Here, we will review the role that viral orf-I protein products play in altering intracellular signaling, protein expression and cell–cell communication in order to escape immune recognition and promote T-cell proliferation. We will also review studies of orf-I mutations found in infected patients and their potential impact on viral load, transmission and persistence. Finally, we will compare the orf-I gene in HTLV-1 subtypes as well as related STLV-1.The human T cell leukemia virus type 1 (HTVL-1), first reported in 1980 by Robert Gallo’s group, is the etiologic agent of both cancer and inflammatory diseases. Despite approximately 40 years of investigation, the prognosis for afflicted patients remains poor with no effective treatments. The virus persists in the infected host by evading the host immune response and inducing proliferation of infected CD4 Delta retrovirus genus, alongside the closely related HTLV-2, -3, and -4 viruses, simian T-cell leukemia viruses (STLV) 1–4, and bovine leukemia virus (BLV)  is homologous to that of NFAT [PXIXIT]  . The p12[PXIXIT] . Besides[PXIXIT] , which c[PXIXIT] .+ vacuolar ATPase, p12/p8 may affect signaling. The amino acids of HTLV-1 p12/p8 were found to be similar to those of the Bovine Papilloma Virus (BPV) E5 proteins. Like the BPV E5 oncoprotein [orf-I expression impairs HTLV-1 replication in dendritic cells [As suggested by their interaction with the Hoprotein , 73, p12oprotein , 55. Whioprotein , 55. V-Aoprotein . By bindoprotein , 76. HTLoprotein –79. Indeic cells .+ T-cells specifically recognize viral peptides via the TCR to destroy infected cells. Consequently, many viruses have evolved proteins whose main function is to interfere with this pathway [2 microglobulin (β2M) protein, with MHC-I-Hc binding affinity to β2M enhanced when in the presence of peptide. In this case, MHC-I-Hc folds and assembles into the peptide-MHC-I-Hc-β2M complex in the lumen of the ER [2-microglobulin necessary to form a mature MHC-I complex  antigen presentation pathway plays a central role in developing host immunity against pathogens. MHC-I molecules are expressed on the surface of all nucleated cells and present peptides to the TCRs of cytotoxic T lymphocytes. Effector CD8 pathway . In MHC-f the ER . The p12Orf-I mRNA is expressed early after virus entry and is critical for establishing and maintaining viral infection in vivo [+ T-cells infected with HTLV-1 molecular clones expressing neither p12 or p8 (p12KO), both p12 and p8 (WT), predominantly p8 (N26), or predominantly p12 (G29S), a decrease in surface MHC-I was only observed in the CD4+ T-cells infected with the G29S virus [+ T-cell clones over time. in vivo , 83, 84.9S virus . However9S virus . Thus, t+ T-cells infected with HTLV-1 [+ T-cells did not express ligands for the NK cell activating receptors, natural cytotoxicity receptors, or NKG2D [orf-I expression was sufficient to decrease ICAM-1 and ICAM-2 expression in primary CD4+ T-cells. Treatment of Tax-expressing HTLV-1 transformed cells with pomalidomide (POM), an immunomodulatory drug used in the treatment of multiple myeloma [orf-I dependent: MHC-I and ICAM-I expression increased in wild type (WT) HTLV-1 immortalized CD4+ T-cells after POM treatment, but their levels did not rise in HTLV-1 orf-I knockout immortalized CD4+ T-cells [While down modulation of MHC-I surface expression may allow infected cells to evade CTL recognition, it makes them susceptible to natural killer (NK) cell lysis. NK cells recognize and destroy cells that express low levels of MHC-I at their surface. Like CTLs, NK cells can kill infected cells directly by mediating cytolysis through perforin and granzyme production (reviewed in ). NK-tarh HTLV-1 . Furtherh HTLV-1 . The major NKG2D . The aut myeloma , led to  T-cells . Thus, porf-I region of HTLV-2, and an additional protein, p11, is encoded by the orf-V region, which overlaps with orf-I. Both proteins were shown to bind the MHC heavy chain, but do not bind the IL2Rβ chain or the 16-kDa subunit of the vacuolar H+ ATPase [Interestingly, additional proteins with functions similar to that of p12 have been identified in HTLV-2 and STLV-3. For instance, the corresponding p10 protein is encoded by the + ATPase . Moreove+ ATPase .Surface adhesion molecules like LFA and ICAM are also important in viral transmission. Cell-free virions are not easily detected in the blood plasma of infected individuals and cell-free virus is poorly infectious for most cell types except dendritic cells , 93–95. Furthermore, the p8 protein was also shown to be transferred to target cells , 110. Usorf-I expression was necessary for HTLV-1 infection in the rabbit model [orf-I in those studies, however, the molecular clone used had a frameshift affecting the gene encoding HBZ  to ACG (threonine) in all Australian HTLV-1C isolates, and to UCG (serine) in the Melanesian isolate [hbz overlaps 303 nucleotides within the orf-I, any changes in the HTLV-1C p12 coding region could also potentially affect HBZ amino acid sequence, expression, and function. Since HBZ and Tax are thought to play distinct but related roles during the multi-step oncogenesis and inflammation caused by the virus, the imbalanced expression of HBZ and Tax in HTLV-1C patients may impact its novel disease progression.Four major geographic subtypes of HTLV-1 have been identified: HTLV-1A, HTLV-1B, HTLV-1C and HTLV-1D , 116, wi and p12 . Interes isolate –120. Mor isolate  from an  isolate , 121. Itrex-orf-I, uses an initiation codon in exon 2 and the acceptor splice site at position 6383 to encode a protein of 152 amino acid referred to as the Rex-orf-I protein of 19 kDa. In this mRNA, the first coding exon of the Rex protein is joined in frame to p12/p8. Interestingly, ectopic expression of the of rex-orf-I cDNA yielded a 12-kDa protein that has the same relative migration as the p12 protein expressed by the singly spliced orf-I RNA. It has been demonstrated that this mRNA could encode p12 since the internal orf-I AUG is present in this mRNA, or p12 could be generated by posttranslational cleavage of the larger precursor protein [rex-orf-I show conservation of the amino acids that constitute the hallmark motifs implicated in p12 function. Thus, the protein products of rex-orf-I could possibly compensate for the role of p12 in viruses that do not contain the canonical initiation codon of p12 . Moreover, it has been suggested that HTLV-1 has a simian origin and was originally acquired by humans through interspecies transmission from STLV-1-infected Old World monkeys. This hypothesis was supported by the high percentage of identity between STLV-1 strains from chimpanzees and mandrills with some HTLV-1 strains present in inhabitants of West and Central Africa. In these specific areas, zoonotic transmission from non-human primates (NHP) infected with STLV-1-to humans is still ongoing today , 130–132Despite its wide geographic distribution and identification in more than 20 Old World primate species in both Asia and Africa, only a few complete STLV-1 genome sequences are available , 133–136orf-I in promoting infected cell proliferation and their evasion from host immune recognition. Further, expression of p12/p8 is necessary in both the macaque and humanized mouse models. One would therefore expect that the viral protein is important for establishing a lifelong infection in humans as well. This hypothesis was supported by a study that looked at 160 HTLV-1-infected individuals (HAM/TSP or carriers) from various geographical regions, in which none of the approximately 1600 orf-I cloned sequences analyzed had a premature stop codon [This review has highlighted the important functions of op codon . Howeverop codon . In a stop codon .Sequence analysis, however, shows that these truncated proteins retain the leucine zipper sites, the dileucine motif, the calcineurin binding sites, and the receptor binding sites for IL-2 beta and gamma chains and both cleavage sites Fig. . As mentorf-I gene was stably maintained over years in these individuals. Moreover, they found one HAM/TSP patient in which a nucleotide substitution from G to A at position 6836 resulted in the destruction of the initiation codon of p12. This virus was also found in two of the patient’s sisters, one carrier and one with HAM/TSP, indicating maternal transmission [orf-I could be expressed by an alternative doubly spliced mRNA in these patients . Torf-I expression is dispensable for HTLV-1C transmission and infectivity. Instead, we favor the hypothesis that an alternatively spliced mRNA may be used to provide the AUG initiation codon for the orf-I encoded protein(s). Interestingly, proteins that show p12 like functions have been identified in HTLV-2 and STLV-3 [rex-orf-I in infection, transmission, and pathogenesis of different HTLV-1 subtypes. Despite having had the complete genomic sequence for HTLV-1 for over 30 years, the mechanisms that govern disease status and host immune response are still unclear. We believe that developing a greater understanding of the complex kinetics, expression level, and function of the genes encoded in the 3′ end of the virus will allow us to develop novel therapeutic approaches for the treatment of HTLV-1 infection.We don’t believe this observation suggests that d STLV-3 , 92. FurAdditional file 1: Figure S1. Structure of the HTLV-1 proviral genome. The proviral DNA with the LTRs, and the unspliced, singly spliced and doubly spliced mRNA transcripts are shown. The names of the gene transcripts are depicted inside each specific box. Solid lines indicate the exons and the dotted lines indicate the introns.


