus infection in aGVHD patients after LT was preliminarily revealed, so a more extensive multicenter study to examine the relationship between HTLV-1 and aGVHD will be beneficial. Simplified strategies for diagnosis of HTLV-1 infection and antiretroviral therapy will play positive roles in the prevention and treatment of aGVHD after LT. At the same time, it explains one of the various viral hazards in humans and calls for more research on virus-related lymphocytic diseases.The confirmation and prevention of HTLV-1 infection depend on the perioperative preparation of transplantation. Viral infection can occur when the number of cells containing the provirus is more than 9 × 10This study was approved by the ethical review board of Renji Hospital, Shanghai Jiao Tong University School of Medicine . All patient samples were obtained with informed consent under the supervision of IRB.N = 4), post-transplant infection patients (N = 3), post-transplant regular recovery recipients (N = 4), and pre-operative patients (N = 6). For patients with aGVHD, post-transplant rejection, or post-transplant infection, samples were collected during the disease progression. Specimens were collected before surgery from pre-operative control patients. The patient IDs, clinical information, and groupings for the patients evaluated in this study are listed in Table The DCD recipients were divided into two groups, the aGVHD and control groups. The aGVHD group consisted of seven patients diagnosed with aGVHD in different years between 2015 and 2020. The control group consisted of 17 recipients, including post-transplant rejection patients  and 17 recipients in the control group Table .N = 4), post-transplant infection patients (N = 3), and post-transplant regular recovery recipients (N = 3). The PBMCs of the aGVHD group were collected at two different time points between onset and death. The PBMCs in the control group were collected only once.For CyTOF and subsequent analysis, PBMC samples were collected from 3 aGVHD patients  and 10 recipients in the control group, including post-transplant rejection patients , and bone marrows obtained from the 7 aGVHD patients Table . The FFPN = 37) were harvested from the aGVHD and control group patients, including skin lesions, peripheral blood, and biopsy tissues of livers, intestines, ventricular walls, and lungs . Real-time RT-PCR assays for HTLV-1 RNA detection were performed using Human T-cell Lymphoma Virus 1 (HTLV-1) Probe qRT-PCR Kit  in an ABI 7500 real-time PCR system  following the kit instruction. Each reaction mixture contained 10 μL buffer, 2 μL enzyme mixture, 2 μL mixture of forward and reverse primer, 1uL probe solution, and 5 μl specimen. The thermal cycling parameters were 30 min at 50 °C for reverse transcription, 10 min at 94 °C for predegeneration, and 40 cycles containing 15 s at 94 °C and 1 min at 60 °C.The antibodies used in this study and the corresponding manufacturers and concentrations are listed in Table 191Ir/193Ir DNA intercalator  with Maxpar Perm-S Buffer  overnight at 4 °C.PBMCs were separated by density gradient centrifugation with Ficoll-Paque PLUS  Fig. . After l191Ir/193Ir DNA intercalator  with DPBS.Tissue sections were cut from FFPE tissue blocks of host livers, skins, bone marrow biopsies, and graft liver biopsies from Renji Hospital Fig. . The tiswww.cytobank.org). A series of gates were used to select single cells and CD45+ cells, as depicted in Fig. 35. ArcSinh transformation, with a scaling factor of 5, was chosen as the transformation method to diminish the noise in these measurements.After acquisition on Helios , data were normalized with four standard EQ beads Fig. . Mass cyImages were acquired with a Hyperion Laser Scanning Module  coupled to Helios mass cytometer Fig. . FluidigSerum samples were collected from the patients (ID:144 and ID:145) after diagnosing aGVHD. Blood samples were centrifuged for 5 minutes to separate serum. The serum was stored at -80°C. Samples were all tested by HTLV-I ELISA Kit . All operations were carried out according to the kit instructions and the optical density was measured at 450 nm using a microtiter plate reader .p value <0.05 was considered statistically significant. In Fig. Fisher’s exact test was adopted to test the differences in HTLV-1 positive rate between groups, and Benjamini-Hochberg adjustment was applied to control the false discovery rate. The adjusted Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


The cortex is a highly organized structure that develops from the caudal regions of the segmented neural tube. Its spatial organization sets the stage for future functional arealization. Here, we suggest using a developmental perspective to describe and understand the etiology of common cortical malformations and their manifestation in the human brain. Understanding the origins of higher cortical functions and their malfunction in developmental diseases requires delineating how brain areas are formed during development. It has been long appreciated that the human brain is composed of defined areal and laminar structures that differ in composition ,2,3,4. TThe first indication of the developing human central nervous system (CNS) appears in the third week of human embryonic development in a process termed neurulation. A plate of thickened ectoderm appears in the mid-dorsal region of the tri-laminar embryo, the neural plate. The formation of the anterior neural plate, which will give rise to the forebrain, is induced by signals from the prechordal plate (PrCP), a cell population that originates from the mesendoderm and migrates rostrally from the primitive node along the midline between the ectoderm and the endoderm layers. In a series of extensive morphogenetic movements, the neural plate bends inwards to generate a tubular structure with open neural folds, then brought together at the dorsal midline to generate the neural tube  and dorsal–ventral (DV) axes of both the spinal cord and the anterior structures of the CNS is governed by the production of instructive molecules—morphogens—that are expressed by small cell populations, often referred to as patterning centers or organizers. A fundamental question is how positional information is conveyed and interpreted by the cells to express subsets of transcription factors that will execute the developmental fate. A model for the interpretation of morphogens areal information into distinct cell fates was proposed by Wolpert (the French flag model) . AccordiThe anterior neural ridge (ANR) is an example of an organizing center that governs