The sensitivity for the post hoc-developed peak attenuation—peak point Laplacian method for classifying pure UA stones was 95%  and the specificity was 99% . In conclusion, quantitative image analysis of thin slice routine single-energy CT images is promising for predicting pure UA content in urinary stones, with results comparable to double energy methods.Urinary stones composed of uric acid can be treated medically. Prediction of uric acid stone type is, therefore, desirable when a urinary stone is diagnosed with unenhanced CT. The purpose of the present study was to describe single-energy thin slice quantitative CT parameters of urinary stones correlated to chemical stone type and to develop a method to distinguish pure uric acid stones (UA) from other stones (non-UA/Mix). Unenhanced thin slice single-energy CT images of 126 urinary stones (117 patients) with known chemical stone type were retrospectively included in the study. Among the included stones, 22 were UA and 104 were non-UA/Mix. The included CT images and Laplacian filtered images of the stones were quantitatively analyzed using operator-independent methods. A post hoc classification method for pure UA stones was created using a combination of cutoff values for the peak attenuation and peak point Laplacian. The stone types differed in most quantitative image characteristics including mean attenuation ( Urinary stone disease is a common urological disease with a life time risk of approximately 10–15% , 3.The role of NECT is not only to diagnose the urinary stone but also to provide information that guides the patient and the referring clinician in management of the disease. The size and location of a ureteral stone predict the chance of spontaneous passage. The stone characteristics of a renal stone are correlated to stone type and prognosis for treatment success , 4–8.The most common urinary stone types are calcium-based (Ca) stones followed by uric acid stones (UA), and infection-related struvite stones. UA stone composition is of particular interest to predict in intra-renal stones, since these stones can be treated through alkalinization of the urine , 9.During the last decade, several studies have investigated the classification of UA stones vs non-UA stones using dual-energy CT (DECT) –16. HoweAlthough it is widely known that the attenuation value of a urinary stone in NECT correlates closely to the stone type, there is an overlap in reported attenuation values, especially for small stones , 16–19. The purpose of the present study was, therefore, to describe single-energy thin slice quantitative CT parameters of urinary stones correlated to chemical stone type and to develop a method to distinguish pure UA stones from non-UA stones.In 108 patients  with single-energy NECT examinations performed between January 2012 and January 2016, 110 stones with known compositions were retrospectively included in the study. The inclusion was based on the register of urinary stones analyzed with infrared (IR) spectroscopy at the institutional laboratory between January 2012 and January 2016. Stones were included according to Fig. Because of the low number of UA stones included according to Fig. The chemical stone composition was determined using Fourier transform IR spectroscopy . The stones were classified as UA, or non-uric acid/mixed (non-UA/Mix) according to Table The DECT reference scan of the additional UA stones was acquired with a 2 × 128 channel dual-source CT scanner . The dual-energy datasets were 100 kV/Sn140 kV, except in one stone where 80 kV/Sn140 kV was used. Color coded images were created using manufacturer default settings of the Kidney stone application in Syngo.Via (14 stones) and in Multi Modality Workplace (2 stones), . A stone was considered as a pure UA stone if ≤1 mm DECT high- and low-energy datasets were available and the color coded ≤1 mm thick images were entirely or almost entirely color coded as UA stone. The determination of stone type with DECT was performed before quantitative analysis of the single-energy images.n = 33), 40 channels, filter B, CTDI 4.9 mGy ; Siemens Somatom Definition AS, (n = 15), 64 channels, filter B20f/I30f2, CTDI 5,7 mGy; Siemens Somatom Definition Flash (n = 78), filter B20f/I30f3, CTDI 4.7 mGy, except one patient, CTDI 6.1 mGy.Only 1 mm contiguous axial images performed with the institutional intermediate dose stone NECT protocol (field of view (FoV) 420 mm, in plane pixel size ~0.8 × 0.8 mm, 120 kVp, 70 mAs/slice) were included. The images were acquired with three different CT systems: Philips Brillance,  and the background, which was approximated to 0 HU. Any hole in the thresholded image mask was filled and considered to be part of the stone.For each segmented stone, histogram statistics were computed (mean attenuation (meanHU), standard deviation (sdHU), kurtosis, and skewness). The stone images were filtered using a scaled 3D Laplacian filter see “” for texThe voxel location corresponding to maxHU was defined as the peak point. The peak point Laplacian (ppLapl) was defined as the voxel in the Laplacian filtered image corresponding to the peak point.The sphericity and volume of the stone were evaluated using an alpha shape that encompassed all segmented voxel locations see “”.The largest diameter of each stone in the axial slice plane was measured with the caliper tool in the PACS workstation using a bone window setting L300/W1120) [ [20].No predefined hypothesis could be set up prior to image analysis because of the limited availability of quantitative CT analysis data in the literature. The study, therefore, consisted of data exploration for discovering the best combination of quantitative image parameters for stone type prediction. Shapiro–Wilks test showed non-normal distribution for a majority of parameters. Consequently, non-parametric Wilcoxon signed-rank tests between the UA group and the non-UA/Mix group were used. The Spearman correlation coefficient versus maxHU was computed for each parameter.A post hoc classification method was created using a combination of cutoff values for maxHU and ppLapl. The sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV) for the peak attenuation—peak point Laplacian method were computed with 95% confidence intervals (CI).Image analysis and statistics were computed using Matlab R2015b . The Regional Research Ethics Board approved the study protocol and waived the informed consent requirement.One stone had ambiguous references and was excluded from the analysis according to Fig. The manually estimated largest diameter ± SD was 6.0 ± 2.4 mm and 5.1 ± 2.9 mm for UA and non-UA/Mix stones, respectively.The quantitative parameters are detailed in Table The histogram-based statistical properties, kurtosis, and skewness showed moderate correlation with maxHU, while the meanLapl showed low correlation with peak attenuation. The ppLapl was not correlated to maxHU.Even though the median results were significantly different between the stone types for almost every parameter according to Wilcoxon signed rank test, no single parameter could completely distinguish pure UA stones from the other stone types. The stone attenuation is, as previously known, the major predictor differentiating pure UA stones from non-UA/Mix stones , 17–19. x-axis were created with volume, meanLapl, kurtosis, skewness, sphericity and ppLapl on the y-axis, respectively. The scatterplots were visually evaluated for optimal clustering effects of UA stones vs the non-UA/Mix stones. The visually best discriminant was the combination of peak attenuation and peak point Laplacian, see Fig. Separate scatterplots with maxHU on the Optimal cutoff values for classification of pure UA stones were created based on visual examination of Fig. With an alternative limit for UA stones (maxHU ≤ 800 HU and ppLapl ≤ 190 HU), the sensitivity was 91% , the specificity was 99%  and the accuracy was 97.6% .The optimal cutoff for maxHU as single parameter was 745 HU. With this cutoff the accuracy for classification of pure UA stones was 96.8% .The role of NECT in urinary stone disease is not only to provide the diagnosis but also to provide information that guides the patient and the clinician in the management of the stone , 3. In tIn almost every analyzed quantitative single-energy parameter, there was a significant difference related to stone type. As previously demonstrated, the stone attenuation is the major predictor of stone type , 17–19.Although the difference in accuracy is small compared to stone type classification based on maxHU alone, a peak attenuation—peak point Laplacian method is suggested based on the clustering demonstrated in Fig. In vivo stone classification using DECT has during the last decade become well established and studies have shown that DECT stone classification can be performed even with low to intermediate dose protocols , 15. HowMaxHU and ppLapl are point estimates that have an important advantage, since they are independent of the segmentation parameters. In contrast, the segmentation-based parameters depend not only on the stone but also on the delineation between the urinary stone and the background, which can be achieved differently with or without human interaction –25.In digital image processing, the Laplacian is a standard image filter which primarily is used for edge enhancement . In the The difference in ppLapl between UA and Ca stones with low maxHU has a logical interpretation. Low attenuating Ca stones are smaller than UA stones with similar maxHU, leading to a larger peakedness for the Ca stones, quantified by ppLapl. Larger Ca stones show a wide high density peak, resulting in high maxHU and low ppLapl, see Fig. Several factors contribute to the high accuracy of NECT classification in the present study compared to previous studies. The automated analysis of the stones without the need for freehand ROIs, the consistent use of thin slice images and the use of point estimates that are independent of segmentation parameters are the strengths in the study. Other contributing factors are related to the limitations of the study—the distribution of stone types that was found in the included cohort and the post hoc-defined cutoffs.Although the number of stones in the present study is larger than in many DECT studies , 14–16, The most important limitation in the present study is that there was no sufficient pre-study quantitative data for setting up a test hypothesis before the study. The sensitivity and specificity is, therefore, likely to be overestimated since the suggested limits were optimized for the existing data.Struvite stones can often be identified based on morphology  and in tQuantitative image analysis is affected by image parameters including kVp, FoV, slice thickness, and reconstruction algorithm . Even thWith the limitations of the study in mind, it can be concluded that quantitative image analysis of thin slice single-energy NECT images is promising for predicting pure UA content in urinary stones, with results comparable to DECT. However, further prospective evaluation of the proposed peak attenuation—peak point Laplacian method is needed.


Mice treated with NaIO3 showed severe RPE and photoreceptor cell death/necroptosis, destruction, oxidative damage, retinal stress, and reduced retinal function. Treatment with anti-thyroid drug protected RPE and photoreceptors from damage/cell death induced by NaIO3, reduced oxidative damage of RPE and photoreceptors, and preserved retinal function. Gene expression analysis showed that the NaIO3-induced RPE/photoreceptor damage/cell death involves multiple mechanisms, including cellular oxidative stress responses, activation of necroptosis/apoptosis signaling, and inflammatory responses. Treatment with anti-thyroid drug abolished these cellular stress/death responses. The findings of this study demonstrate a role of TH signaling in RPE and photoreceptor cell death after oxidative stress challenge, and support a role of TH signaling in the pathogenesis of AMD.Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Dry AMD is characterized by a progressive macular degeneration of the retinal pigment epithelium (RPE) and photoreceptors, and the RPE oxidative damage/dystrophy is at the core of the disease. Recent population/patients-based studies have shown an association of high free serum thyroid hormone (TH) levels with increased risk of AMD. This work investigated the effects of TH signaling inhibition on RPE and photoreceptor damage/cell death in an oxidative stress-induced mouse model of AMD. TH signaling inhibition was achieved by anti-thyroid drug treatment and oxidative stress was induced by sodium iodate (NaIO There are two types of AMD, the dry and wet forms. Dry AMD, also known as geographic atrophy, is a form of slowly progressing geographic atrophy of the macula, and comprises a majority of AMD cases (∼90%), whereas wet AMD rapidly progresses to blindness and involves the abnormal formation of blood vessels in the macula. Dry AMD is characterized by a progressive macular degeneration of the retinal pigment epithelial (RPE) cells and photoreceptors, lipofuscin (A2E) accumulation, and drusen formation. It is generally recognized that multiple factors, including aging, oxidative stress, chronic inflammation, and genetic defects, are involved in the RPE and photoreceptor dystrophies/AMD lesions. However, oxidative stress/damage to the RPE has been recognized as the core pathogenic lesion of the disease4.Age-related macular degeneration (AMD) is the leading cause of blindness in elderly, exhibiting complex interplay of genetic and environmental factors6 and cone photoreceptor viability10. Recently, TH signaling has been implicated in the pathogenesis of AMD. The prospective population-based studies showed that higher free serum TH values were associated with increased risk of AMD13. The patient population-based study also showed a high association between thyroidopathy and AMD15. These findings suggest an association of TH signaling with AMD. Indeed TH signaling has been linked to other types of neurodegenerative conditions, including Alzheimer’s disease17. The present work investigated the effects of TH signaling inhibition on RPE and photoreceptor damage/cell death in a mouse model of AMD induced by sodium iodate (NaIO3). We found that treatment with anti-thyroid drug protected RPE and photoreceptors from oxidative damage and cell death/necroptosis induced by NaIO3 and preserved retinal function. Moreover, treatment with anti-thyroid drug abolished NaIO3-induced upregulation of the genes involved in cellular stress responses, inflammatory responses, and cell death signaling. The findings of this study demonstrate a role of TH signaling in RPE and photoreceptor cell death induced by oxidative stress challenge and support a role of TH signaling in the development and progression of AMD.Thyroid hormone (TH) signaling regulates numerous physiological functions, including cell growth, differentiation, and metabolic homeostasis. In the eye, TH signaling regulates cone opsin expression3 induces RPE and photoreceptor oxidative damage/cell death, mimicking the feature and progression of AMD20. This model has been commonly used to study RPE and photoreceptor oxidative damage in AMD. To determine the role of TH signaling in oxidative stress-induced damage and cell death, we examined the effects of anti-thyroid treatment in mice that have been treated with NaIO3. C57BL/6J mice received anti-thyroid treatment via drinking water (1% sodium perchlorate monohydrate and 