forebrain patterning via positional information. The ANR is a temporary structure located at the most-rostral edge of the neural plate a. The ANSimilarly, ablation of the ANR or loss of Fgf8 in the anterior neural plate cells of zebrafish embryos resulted in a distorted telencephalon with abnormal morphology, altered gene expression, and axonal misprojection . FurtherFollowing the formation of the left and right telencephalic vesicles, the mediolateral axis of their dorsal cortical primordium develops into two signaling centers: the hem and the anti-hem. Both centers flank the cortical neuroepithelium and express different types of signaling molecules. The cortical hem expresses Wingless (Wnt) genes  and bone morphogenetic protein (BMP) genes  and serves as the organizer of the hippocampus. Indeed, the induction of ectopic hem structures adjacent to Lhx2 null cells can produce multiple hippocampal fields ,28. The In addition to FGF, BMP, and Wnt signals governing the AP and mediolateral patterning of the dorsal telencephalon, additional signaling factors participate in the patterning of the ventral and the lateral telencephalon. Sonic Hedgehog (Shh), a morphogen known for its graded function in the ventral spinal cord, has been shown to work in two phases during forebrain development. In the early stage, Shh is expressed in the PrCP and mediates the initiation of forebrain development from the anterior neural plate as well as the subdivision of the anterior prosencephalon into left and right vesicles. Next, the early Shh signal triggers a secondary induction activity in the forebrain, where it will sequentially specify the progenitors of the medial ganglionic eminence (MGE) and, later, of the lateral ganglionic eminence (LGE) c 29]..29].Retinoic acid (RA), a metabolite of retinol (vitamin A), functions as a ligand for nuclear retinoic acid receptors (RARs), which regulate the expression of numerous downstream target genes during both early embryogenesis and adulthood. Early in development, RA appears in a morphogenic gradient  established by an interplay between diffusion gradients and localized RA metabolism, thereby allowing RA to control the patterning of various structures . RA is lAn essential principle in forebrain induction and patterning is that signaling molecules, such as those mentioned, do not work independently but rather display an intricate regulatory network. Different secreted morphogenetic molecules in and around the patterning centers govern direct or indirect interactions between multiple signaling pathways and their downstream transcription factors. For instance, Fgfs, expressed at the ANR, are regulators of mid-line patterning and antagonize Wnt and BMP of the cortical hem . At lateFinally, while the local AP planar induction sets up a transverse regionalization of the major forebrain parts, DV patterning along the neural tube is also fundamental for defining longitudinal zones. The best-studied DV patterning of cellular and molecular events in the CNS is that that shapes the hindbrain and the spinal cord DV axis, in which distinct neuronal subtypes emerge from pre-patterned progenitor domains at a defined location along the DV axis ,38,39,40The cerebral cortex has an intrinsic functional architecture critical for its proper performance. It is derived from the dorsal and ventral telencephalon. The dorsal telencephalic domain gives rise to the significant neuronal population that will occupy the brain, the excitatory neurons, while the ventral telencephalon includes the ganglionic eminences (GE) from which the minor population of GABAergic interneurons originates. The emergence of different functional areas results from orchestrated events discussed elsewhere ,44.Cortical development involves several modes of migration during which cells move away from their place of birth. Migratory events are generally categorized based on their general movement pattern  or by the characteristic morphological or dynamic behavior of the migrating cells . An important example of how cell migration shapes the brain is that of the early-born neurons, the Cajal–Retzius cells (CRs). CRs are one of several transient cell populations in the developing brain  and spreThe primordial progenitors of the cortex are the neuroepithelial cells (NECs). NECs are elongated cells that span the entire thickness of the neuroepithelium from the ventricular  surface to the laminal  side ,49 Figu. During This cyclic motion, termed interkinetic nuclear migration or motion, was first discovered by Schaper in 1897 and re-discovered by Sauer in 1935 ,53,54. LNotch is a central signaling pathway that acts at several key time points during corticogenesis, ensuring progenitor pool expansion and maintenance, differentiation and neurogenesis, as well as folding of the cortex. The Notch pathway was first identified in the fruit fly and is highly conserved . DecadesEarly studies showed that forced activation of Notch signaling (NICD expression) in the mouse forebrain promoted radial glia identity , pointinNotch activity suppresses neurogenesis and promotes the maintenance of neural precursors by activating canonical Notch target genes, Hairy/Enhancer of Split (HES), and Hairy/Enhancer of Split related to the YRPW motif (HEY) . This weFinally, Notch activity was linked to the evolutionary expansion and folding of the human cortex. Disturption of neurogenic symmetry, regardless of bRG expansion, is suggested to allow cortical folding. This is achieved by local disruption of Notch activity while maintaining niches of sustain Notch activity . Three pAs radial migration proceeds, early-born neurons will generally contribute to the deep layers, whereas late neurons will migrate through the earlier neurons to form the more superficial layers . This orOne group of human genetic diseases that exhibits aberrant signaling related to brain patterning is holoprosencephaly (HPE) ,91,92,93The genetic etiology of HPE is highly heterogenous and may exhibit a highly variable phenotype. Different cytogenic abnormalities are a leading cause of HPE among live-born patients, and the most prominent ones are trisomy 13 and trisomy 18 . MutatioThe four major mutated genes in HPE are SHH, ZIC2, SIX3, and FGFR1 . SHH wasMutations in the transcription factor ZIC2 are common in HPE. Mice with hypomorphic alleles exhibit middle interhemispheric variant HPE ,121.ZIC2 has been suggested to act downstream of NODAL, a secreted member of the transforming growth factor ß (TGF-ß) family, as it is essential for stabilizing the epiblast state and the specification