0.05% methomazole), beginning at postnatal day 20 (P20), received a single injection of NaIO3  at P30, and were then analyzed for RPE and photoreceptor damage/cell death at 3 days post-NaIO3 injection. ELISA analysis showed that the serum triiodothyronine (T3) level in anti-thyroid-treated mice were reduced by about 70%, compared with untreated controls  and outer segment (OS) areas, reduced numbers/thickness of the ONL, and shortened OS  staining has previously indicated NaIOing Fig. . Treatmeion Fig. . Retinaling Fig. .Fig. 3Tr3 challenge, as described above, and were analyzed for RPE and photoreceptor oxidative damage at 3 days post-NaIO3 injection. RPE oxidative damage were assessed by immunofluorescence labeling of the DNA double strand break/damage markers p-γH2AX and 8-OHdG on the RPE whole mounts22. Mice that have been treated with NaIO3 showed significantly increased labeling of p-γH2AX, compared with untreated controls  in intermediate filaments. In this study, we examined the effects of anti-thyroid treatment on Müller glia activation. Mice received anti-thyroid treatment and NaIOion Fig. .Fig. 5Tr3 challenge, as described above, and were analyzed for retinal function by electroretinogram (ERG) recordings at 3 days post-NaIO3 injection. NaIO3 treatment reduced scotopic a- and b-wave amplitudes by about 55 and 44%, respectively, compared with untreated controls, and treatment with anti-thyroid drug significantly preserved scotopic b-wave but not a-wave responses  selectively induces RPE oxidative damage in experimental animals in a concentration-dependent manner, with more severe damage in the central and middle regions of the RPE25. Photoreceptors degenerate as results of RPE cell loss and the drug’s direct action. NaIO3 challenge has been used in various animal models, including mouse28, rat30, rabbit31, sheep32, cat33, and swine35, to study RPE/photoreceptor oxidative damage in AMD, and evaluate the novel therapeutic interventions. In the present study, we used this model to investigate the effects of TH signaling inhibition. As expected, a single injection of NaIO3 caused severe damage of RPE and photoreceptors, activated multiple cellular stress/death pathways, and impaired retinal function. Treatment with anti-thyroid drug nearly completely preserved RPE and photoreceptors from damage/cell death, reversed gene expression alterations, and partially preserved retinal function. These findings demonstrate a role of TH signaling in RPE and photoreceptor cell damage/death induced by NaIO3/oxidative stress. The observed effects from anti-thyroid treatment likely resulted from the reduced TH levels in the circulation  mechanisms40. Inhibitors of caspases and necroptotic signaling pathways inhibit RPE and photoreceptor cell death induced by NaIO3 in vivo and in vitro39. Consistent with previous reports, this study shows that a single injection of NaIO3 induced upregulation of the genes involved in oxidative stress responses, inflammatory responses, and cellular necroptotic/apoptotic signaling. The local inflammatory responses/innate immune responses were also shown by activation of Müller glia/up-regulation of GFAP. There are a few interesting findings in the gene expression alterations: the apoptotic genes were similarly upregulated in the RPE and retinas; more oxidative stress response genes were upregulated in the RPE, relative to that in the retina; and more significant elevation of the necroptosis genes and inflammatory genes was observed in the retina, relative to that in the RPE. These observations support the view that the oxidative stress responses are the predominant reactions in the RPE whereas the necroptosis/inflammatory responses are the predominant reactions in the retinas. Nevertheless, treatment with anti-thyroid drug effectively suppressed expression of these genes in both RPE and retinas and abolished Müller cell activation. Thus, TH signaling inhibition-induced RPE and photoreceptor protection was likely achieved via multiple mechanisms, including suppression of oxidative stress responses, cell death signaling activity, and inflammatory responses. The question of how inhibition of TH signaling leads to suppression of these different cellular stress responses/death activities remains to be addressed. Because TH signaling plays a pivotal role in mitochondrial metabolism/homeostasis and reactive oxygen species production, one would expect that the anti-oxidative stress effects/protection of mitochondrial homeostasis might be at the core of the TH signaling inhibition-induced protection. Investigations on the TH regulation of RPE and photoreceptors with a focus on the mitochondrial homeostasis/stress might be particularly significant.Although oxidative stress/damage serves as an initiating factor, NaIOAMD is a multifactorial disorder, involving apoptosis/necroptosis of both RPE and photoreceptors, triggered by oxidative stress and worsened by inflammatory responses. This work shows that TH signaling inhibition protected RPE and photoreceptor cells from oxidative damage/cell death in an oxidative stress mouse model of AMD, accompanied by suppression of the upregulation of the genes involved in oxidative stress and inflammatory responses. Results from this animal model study are in line with the clinical findings showing a correlation of high free serum TH levels with increased risk of AMD, and support a role of TH signaling in the pathogenesis of AMD. Because both RPE and photoreceptors are involved in the disease pathogenesis and are protected by anti-thyroid treatment, inhibition of TH signaling may provide dual benefits in the management of AMD.3-induced mouse model of AMD. We show that the anti-thyroid treatment reduced RPE/photoreceptor oxidative damage/cell death, protected retinal function, and suppressed upregulation of the genes involved in cellular oxidative stress responses, cell death pathways, and inflammatory responses. The results of this study demonstrate a role of TH signaling in the RPE/photoreceptor cell death induced by oxidative stress challenge, and support a role of TH signaling in the pathogenesis of AMD. Further investigation on the regulation of TH signaling in the RPE and photoreceptor survival will help understand how suppression of TH signaling leads to protection and whether targeting TH signaling has therapeutic significance for AMD.In summary, this work investigated the effects of TH signaling inhibition on RPE/photoreceptor cell death and retinal function in an NaIOC57BL/6J mice were obtained from the Jackson Laboratory and used in this study. Mice were maintained under cyclic light (12-h light–dark) conditions. Cage illumination was 7-foot-candle during the light cycle. All animal maintenance and experiments were approved by the local Institutional Animal Care and Use Committee  and conformed to the guidelines on the care and use of animals adopted by the Society for Neuroscience and the Association for Research in Vision and Ophthalmology. Mice of either sex were used in the experiments. Mice were randomly assigned, within a litter, for the drug treatment or vehicle/untreated experiments; littermate controls were used whenever possible; and no animals were excluded from the analysis. No blinding was carried out for animal experiments.® 594 phalloidin  and Alexa Fluor® 488 donkey anti-rabbit IgG  were purchased from Life Technologies; DAPI , NaIO3 , and PI  were purchased from Millipore Sigma; biotinylated PNA  was purchased from Vector Labs; GFAP antibody  was purchased from DAKO; p-γH2AX antibody  was purchased from Novus Biologicals; and 8-OHdG (E8) antibody  was purchased from Santa Cruz Biotechnology, Inc.Alexa Fluor3 injection were performed as described previously37. Briefly, mice received anti-thyroid treatment via drinking water (1% sodium perchlorate monohydrate and 0.05% methomazole), beginning at P20, and received a single injection of NaIO3  at P30. These mice were then analyzed for RPE and photoreceptor damage/cell death and retinal function at 3 days post-NaIO3 injection, and for gene expression alterations at 1 day post-NaIO3 injection.Anti-thyroid treatment and NaIO8. Briefly, 25 μL of serum samples and standards with different T3 concentrations were added into the assigned wells, the assays were performed by following the manufacturer’s instruction, and the absorbance of each well was read at 450 nm . The standard curve was generated by using a three-parameter exponential nonlinear regression in Sigma-Plot software, and the sample T3 concentration was then calculated according to the three-parameter exponential equation.Serum T3 levels were analyzed using a mouse/rat T3 ELISA kit  with a total T3 detection limit at 0.25 ng/mL, as described previously® 594 phalloidin (1:40) for 30–45 min at room temperature and DAPI (1 ng/mL) for another 30 min at room temperature, followed by wash . The RPE whole mounts were made by transferring the sheets onto the slides, followed by mounting with Hard medium .The RPE whole mounts were prepared for immunofluorescence labeling. Briefly, eyes were enucleated and fixed in 4% paraformaldehyde  for 1 h at room temperature, followed by removal of the cornea, lens, muscles, and retina. The RPE sheets (the sclera-choroid-RPE sheets) were then fixed in 4% PFA for another 1 hour at room temperature, followed by wash  and blocking with 10% FBS in 0.5% Triton X-100 in PBS for 1 hour at room temperature. The RPE sheets were then stained with Alexa Fluor8. For retinal whole mount preparations, eyes were enucleated, marked at the superior pole with a green dye, and fixed in 4% PFA for 30 min at room temperature, followed by removal of the cornea and lens. The eyes were then fixed in 4% PFA in PBS for 4–6 h at room temperature, and retinas were isolated and the superior portion was marked for orientation with a small cut. For retinal cross sections, eyes were enucleated (the superior portion of the cornea was marked with green dye prior to enucleation) and fixed in Prefer (Anatech Ltd.) for 25–30 min at room temperature. Paraffin sections (5-µm thickness) passing vertically through the retina  were prepared using a Leica microtome (Leica Biosystems). Immunofluorescence labeling was performed as described previously8. Briefly, retinal whole mounts or sections were blocked with Hanks’ balanced salt solution containing 5% BSA and 0.5% Triton X-100 for 1 h at room temperature or overnight at 4 °C. Prior to blocking, antigen retrieval was performed in 10 mM sodium citrate buffer (pH 6.0) for 30 min in a 70 °C water bath. Primary antibody incubation  was performed at room temperature for 2 h, followed by incubation with AlexaFluor-488 or −568, or FITC-conjugated secondary antibody. PNA immunohistochemistry was performed using biotinylated PNA (1:250) and streptavidin-Cy3 (1:500).The retinal whole mounts and cross sections were prepared for immunofluorescence labeling, as described previouslyhttps://imagej.net/) was used to analyze the damaged area on the RPE whole mounts. For quantification of RPE cell numbers and RPE nuclear numbers, images from four quadrants in the central, middle and peripheral regions were counted and normalized to the number in one square millimeter. Evaluation of cone density on retinal whole mounts was performed as described previously41. For retinal morphometric analysis, retinal cross sections stained with hematoxylin and eosin (H&E) were used for morphometric analysis to evaluate ONL integrity/rod survival, as described previously42.Low magnification images were taken under the Olympus MVX10 dissection microscope equipped with Image-Pro 6.3 software  and high magnification images were taken with an 60X objective on the FV1000 confocal laser scanning microscope equipped with FluoView imaging software . ImageJ software (18. PI (0.5 μg) in 50 μl PBS was delivered through retro-orbital injection at 15 min before sacrificing the mice. The enucleated eye globes were fixed in 4% PFA for 1 hour, anterior part was removed, and the sclera-RPE was fixed in 4% PFA for one additional hour, followed by DAPI staining and fluorescence microscopy.PI staining of RPE whole mounts was performed as described previously43. Immunofluorescence labeling was imaged using an Olympus FV1000 confocal laser-scanning microscope, and TUNEL-positive cells in the outer nuclear layer passing through the optic nerve were counted and averaged from at least 3 sections per eye from 3–5 mice per condition. TUNEL was also performed on RPE whole mounts. Briefly, eyes were fixed in 4% PFA for 1 hour. The sclera-RPE were then dissected and fixed in 4% PFA for one additional hour. After antigen retrieval performed in 10 mM sodium citrate buffer (pH 6.0) for 30 min at 70 °C, the sclera-RPE were permeabilized in 1% Triton X-100 in 10% FBS for 2 h at room temperature, followed by labeling using the in situ cell death fluorescein detection kit.Terminal deoxynucleotidyltransferase dUTP nick-end labeling (TUNEL) was performed on paraffin-embedded retinal sections, using an in situ cell death fluorescein detection kit , as described previously44. Briefly, after overnight dark adaptation, animals were anesthetized by intraperitoneal injection of 85 mg/kg ketamine and 14 mg/kg xylazine. ERGs were recorded using an LKC system . Potentials were recorded using a platinum wire contacting the corneal surface through a layer of 2.5% methylcellulose. For assessment of scotopic responses, a stimulus intensity of 1.89 log cd s m−2 was presented to dark-adapted dilated mouse eyes in a Ganzfeld . To evaluate photopic responses, mice were adapted to a 1.46 log cd s m−2 light for 5 min, then a light intensity of 1.89 log cd s m−2 was administered. Responses were differentially amplified, averaged, and stored using a Nicolet Compact-4® signal averaging system.Full-field ERG testing was carried out as described previously45. Total RNA preparation and reverse transcription were performed as described previously46. The gene encoding the mouse hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1) was included as an internal control. Supplemental Table 46.The mouse RPE cells were isolated as describedt-test at a 0.05 alpha level. One-way ANOVA was used to analyze for significance within sets of data, and two-tailed Student’s t-test was used for differences between two groups of data. Differences were considered statistically significant when P < 0.05. Statistical tests for every figure are justified as appropriate. Data were analyzed and graphed using GraphPad Prism® software .Results are expressed as means ± SEM of number of mice. Power analysis was performed to choose the sample size. The analysis indicates that a sample size of 3–6 mice/group for evaluations of retinal degeneration in the mouse retinas will provide at least 80% power (1-β) for a two-sided, two-sample Supplementary InformationSupplementary Table 1Supplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4