of mesoderm and endoderm, including the PrCP mesoderm, whose instructive functions were discussed. Nodal signals as a heterodimer with TGF-ß family members GDF1/3 and the activated receptor complex phosphorylates the signal transducers Smad2/3 at their C-terminal. ZIC2 possibly acts by direct interaction with SMAD2 and SMAD3 . In addiMutations in SIX3 are another leading cause of HPE . SIX3 isMicrodeletions and missense mutations in 5′-TG-3′-interacting factor (TGIF) also result in HPE. TGIF interacts with several pathways that are important in the developing brain. TGIF regulates the TGFb/Nodal signaling pathway and SHH signaling independently . In addiBMP signaling has a clear role in brain patterning. Nevertheless, so far, there are no specific mutations in HPE patients related to this pathway. However, several mouse mutants are exhibiting HPE-like phenotypes with mutations in genes associated with this signaling pathway. These mutations include BMP receptors , chordinMutations in additional genes causing HPE include proteins with several known functions that are likely to regulate the signaling pathways, as mentioned earlier. However, direct connections are yet to be identified. Mutations in CNOT1 that encode for a subunit of the CCR4–NOT Transcription Complex Subunit 1 result in HPE ,144. MutConsistent with the known functions of RA, loss of RA signaling in the developing fetus has detrimental effects on early arealization and later neurodevelopmental processes. Mutations in genes involved in RA signaling have been implicated in a wide range of diseases, including cancer, metabolic disorders, eye development, retinal function, and neurodegenerative diseases ,152. MutInterestingly, abnormal RA signaling is not exclusively the result of a genetic mutation. Exposure of developing fetuses to alcohol is known to cause congenital disabilities and intellectual and neurodevelopmental disabilities. Fetal alcohol syndrome (FASD) was first described in the early 1970s as a specific cluster of congenital disabilities resulting from chronic prenatal alcohol exposure. In the case of the severe form of FASD, the patients have craniofacial malformations , a high prevalence of microcephaly, prenatal and postnatal growth restriction, and central nervous system neurodevelopmental abnormalities . FASD isThe connection between ethanol metabolism and retinol arises from the biochemical similarity between ethanol clearance from the body and RA biosynthesis ,159. EthIn the mature brain, the areal organization can be manifested in differences in the cytoarchitecture, cell number, cell density, and lamination in different brain areas. Histological differences define anatomical subdivisions rooted in the developmental phases of forebrain development. They may result in differential sensitivity to pathological mutations or variations in gene dosage that dictate the disease phenotype.The lissencephaly–pachygyria spectrum of diseases defines a variety of brain malformations that cause relative smoothness of the brain surface and includes lissencephaly (smooth brain surface), agyria (no gyri), and pachygyria (broad gyri) . These bSeveral of the lissencephalies exhibit area-specific severity of the phenotype ,162. In LIS1, Lissencephaly 1 [DCX, Doublecortin [LIS1 mutations result in a more severe phenotype in the parietal and occipital cortex, whereas in the case of mutations in DCX, the malformation is more pronounced in the frontal cortex [ARX [KIF2A [MACF1 [DYNC1H1 [TUBA1A [TUBG1 [TUBB2B, TUBB3 [TUBB5 [TUBA8 [Regional severity has been noted in lissencephaly caused by mutations in ephaly 1 , or DCX,lecortin ,166. LISl cortex ,168. A stex [ARX ,170, KIFX [KIF2A , MACF1 [A [MACF1 , DYNC1H1[DYNC1H1 ,174,175. [TUBA1A ,177,178,A [TUBG1 , TUBB2B,B, TUBB3 , TUBB5 [3 [TUBB5 , TUBA8 [5 [TUBA8 . Mutatio5 [TUBA8 . A diffe5 [TUBA8 .ACTG1 [ACTB [CRADD [RTTN [Anterior-to-posterior increased severity also involves genes of the actin superfamily of proteins: ACTG1 , ACTB [1G1 [ACTB ,186, CRAB [CRADD , and RTTDD [RTTN .Understanding and identifying developmental principles and signaling pathways that shape the anterior CNS have progressed considerably. The areal organization of the human brain is tightly linked to brain function. Thus, the proper progression of the developmental plan is critical for brain functions. A deeper understanding of the etiology of neurodevelopmental conditions is rooted in uncovering the essential steps during brain patterning. This may be true not only when gross brain abnormalities are seen but also when abnormalities are manifested in subtle arealization defects that are not always appreciated but can profoundly influence an individual’s cognitive and social skills.


Second, we studied the association between 18F-fluorodeoxyglucose PET and lower dopamine transporter availability in the putamen, another hallmark of synucleinopathies. Patients with isolated rapid eye movement sleep behaviour disorder from the Mayo Clinic Alzheimer’s Disease Research Center and Center for Sleep Medicine (n = 22) and age-and sex-matched clinically unimpaired controls  from the Mayo Clinic Study of Aging were included. All participants underwent 18F-fluorodeoxyglucose PET and dopamine transporter imaging with iodine 123-radiolabeled 2β-carbomethoxy-3β-(4-iodophenyl)-N-(3-fluoropropyl) nortropane on single-photon emission computerized tomography. A subset of patients with isolated rapid eye movement sleep behaviour disorder with follow-up evaluations (n = 17) was classified as isolated rapid eye movement sleep behaviour disorder progressors (n = 7) if they developed mild cognitive impairment or Parkinson’s disease; or isolated rapid eye movement sleep behaviour disorder stables (n = 10) if they remained with a diagnosis of isolated rapid eye movement sleep behaviour disorder with no cognitive impairment. Glucose metabolic abnormalities in isolated rapid eye movement sleep behaviour disorder were determined by comparing atlas-based regional 18F-fluorodeoxyglucose PET uptake between isolated rapid eye movement sleep behaviour disorder and clinically unimpaired. Associations between 18F-fluorodeoxyglucose PET and dopamine transporter availability in the putamen were analyzed with Pearson’s correlation within the nigrostriatal pathway structures and with voxel-based analysis in the cortex. Patients with isolated rapid eye movement sleep behaviour disorder had lower glucose metabolism in the substantia nigra, retrosplenial cortex, angular cortex, and thalamus, and higher metabolism in the amygdala and entorhinal cortex