Oxidative post-translational modifications (oxPTM) of receptors, enzymes, ion channels and transcription factors play an important role in cell signaling. oxPTMs are a key way in which oxidative stress can influence cell behavior during diverse pathological settings such as cardiovascular diseases (CVD), cancer, neurodegeneration and inflammatory response. In addition, changes in oxPTM are likely to be ways in which low level reactive oxygen and nitrogen species (RONS) may contribute to redox signaling, exerting changes in physiological responses including angiogenesis, cardiac remodeling and embryogenesis. Among oxPTM, S-glutathionylation of reactive cysteines emerges as an important regulator of vascular homeostasis by modulating endothelial cell (EC) responses to their local redox environment. This review summarizes the latest findings of S-glutathionylated proteins in major EC pathways, and the functional consequences on vascular pathophysiology. This review highlights the diversity of molecules affected by S-glutathionylation, and the complex consequences on EC function, thereby demonstrating an intricate dual role of RONS-induced S-glutathionylation in maintaining vascular homeostasis and participating in various pathological processes. Covering vascular lumen walls, endothelial cells (ECs) are a central component building the interface between blood and underlying tissue. ECs act on multiple processes to ensure the maintenance of systemic vascular homeostasis, including the regulation of blood pressure, haemostasis, tissue vascularization and inflammation. Endothelial dysfunction is thus involved in a wide range of pathologies, especially cardiovascular diseases (CVDs) such as diabetes, hypertension and peripheral arterial disease. Importantly, the direct contact of ECs with blood circulation makes them highly sensitive to local oxygen levels. The role of oxidative stress and high levels of reactive oxygen and nitrogen species (RONS), resulting from ischemia/reperfusion, is now widely recognized in the onset of CVDs ,2. RONS The harmful effects of oxPTM have been widely related to pathological processes. However, attempts to develop antioxidant therapies to reduce oxidative damages and resolve CVDs have not met the success expected . MoreoveIn this context, this review will summarize the latest insights on the role of glutathionylated proteins in the regulation of major EC pathways, and their implications in the main physiological and pathological processes linked with vascular homeostasis.After translation, proteins undergo various modifications including palmitoylation, phosphorylation or hydroxylation, which alter protein function through changes in cellular location, activity or stability. Cysteinyl residues are especially sensitive to levels of oxygen and nitrogen species (RONS), resulting in a range of oxidative post-translational modifications (PTM) including S-nitrosylation, S-sulfenylation and S-glutathionylation.Under physiological RONS levels, the antioxidant molecule glutathione (GSH) can bind to specific reactive cysteinyl residues via the creation of reversible disulfide bonds depending on the cysteine position and redox potential. This process of S-glutathionylation can be reversed by thiol modifying enzymes, predominantly glutaredoxin (Grx), and appears as a protective mechanism against permanent protein damage following irreversible cysteine oxidation, summarized in The different molecular mechanisms involved in S-glutathionylation PTM have been extensively reviewed ,9. BeyonEndothelial cells (ECs) are a specialized tissue lining the lumen of all blood vessels. At the interface between circulating blood and underlying tissues, ECs have multiple functions  includinThe importance of epigenetics in EC function and related complex diseases is meeting a growing interest, highlighted by the recent emergence of whole genome studies. The modification of histone methylation state plays a key role in EC function by acting upstream of gene transcription, and its implications in cardiovascular pathologies . ImportaIn eukaryotes, the chromatin is packaged in individual units called nucleosomes. Histones, the building bricks of nucleosomes, are subject to various PTM which directly impact the chromatin structure and, as a result, gene expression . AlthougThe effect of S-glutathionylation on the modulation of chromatin structure can be appreciated indirectly, via the modification of histone-modifying enzymes. Particularly, RONS are known to decrease histone deacetylase (HDAC) activity, notably via nitrosylation, favoring gene transcription ,87. HoweS-glutathionylation regulates various transcription factors in ECs, most of the time by inhibiting their activity and, therefore, silencing the expression of downstream targets a. IntereNFκB transcription factor complex is mostly known for its pro-inflammatory properties, characterized in ECs by a promotion of vascular permeability and leukocyte recruitment . BesidesS-glutathionylation regulates various components of the NFκB pathway. NFκB major subunits p50 and p65 activity are inhibited by S-glutathionylation, preventing DNA-binding or nuclear translocation and downstream gene transcription ,19. S-glAs a component of the AP-1 transcription factor complex, c-Jun protein promotes cell proliferation and angiogenesis in ECs. However, its precise roles in cell survival are still controversial. The signaling cascade of c-Jun is known to be susceptible to redox regulation ,99,100. p53 is gatekeeper of a wide range of cellular pathways, mostly known for its antitumorigenic properties preventing abnormal cells proliferating. However, the role of p53 in non-cancerous cells such as ECs has recently attracted interest due to its crucial implications in angiogenesis, apoptosis and vascular dysfunction . p53 S-gNuclear factor (erythroid-derived 2)-like 2 (Nrf2), a basic redox-regulated leucine zipper protein, is another major transcriptional factor that plays fundamental roles in EC functions including essential anti-angiogenic activity. In response to oxidative stress, Nrf2 mediates antioxidant and anti-inflammatory signals providing crucial cytoprotective effects in many cells types including the endotheliuma 102]. . 102]. THypoxia-inducible factor 1-alpha (HIF-1α) is the major regulator of oxygen homeostasis in ECs, and promotes angiogenic signaling in response to hypoxia . AlteratThe JAK/STAT signaling pathway is activated in response to cytokines and growth factors and stimulates a wide range of physiological processes including inflammation, apoptosis, differentiation, cell migration and proliferation. In this pathway, Signal Transducer and Activator of Transcription (STAT) family proteins are intracellular transcription factors phosphorylated by JACK, and translocated to the nucleus to promote gene expression a 106]. . 106]. TS-glutathionylation of STAT3 prevents phosphorylation inhibiting DNA-binding capacities , and a mProtein phosphorylation is a pivotal post-translational modification regulating cell signaling pathways. Phosphatase and kinase enzymes regulate the phosphorylation state of major proteins in EC signaling as receptors and downstream targets a. While Protein tyrosine phosphatases (PTP) down-regulate key receptors by dephosphorylating kinase domains.