compared with clinically unimpaired. Patients with isolated rapid eye movement sleep behaviour disorder who clinically progressed over time were characterized by higher glucose metabolism in the amygdala and entorhinal cortex, and lower glucose metabolism in the cerebellum compared with clinically unimpaired. Lower dopamine transporter availability in the putamen was associated with higher glucose metabolism in the pallidum within the nigrostriatal pathway; and with higher 18F-fluorodeoxyglucose uptake in the amygdala, insula, and temporal pole on a voxel-based analysis, although these associations did not survive after correcting for multiple comparisons. Our findings suggest that cerebral glucose metabolism in isolated rapid eye movement sleep behaviour disorder is characterized by hypometabolism in regions frequently affected during the prodromal stage of synucleinopathies, potentially reflecting synaptic dysfunction. Hypermetabolism is also seen in isolated rapid eye movement sleep behaviour disorder, suggesting that synaptic metabolic disruptions may be leading to a lack of inhibition, compensatory mechanisms, or microglial activation, especially in regions associated with nigrostriatal degeneration.Alterations of cerebral glucose metabolism can be detected in patients with isolated rapid eye movement sleep behaviour disorder, a prodromal feature of neurodegenerative diseases with α-synuclein pathology. However, metabolic characteristics that determine clinical progression in isolated rapid eye movement sleep behaviour disorder and their association with other biomarkers need to be elucidated. We investigated the pattern of cerebral glucose metabolism on  et al. report a pattern of hypo- and hyper-cerebral glucose metabolism in patients with isolated rapid eye movement sleep behaviour disorder. Hypermetabolism in certain regions was associated with nigrostriatal degeneration. This pattern also differed between patients with isolated rapid eye movement sleep behaviour disorder who clinically progressed and those who remain stable over time.Diaz-Galvan   All patients underwent FDG PET and DaTscan imaging between 2013 and 2021. Diagnosis of iRBD was made according to the International Classification of Sleep Disorders-second edition (ICSD-II) criteria.26 These criteria required a clinical history of dream enactment behaviour. All patients underwent a polysomnographic recording which documented REM sleep without atonia, the electrophysiologic substrate for RBD. We excluded patients with psychiatric and/or neurological comorbidity, the presence of any other sleep disorder, and the use of medication, and/or substance abuse that may better explain the RBD symptoms. Clinically unimpaired participants  without cognitive, motor, or sleep disorders were also included as the control group and matched 2:1 to the iRBD patients on age, sex, and length of follow-up. The clinically unimpaired group was selected from the Mayo Clinic Study of Aging (MCSA), and participants were characterized as clinically unimpaired by a consensus of neurologists and neuropsychologists after a comprehensive in-person evaluation.27This study included patients with iRBD (n = 17) was also followed over a range of 2 to 6 years  with clinical evaluations performed at approximately annual intervals. We classified the patients into iRBD progressors (n = 7) if they developed cognitive impairment and/or PD, or as iRBD stables (n = 10) if their cognitive status remained unimpaired and no PD evolved throughout the follow-up. Among the iRBD progressors, six progressed to MCI-LB and only one to PD. Diagnoses of MCI-LB and PD were established by a team of neurologists according to published criteria after an in-person examination that included a comprehensive neuropsychological assessment.29 This subset of iRBD patients with follow-up (n = 17) were also age-, sex-, and follow-up time-matched to a subset of clinical unimpaired controls from the baseline control cohort .A subset of iRBD patients 31 were used to assess the global cognitive status. Clinical features of DLB were assessed as described in previous reports from the DLB cohorts, such as in Choudhury et al. (2021).32 Briefly, parkinsonism was determined in the neurological examination as having at least two of the four cardinal features: tremor, rigidity, bradykinesia, and postural instability. The severity of parkinsonism was quantified with the Unified Parkinson’s Disease Rating Scale Part III (UPDRS-III).33 Visual hallucinations were considered to be present when they were fully formed, not restricted to a single episode, and not related to another medical issue, dementia, or treatment. The presence of fluctuations was determined by a score of 3 or 4 on the four-item Mayo Fluctuations Scale.34Clinical information was obtained through a neurological interview and examination, and a neuropsychological assessment. The Mini-Mental State Examination (MMSE)The study was approved by the Mayo Clinic Institutional Review Board and informed consent was obtained from all participants.3 of the resolution was performed for anatomical segmentation and labelling of the FDG PET images. PET imaging was performed with PET/CT scanners  operating in 3D mode. Patients were injected with an average of 348 MBq of 18F-FDG . After a 30-min uptake period, we obtained four 3.75-minute dynamic frames. A CT image was obtained for attenuation correction. DaTscan was performed at a 123I-FP-CIT SPECT  according to a previously published protocol.35 In brief, at least 1 h before the injection of 123I-ioflupane, a 100 mg Lugol solution was given, and then the recommended 123I-ioflupane dose of 111–185 MBq (3–5 mCi) was slowly administered intravenously. SPECT imaging occurred 3–6 h after injection. GE D670/D630 SPECT systems with ultrahigh-resolution fan beam collimators and an energy setting of 159 keV 20% windows were used on all patients. Data were reconstructed with an ordered subset expectation maximization method; the planar images were prefiltered with a Butterworth filter . No attenuation correction was used. Projection images were used for the quantitative analysis.MRI exams were performed at 3 Tesla. A 3D high-resolution magnetization prepared rapid gradient echo  with approximately 1 mm36 and DISTAL atlas for the substantia nigra;37 (iii) grey matter plus white matter sharpening to remove the effects of atrophy on regional FDG uptake; (iv) and calculation of the FDG standardized uptake value ratio (SUVr) in each voxel and ROIs. We used the median value of the pons uptake as the internal reference.FDG PET images were analyzed using an automated image analysis pipeline on SPM12. The pipeline consists of the following steps: (i) registration of the PET image volumes of each participant to their own T1-weighted MRI using 6 degrees of freedom affine registration with mutual information cost function; (ii) atlas-based segmentation and parcellation of FDG images into the region of interest (ROI) in each participant’s T1-weighted MRI space. We used the Mayo Clinic Adult Lifespan template (MCALT) for the parcellation of 49 grey matter ROIs,123I-FP-CIT uptake on SPECT were performed by DaTQUANT™ software, Version 2 . The volumes of interest (VOIs) of DaTQUANT of fixed size were semi-automatically placed over the right and left putamen in the transaxial slice showing the most intense tracer uptake. Another VOI was placed over the occipital lobe representing the cortical background. The software automatically placed the same VOIs in the adjacent previous and following slices such that data from three contiguous slices were used. Then, the left and right striatum-to-background ratio (SBR) of the putamen was automatically calculated. Each SBR was classified as minimum, maximum, and average for raking purposes. Minimum SBR corresponds to the lowest value, maximum SBR corresponds to the highest value, and average SBR corresponds to the average between maximum and minimum SBRs. For this study, we used the average SBRs of the left and right putamen. We chose to study putamen SBR because we have previously shown that DaTQUANT scores from the putamen have the best discrimination between patients with autopsy-confirmed LB disease and those without LB disease.35Semiquantitative calculations of putaminal t-tests for continuous variables or X2 tests for categorical variables. The area under the receiver operation curves (AUROC) was calculated to measure the ability of FDG SUVr in each atlas-based ROI (50 ROIs) to distinguish iRBD patients from CU controls in the whole sample, or the subgroups of iRBD stables and iRBD progressors from CU in the subset with follow-up data. AUROCs were generated using a weighted two-stage parameter estimation approach accounting for matching on age, sex, and length of follow-up.38 ROI analysis using weighted AUROC for FDG SUVr was not corrected for multiple comparisons in order not to inflate the probability of Type II error. Unadjusted Pearson correlations were used for the associations of RBD onset age and RBD duration with FDG PET. Finally, we approached the study of the association between putamen 123I-FP-CIT SPECT and FDG PET in two ways: (i) we investigated the relationship between the putamen 123I-FP-CIT uptake and FDG SUVr in the nigrostriatal pathway nuclei using age-adjusted Pearson correlations and (ii) we investigated the putamen 123I-FP-CIT uptake and cortical FDG SUVr using a voxel-based approach. Voxel-based analyses were conducted using a regression model including putamen 123I-FP-CIT SPECT as a predictor and age as a covariate. All the voxel-based analyses were performed within the general linear model (GLM) framework of SPM12, and statistical maps were displayed at a significance of P < 0.001.Clinical and demographic characteristics were reported for the CU participants and iRBD patients as means and standard deviations for continuous variables, and counts (%) for categorical variables. Pairwise characteristic comparisons between CU and iRBD patients in the cross-sectional and longitudinal samples were done using conditional logistic regressions that account for the matching. The characteristics comparing iRBD stables and iRBD progressors were done using The data that support the findings of this study are available from the corresponding author upon reasonable request.n = 17; 77%) and the mean (SD) age was 65.4 (8.0) years. The CU group was similar to iRBD on age and sex by design. CU and iRBD groups did not differ on MMSE and CDR-SOB, consistent with the expected cognitive profile of iRBD. The duration of iRBD ranged from one year up to 25 years. A small proportion of iRBD patients showed additional core clinical features of DLB , such as mild parkinsonism , visual hallucinations (n = 1 progressor), and cognitive fluctuations (n = 1 progressor).The baseline characteristics of the entire cohort are displayed in AUROC value, which measures the ability to distinguish iRBD and CU groups. Patients with CU were the reference group, such that AUROC values greater than 0.5 indicated higher FDG uptake in iRBD compared with CU. Conversely, AUROC values lower than 0.5 indicated lower FDG uptake in iRBD compared with CU group. Among the 50 atlas-based regions that we explored, results showed that patients with iRBD had lower FDG uptake in the substantia nigra , restrosplenial cortex , thalamus , and angular gyrus  compared with CU. On the contrary, patients with iRBD had higher FDG uptake in the amygdala , and entorhinal cortex  compared with CU. AUROC plots from all the regions included in the analysis are summarized in Atlas-based regional analyses were performed using the r = 0.48, P = 0.018) and later RBD onset . Lower FDG PET SUVr in the angular cortex was also associated with a shorter disease duration of RBD . Pearson correlations between symptom duration or age of RBD onset and FDG PET SUVr were not significant in the rest of the regions: the substantia nigra , retrosplenial cortex , entorhinal cortex , and amygdala .We also explored the association of regional FDG SUVr in the amygdala, entorhinal cortex, thalamus, retrosplenial cortex, angular cortex, and substantia nigra with disease duration and age of RBD onset. Only lower FDG PET SUVr in the thalamus was associated with a shorter duration of RBD (n = 7) of the iRBD patients cognitively progressed and evolved to MCI-LB or developed PD, while 58% (n = 10) remained stable. Clinically, four iRBD patients had the core clinical features of DLB .A subset of 17 iRBD patients was followed up for a range of 2.4 to 6.4 years. Forty-one percent  and the amygdala , and lower FDG uptake in the cerebellum . Compared with CU, iRBD stables had higher FDG uptake in the amygdala , cerebellum , entorhinal cortex , and fusiform gyrus .Regional FDG PET characteristics of iRBD progressor and iRBD stable groups were determined by using 123I-FP-CIT SPECT uptake and FDG PET was approached in two ways: (i) the relationship between putamen 123I-FP-CIT uptake and FDG PET SUVr in the nigrostriatal pathway nuclei was investigated with age-adjusted Pearson correlations; (ii) putamen 123I-FP-CIT uptake and cortical FDG PET SUVr was investigated