-, highlighting its role in a negative feedback loop central to VEGF signaling.Low-molecular-weight protein tyrosine phosphatase (LMW-PTP) family proteins inhibit growth factors regulating cell growth and angiogenesis, but also inhibit focal adhesion kinase regulating cell migration. LMW-PTP, characterized by the presence of two reactive cysteines located in their catalytic domain, are widely recognized as a redox-dependent “molecular switche” in many cell types ,116,117.PTP1B is a major regulator of EC proliferation through its binding and inactivation of the VEGFR2 receptor. Its phosphatase activity on tyrosine domains also applies to the VE-cadherin receptor, stabilizing cell–cell adhesion and, therefore, limiting vascular permeability . S-glutaGTPases, hydrolyze GTP to provide the energy required for central physiological processes, play an important role in the regulation of vascular permeability and RONS production. Defects in vascular barrier function are a hallmark of vascular dysfunction, promoting leukocyte transmigration and chronic inflammation, and have been associated with various severe pathologies such as diabetes ,122 or pSmall Rho GTPases act in a complex interplay to modulate the dynamics of the actin cytoskeleton, a central feature for EC barrier function and migration ,125. GTPBeyond the regulation of vascular barrier integrity, further understanding of Rho GTPase redox regulation could provide important insights on EC response to oxidative stress through NADPH oxidase and eNOS activation ,125,126 Kinase proteins are now widely recognized as redox-regulated, and more specifically subject to inhibition by S-glutathionylation due to the presence of a reactive cysteine residue in their catalytic domain . It is nAlthough precise biological implications of protein kinase redox regulation have not been extensively explored in ECs, the results showing that their enzymatic activities are importantly altered by S-glutathionylation could have crucial impacts for EC physiology. Indeed, the role of various kinases in key EC functions, especially the regulation of vascular barrier function and blood pressure, is well established ,129.2−) and hydrogen peroxide from NOX regulates multiple redox-dependent pathways, especially those involved in the regulation of vascular tone and angiogenesis through NO inhibition i), tightly regulated by calcium-dependent channels located at the surface of the endoplasmic reticulum (ER), mitochondria and plasma membrane  conreported ,144, sug2+ ATPase (PMCA) by S-glutathionylation have an effect on Ca2+ oscillatory patterns and [Ca2+]i i . Interes cytosol . This la cytosol . Despite cytosol , whether2+ uptake in ER stores in ECs and SMC, directly regulating downstream processes involved in the maintenance of vascular homeostasis . . 163]. IIntegrins are the major points of contact between ECs and their extracellular microenvironment, driving cell migration, proliferation and regulating vessel permeability. In addition to promoting cell adhesion and migration within the ECM, integrins act as receptors to trigger specific proangiogenic signaling pathways a 169]. . 169]. IVascular cell adhesion protein 1 and Intercellular Adhesion Molecule 1 (VCAM-1 and ICAM-1) are mainly expressed in ECs in response to pro-inflammatory cytokines and RONS. Adhesion proteins play a pivotal role in EC activation and binding to leukocytes, mediating leukocyte transmigration during inflammation and are, therefore, involved in inflammatory diseases such as rheumatoid arthritis ,174. GroCytoskeletal dynamics, a key feature driving vascular angiogenesis and barrier function, are governed by polymerization and depolymerization of the two main component units, actin and tubulin. Both proteins are highly redox-sensitive and S-glutathionylation is now recognized to have a major impact on the regulation of polymerization-depolymerization cycles in various cell types ,136,176.S-glutathionylation has emerged as a novel redox switch altering the functions of EC through the modulation of key signaling pathways. Its importance and complexity in EC homeostasis and cardiovascular physiopathology is highlighted by recent insights on how S-glutathionylated protein functions are altered in interlinked molecular pathways at multiple levels .It is now apparent that S-glutathionylation can coordinate gene transcription by modulating epigenetics and transcription factors. Studies investigating the impact of S-glutathionylation in epigenetics are still at their early stage, and further work is required to fully understand how it alters gene expression. However, current insights demonstrate that both histone proteins and histone-modifying enzymes are prone to S-glutathionylation, altering chromatin structure and gene expression. Given the number of various genes regulated by those processes, novel insights on the links between oxidative stress and epigenetics would allow a better understanding of EC signaling in response to RONS, and highlight novel factors involved in complex diseases for which genetic factors alone are not sufficient to explain. The effect of S-glutathionylation on transcription factors suggest differential effects on their activity depending on the protein targeted. Accordingly, it is difficult to define a general effect of this modification on EC physiology. However, the redox regulation of VEGF signaling appears central to the activation of downstream transcription pathways and the alteration of resulting EC functions.S-glutathionylation exhibits a general inhibitory effect on enzymes by altering the structure of their catalytic site and impairing their activity. This has important repercussions when applied to phosphatases, GTPases and kinases, which are key signal transducers in ECs. The multiplicity of pathways regulated by enzymatic activity underlines the crucial importance of understanding the redox mechanisms dictating their behavior to better understand EC responses to oxidative stress.As upstream mediators of S-glutathionylation, RONS themselves are subject to redox regulation. The two main RONS sources in EC, NOX and eNOS, are under tight redox control. The different effects of S-glutathionylation on RONS-producing systems have a general tendency to promote RONS production. This suggests a retroactive system, where RONS sustain their own production, which could importantly participate in oxidative stress-mediated EC dysfunction.2+ homeostasis in EC, and that a tight regulation of RONS levels is required to ensure physiological Ca2+ levels in cellular stores allowing proper EC functions.In respect to calcium signaling, S-glutathionylation tends to favor ion entry into the cell cytosol through reversible alteration of various player functions, except for the SERCA channel. Those results confirm that this modification plays a central role in CaThe opposing effects of S-glutathionylation on different apoptotic factors may explain the complex and diverse effect of oxidative stress on EC apoptosis.The role of S-glutathionylation in EC autophagy is less studied, and we expect future work will target this pathway.Beyond signaling molecules, the influence of S-glutathionylation on cell structure components is another example of the duality of S-glutathionylation effects. While this modification tends to lower cell growth and motility by inhibiting cytoskeleton polymerization as well as surface adhesion molecules, the positive effects observed on metalloprotease proteins favor cell migration within extracellular tissues. S-glutathionylation induced degradation of adhesion molecules might also play an important role in the regulation of leukocyte transmigration and inflammation.All modifications depicted in this review are integrated into a diagram to reflect the complexity of S-glutathionylation redox switch in the modulation of EC signaling pathways . AltogetHowever, the difficulty lies in finding a consistent effect of S-glutathionylation and appreciating the complex mechanism of RONS/antioxidant balance during physiology and pathophysiology. The importance of basal redox signaling in normal physiology could provide a better understanding of why antioxidant therapies failed to meet expectations. Greater appreciation of redox signaling in physiology is of upmost importance since antioxidant treatments in clinical trials have caused unexpected adverse effects. Studies on S-glutathionylation are still at their relatively early stage compared to other PTMs. Another aspect which requires further investigation to appreciate the implications in cardiovascular pathophysiology is how S-glutathionylation interrelates with other oxPTM. A better understanding of the S-glutathionylation process in EC could illuminate the complicated relations linking oxidative stress/redox signaling with CVDs and may also be relevant for other pathologies such as cancer and inflammatory diseases.