using a voxel-based analysis.The investigation of the association between putamen 123I-FP-CIT uptake with FDG PET SUVr in the nigrostriatal pathway nuclei. Because there were no differences identified between the right and left nuclei, we display the results with the averaged FDG SUVr of both hemispheres with the average 123I-FP-CIT SBR. Lower putamen 123I-FP-CIT uptake was significantly associated with higher FDG SUVr in the pallidum . The putamen123I-FP-CIT uptake was not significantly associated with FDG SUVr in the other nigrostriatal nuclei.123I-FP-CIT uptake and cortical FDG SUVr. Since there were no significant associations after correction for multiple associations, we display the uncorrected results (P < 0.001). Lower putamen 123I-FP-CIT uptake was associated bilaterally with higher FDG SUVr in the amygdala, insula, and superior temporal gyrus. Lower 123I-FP-CIT uptake in the putamen was also associated with higher FDG PET SUVr in the right pallidum (P < 0.001).16 However, patients with iRBD also show hypermetabolism in the amygdala and the entorhinal cortex. Moreover, increased glucose metabolism in the amygdala and entorhinal cortex, together with decreased glucose metabolism in the cerebellum, characterized those patients who became cognitively impaired or developed PD during follow-up (i.e. iRBD progressors). The hypermetabolism observed in the amygdala was associated with the loss of dopaminergic activity in the putamen, measured by dopamine transporter availability on 123I-FP-CIT SPECT. This loss of dopaminergic activity in the putamen was also associated with glucose hypermetabolism in the pallidum, amygdala, insula, and superior temporal cortex. Altogether, our data indicate FDG PET hypometabolism in iRBD that may reflect synaptic dysfunction, but also hypermetabolism, which happens simultaneously and is in part associated with nigrostriatal dopaminergic deficiency and clinical progression.Our findings demonstrate that cerebral glucose metabolism in iRBD is characterized by both hypometabolic and hypermetabolic patterns on FDG PET. The topographic pattern of hypometabolism involves the substantia nigra, the retrosplenial cortex, the thalamus, and the angular gyrus, which is consistent with FDG PET studies in MCI-LB.40 Interestingly, we found increased glucose metabolism in the amygdala and entorhinal cortex also in those patients who progressed during follow-up. In synucleinopathies where RBD is frequent, such as PD or DLB, pathological changes are common in the basal forebrain and limbic structures including the amygdala.42 Furthermore, the amygdala is one of the anatomic structures implicated in the generation of REM sleep through both direct and indirect projections from the brainstem.44 However, little is known about the involvement of the amygdala and limbic system in RBD, so the mechanism underlying the observed metabolic hyperactivity is still unclear.In this study, we have shown that FDG alterations in iRBD are characterized by hypermetabolism in limbic structures. Regional hypermetabolism has been observed in previous studies in PD and iRBD, indicating increased glucose metabolism in the cerebellum, basal ganglia, and frontal cortical regions.45 In normal conditions, REM-ON cells in the amygdala are inhibited by serotonin (and possibly GABA) input from the dorsal raphe nucleus in the brain stem. Therefore, the hyperexcitability of the amygdala observed in RBD might be the result of a lack of inhibitory input from different groups of cells in the brainstem. These groups of cells in the brain stem are amongst the earliest and most severely affected regions by Lewy body pathology.46One explanation for the limbic hypermetabolism in iRBD might rely on the dysfunction of reciprocal connections between the amygdala and brain stem nuclei that generate and modulate REM sleep, such as coeruleus/subcoeruleus complex or raphe nuclei. A previous animal study identified REM-ON cells in the central nucleus of the amygdala.et al.47 for a systematic review). In AD, the increase in glucose metabolism has been interpreted as a compensatory response that promotes resilience to pathology progression at early stages, delaying the conversion from MCI to dementia.48–50 However, as the disease progresses and the pathology load worsens, the hypermetabolism is followed by a decline in brain metabolism.51 From these data, we hypothesize that compensatory hypermetabolism might be happening in synucleinopathies when patients are diagnosed with iRBD as an early outcome of increasing α-synuclein burden, which begins to accumulate in the olfactory cortex and brain stem.46 However, it is unclear whether limbic hypermetabolism is an indication of progression to MCI or dementia in iRBD patients. In fact, our findings indicate that those iRBD patients who progressed over time (i.e. iRBD progressors) show hypermetabolism in the amygdala and the entorhinal cortex. On the contrary, those iRBD patients who remained stable over time (i.e. iRBD stables) did not show any hypermetabolic activity compared with controls; whether this stable group represents those with iRBD who are early in the course of synucleinopathy evolution and hence many years or decades from progressing to MCI and/or parkinsonism, or have a non-degenerative aetiology for the RBD, will require more longitudinal assessments. Previous works in AD have shown that higher regional brain metabolism is associated with higher tau deposition on PET and with episodic memory impairment in MCI,52 and that neuronal hyperexcitability may drive tau release, propagation, and spread at the initial phases of the disease.54 This phenomenon may also occur in other neurodegenerative diseases, such as Parkinson’s disease or dementia of Lewy bodies. Hence, it is possible that early hypermetabolism leads to an exacerbation of neuronal damage and represents a maladaptive, detrimental event, rather than a beneficial compensatory response.55Another possible explanation for the limbic hypermetabolism in iRBD relies on compensatory mechanisms occurring as a response to initial neuronal damage during the early stages of neurodegenerative diseases. For example, in Alzheimer’s disease (AD), compensatory mechanisms have been documented over the years and comprise a wide spectrum of biological processes, including increased brain glucose metabolism in selective cortical areas .61 In AD, this activation of the microglia favours an anti-inflammatory phenotype in response to increasing amyloid load.62 Recently, Xiang et al.63 showed that FDG PET uptake is strongly influenced by microglial activity in patients with neurodegenerative diseases. Furthermore, their mouse model showed that elevated microglial FDG uptake correlated with increased expression of the 18-kDa translocator protein (TSPO) on PET, which has been consistently associated with neuroinflammation.56 Overall, their results suggest that microglia activation occurring at early stages of neurodegenerative diseases, most probably because of an inflammatory process, stimulates glucose uptake and thus drives the increase of FDG PET signal (i.e. hypermetabolism). In Lewy body disease, one of the earliest and most severe Lewy body pathologic changes in the cerebral gray matter occurs in the amygdala and entorhinal cortex.46 The increasing α-synuclein accumulation triggers neuroinflammation, which leads to microglial activation, which can potentially increase FDG uptake in these regions. In summary, there are multiple mechanisms that could explain the increased glucose metabolism in the amygdala and entorhinal cortex in iRBD, perhaps occurring independently or even co-existing. Further research is needed to elucidate these mechanisms and their contribution to iRBD progression.Finally, one of the biological events occurring during the earliest stages of neurodegenerative diseases is neuroinflammation,64 A pattern of reduction of glucose metabolism in the substantia nigra has been reported previously in PD and MCI-LB, and it has additive value in distinguishing patients with prodromal dementia of Lewy bodies from those with prodromal AD.65 Current results provide evidence that metabolic alterations in the substantia nigra occur in iRBD as an early biomarker of Lewy body disease. More research is needed to elucidate if the hypometabolism in the substantia nigra is a feature of those patients who will develop parkinsonism. Considering the low proportion of iRBD patients with features of parkinsonism in our cohort , the current study suggests that hypometabolism in the substantia nigra is a metabolic alteration that characterizes patients within the whole spectrum of Lewy body pathology. A consistent pattern of hypometabolism involving the occipital and parietal cortex has been observed in iRBD.40 In the current study, we also found hypometabolism, but it was constrained to the retrosplenial cortex and angular gyrus. The retrosplenial cortex is one of the few limbic regions activated during REM sleep.66 During REM sleep, the retrosplenial cortex is activated by glutamatergic neurons of the claustrum, which are involved in sleep by promoting slow wave activity.67 The activation of glutamatergic neurons in the retrosplenial cortex leads to the inhibition and control of several motor regions that control the muscle atonia during REM sleep. Therefore, the inactivity in the retrosplenial cortex in iRBD might be explained by the dysfunction of the REM sleep circuit itself because of a reduction of glutamatergic input from REM sleep centres in the brainstem. However, decreased metabolism in the retrosplenial cortex and angular gyrus could also be explained by the pathologic spreading of α-synuclein. Patients with DLB have shown greater hypometabolism involving retrosplenial cortex and angular gyrus, together with other posterior temporal, parietal, and occipital regions.68 This hypometabolic pattern is thought to be related to impaired cholinergic synaptic dysfunction as a result of the accumulation of α-synuclein in the basal forebrain,70 which occur relatively early in the progression of the LB disease (i.e. consistent with Stage 4 in Braak’s α-synuclein brain pathology staging model).46Our findings also indicate a hypometabolic pattern characterized by lower FDG uptake in the substantia nigra, retrosplenial cortex, thalamus, and angular gyrus. Neurodegeneration of the substantia nigra in association with a decreased dopaminergic input to the striatum is a hallmark of Lewy body disease.68 On the contrary, studies in Parkinson’s disease and iRBD have found hypermetabolism in the thalamus, instead of hypometabolism.71 These conflicting findings may be explained by differences in study design. While most of the previous studies in iRBD have been focused on detecting the PD-related metabolic pattern,73 in the current study we followed an unsupervised method to describe overall metabolic alterations. Therefore, the metabolic pattern found in the current study might reflect glucose metabolic alterations in iRBD during the very early stages in the continuum of DLB instead of PD. In fact, the proportion of patients with parkinsonism was low (9%) and, among those who progressed over time, only one patient progressed to PD, while the rest received a diagnosis of MCI-LB (n = 6). Metabolic patterns found in iRBD patients may vary by different pathological pathways associated with DLB, PD, or MSA. More longitudinal studies with larger cohorts are needed to test these hypotheses. Following a sequence of metabolic events of hypermetabolism preceding hypometabolism,51 it is also possible that the thalamic hypometabolism observed in the current study reflects a later stage in iRBD. This is supported by the proportion of progressors (30%) who developed MCI-LB or Parkinson’s disease within an average of five years. This high proportion of progressors suggests that most of the patients in this study were already in a later stage of iRBD. A good example of this alteration in the metabolic pattern by disease progression is amygdalar hypometabolism observed in MCI-LB and dementia of Lewy bodies,16 but hypermetabolism observed in iRBD patients.The thalamic hypometabolism observed in our iRBD patients is consistent with the findings in MCI-LB and DLB.74 However, the metabolic pattern in the cerebellum in iRBD is not fully understood. Contrary to our study, Meles et al.20 found increased metabolic activity in the cerebellum, while Carli et al.75 tested individual metabolic patterns in patients with iRBD and found that a high proportion of them had occipito-cerebellar hypometabolism. This heterogeneity may be explained again by the variation in iRBD patients at different stages of the iRBD pathophysiology. Furthermore, other neuroimaging studies in iRBD have also shown loss of grey matter volume and perfusion abnormalities in the cerebellum. While some studies include cohorts with a more recent diagnosis of iRBD, other cohorts may include patients who have more advanced diseases, such as the group of progressors in our cohort. On the one hand, this reduction in cerebellar metabolic activity in iRBD might be explained by the loss of neural tissue and perfusion observed in previous studies,76 and it may be due to a vulnerability of the cerebellum to Lewy body pathology. This cerebellar hypometabolism may also reflect a noradrenergic dysfunction due to the Lewy body pathology in the locus coeruleus. Locus coeruleus is affected by Lewy pathology during the earliest phases of the disease,77 which may reduce its noradrenergic connections to the cerebellum and facilitate hypometabolic alterations.78Hypometabolism was also found in the cerebellum, but only for iRBD patients who progressed to MCI-LB or Parkinson’s disease over time. Altered metabolic connectivity in the cerebellum is a common finding in patients with either PD or DLB, suggesting that the cerebellum is a vulnerable region to Lewy body pathology.79–85 Interestingly, dopamine transporter abnormalities have been associated with more severe RBD symptoms and an accelerated rate of progression to dementia.86–88 However, very little is known about the interplay between 123I-FP-CIT SPECT and FDG PET findings in iRBD. Only Meles et al.73 reported data from both modalities in the same study. They showed that the Parkinson’s disease-related FDG pattern can be identified in patients with iRBD compared with healthy individuals and that the expression of this pattern was higher in iRBD patients with abnormal dopamine transporter SPECT scan. Nonetheless, regional associations of both modalities still need to be elucidated. Within the nigrostriatal pathway, we found that decreases in dopamine transporter availability in the putamen were associated with glucose hypermetabolism in the pallidum. The hypermetabolism in the pallidum associated with the reduction of dopamine transporter activity in the putamen may reflect a disruption of the inhibitory input from the striatum to the pallidum, which occurs early in synucleinopathies.9064 We hypothesize that a lack of inhibitory input from the putamen to the pallidum due to the degeneration of the substantia nigra and nigrostriatal pathway connections most likely leads to hypermetabolism in the pallidum. However, other mechanisms could contribute to the hypermetabolic activity observed in the pallidum. For example, the substantia nigra has also direct connections to the pallidum, so it is possible that metabolic alterations in the pallidum are consequences of the dysfunction of direct dopaminergic innervation from the substantia nigra to the pallidum. Hypermetabolism in the pallidum might be also due to compensatory mechanisms or neuroinflammation. Further data are needed to test these hypotheses in future research. On a voxel-based analysis throughout the cortex, we also found that lower dopamine transporter availability in the putamen was associated with higher FDG uptake in the amygdala, the insula, and the superior temporal gyrus. The association of glucose hypermetabolism in these cortical regions with less dopamine transporter availability in the putamen might be explained by the dysfunction of some indirect connections between them. However, these significant associations might also be explained by the Lewy body-related pathological processes that are occurring simultaneously in the brain. Previous studies have already shown that pathological changes in prodromal DLB and DLB involve limbic structures such as the amygdala.42 Some metabolic and structural changes in the insula and lateral temporal cortex have been also reported even in patients with iRBD in association with lower performance in visuoperceptive and visuospatial functions.91 Further research is needed to understand whether and how iRBD-related metabolic alterations in the cortex are mediated by a nigrostriatal dopaminergic deficiency in conjunction with α-synuclein inclusions.Dopamine transporter abnormalities have been consistently identified in iRBD.5 the origin of this gender predominance may be motivated by both biological reasons and other explanations such as a women’s tendency not to seek medical attention for iRBD. Hence, the power to investigate differences in the FDG PET profile between men and women was limited in this study. Furthermore, our data are cross-sectional and cannot provide information on the temporal evolution of FDG PET pattern and its longitudinal association with dopamine biomarkers in iRBD. In consequence, we still cannot conclude on the cause-and-effect relationship between neurodegeneration and dopaminergic deficit in iRBD. Further investigations in larger cohorts and with a longitudinal design are needed to investigate the trajectory of metabolic alterations in iRBD together with its association with other biomarkers and disease progression.We acknowledge several limitations of our study. One limitation was a relatively small sample size, especially for those patients with follow-up data. Because the study cohort was very small for those who progressed to MCI-LB or PD, the FDG PET signatures found in the subgroup of iRBD progressors still need to be tested and corroborated in larger and independent cohorts, so we can determine their utility to predict clinical progression. In addition, our cohort comes from an ADRC and Center for Sleep Medicine, therefore movement disorders, such as PD, might be underrepresented as a possible endpoint of iRBD progression. Larger cohorts with a wide representation of these conditions and longer follow-up intervals are still needed in the study of biomarkers in iRBD. Another limitation was that most of the iRBD patients in our study were men. Although men are frequently over-represented in iRBD cohorts,123I-FP-CIT SPECT validate previous studies on nigrostriatal dopaminergic degeneration and provide useful information on networks that are directly and indirectly affected in iRBD.The next challenge in the treatment of synucleinopathies is to test disease-modifying therapies in people with iRBD with the aim of slowing, or even preventing, the full manifestation of the Lewy body diseases. With this purpose, it is important to enrich target iRBD populations with biomarkers that improve our understanding of the biological alterations and neurodegeneration in synucleinopathies. This study provides data about glucose metabolic alterations that are indicative of both hypometabolism and hypermetabolism in iRBD. These data can guide clinical decisions on combined treatments that target both mechanisms. Another priority in future clinical trials is the combination of biomarkers that span multiple modalities. Our data on the association between FDG PET and fcad021_Supplementary_DataClick here for additional data file.