Mechanistic target of rapamycin (mTOR) is a master signaling pathway that regulates organismal growth and homeostasis, because of its implication in protein and lipid synthesis, and in the control of the cell cycle and the cellular metabolism. Moreover, it is necessary in cerebellar development and stem cell pluripotency maintenance. Its deregulation has been implicated in the medulloblastoma and in medulloblastoma stem cells (MBSCs). Medulloblastoma is the most common malignant solid tumor in childhood. The current therapies have improved the overall survival but they carry serious side effects, such as permanent neurological sequelae and disability. Recent studies have given rise to a new molecular classification of the subgroups of medulloblastoma, specifying 12 different subtypes containing novel potential therapeutic targets. In this review we propose the targeting of mTOR, in combination with current therapies, as a promising novel therapeutic approach. The mechanistic  target of rapamycin (mTOR) is a master signaling pathway that regulates organismal growth and homeostasis. This pathway is not only implicated in physiological conditions but it is also central in several pathological conditions . mTOR isOne of the most important downstream effects of mTORC1 is the upregulation of protein synthesis. It phosphorylates several translation regulators, including eukaryotic translation initiation factor 4E (eIF4E)-binding proteins  . The phoIt is more and more obvious that the mTOR signaling pathway has an important role in essential cellular functions. The key role of mTOR in controlling cell proliferation has raised the interest in rapamycin for cancer therapy. This drug disrupts mTORC1 protein complex formation, whereas mTORC2 is quite insensitive to the drug, and long-term treatments are required in order to inhibit its assembly  Figure .mTOR plays an important role in the development of an organism because of its implication in the growth, proliferation, and migration of every cell during normal brain development . AccordiNowadays, it is known that the deregulation of mTOR signaling is associated with many brain diseases, including neurological diseases, psychiatric diseases, or pediatric brain tumors ,14. The When forming the cerebellum, cerebellar granule neuron precursors (CGNPs) undergo a rapid expansion in the external granule layer (EGL) on the dorsal surface of the cerebellum. Afterwards, they exit the cell cycle, migrate internally, and differentiate into interneurons . In the Medulloblastoma (MB) is the most frequent pediatric solid tumor, representing around 20% of the tumors of the CNS in childhood . This tuThe progress in MB therapy has increased the survival rate of the patients, although it is very variable depending on the tumor subtype. The strategy that is used nowadays for MB patients is based on maximal tumor resection, followed by chemotherapy and craniospinal radiotherapy . The proMoreover, almost 30% of the patients present with disseminated tumor at the moment of diagnosis . MBs sprThe first classification of MBs was based on their histopathological features. Until 2007, three main subgroups were defined , when the WHO classification defined four different histological subgroups, namely, desmoplastic-nodular, large cell, anaplastic, and MB with extensive nodularity (MBEN), the first one being the one with the best prognosis . This clThis subgroup is characterized by the aberrant activation of the WNT/β-catenin signaling pathway and the good prognosis of the patients . In 2017Table representing key histological and clinical data of two different MB classifications, as well as genetic alterations, metastasis rates, age, and survival data of the Cavalli classification. The mTOR implication in some of the subgroups is also presented. The age groups are infant (0–3 years), children (>3–10 years), adolescent (>10–17 years), and adult (>17 years). Ado—adolescent; amp: amplification; child—children; dup—duplication; LCA—large cell/anaplastic; MBEN—medulloblastoma with extensive nodularity; mut—mutations; ↑—activation.This subgroup is characterized by the aberrant activation of the SHH signaling pathway that gives rise to the disease. Different genes belonging to this signaling pathway can be mutated, such as SHH, PTCH, SMO, SUFU, GLI1, or GLI2. The patients that are classified in the SHH subgroup have an intermediate prognosis, except the infants who have a good prognosis. This information has recently been complemented with the four subtypes that are defined within the SHH group: SHH α, SHH β, SHH γ, and SHH δ  Table 1Table 1. This subgroup presents the worst survival rate  and to dRegarding to the implication of the signaling pathways, the Group 3α tumors are enriched in the expression of the photoreceptor, muscle contraction, and primary cilium-related genes, while Group 3β and 3γ present the enrichment of protein translation pathways. Furthermore, Group 3γ is also enriched in the expression of genes that are related to telomere maintenance .This is the most prevalent group; almost 40% of the MBs are included in this subgroup. As in Group 3, the deleterious signaling pathway that causes the disease has not been identified. There are three different subtypes that have been described within this subgroup: Group 4α, Group 4β, and Group 4γ. The main characteristics of the Group 4α tumors are the v-myc avian myelocytomatosis viral-related oncogene, neuroblastoma-derived (MYCN) and cyclin dependent kinase 6 (CDK6) amplifications, 8p loss and 7q gain. The Group 4β tumors are characterized with the synuclein alpha interacting protein (SNCAIP) duplications and ubiquitous i17q. Finally, the Group 4γ tumors present CDK6 amplifications, 8p loss, and 7q gain, as Group 4α tumors, but with the absence of MYCN amplifications  Table 1Table 1. The experiments that were performed by Cavalli and colleagues have identified differentially activated pathways for each subtype, supporting the existence of the three independent subtypes. The pathways that were identified were the activation of migration pathways in Group 4α, activation of mitogen activated kinase-like protein (MAPK) and fibroblast growth factor receptor 1 (FGFR1) signaling pathways in Group 4β, and activation of PI3K/AKT/mTOR and erb-b2 receptor tyrosine kinase 4 (ERBB4)-mediated nuclear signaling pathways in Group 4γ .It is of relevance to underscore that in 2010, Gibson and colleagues demonstrated that the distinct subgroups of MB have different developmental origins, discovering that some WNT MBs were arisen from the cells in the dorsal brainstem . Later, The cancer stem cell (CSC) hypothesis explains the existence of a small fraction of tumor cells that have stem properties and the ability to proliferate and maintain the tumor growth . These cThe activation of the mTOR pathway plays a key role in the development of several cancer types because of its importance in controlling cell growth and metabolism . AberranThe PI3K/AKT signaling pathway is found to be deregulated through a variety of mechanisms in many human cancers . MutatioDownstream of mTORC1, the overexpression of S6K1, 4EBP1, and eIF4E has been associated to cellular transformation . eIF4E oDuring brain development, the mTOR-mediated signaling pathway masters the differentiation of neurons and glia, as well as the maintenance of the stemness of NSCs . In the IGF positively regulates the mTOR pathway, which is frequently activated in malignant brain tumors, including MB . Such anAs a result of its role in the canonical SHH signaling pathway, mTORC1 seems to be a potentially important molecular target for treating SHH MBs . HoweverRegarding the role of the PI3K/AKT/mTOR signaling pathway, its activation occurs in subgroup 4γ, the most prevalent subtype . MoreoveLike all CSCs, the MBSCs possess the ability of self-renewal and differentiation, increasing the oncogenic potential of the heterogeneous tumor . The traThe fact that PI3K inhibition has a heavy impact on the cell number of primary MB cells has already been demonstrated . FollowiAs mentioned above, the current therapy is not enough to cure all of the MB patients, and the high doses of chemotherapy and radiotherapy that are needed induce severe side effects. This is the reason that recent investigations are directed toward improving targeted therapy in MB subgroups. There are two main objectives; on the one hand, to discover new drugs, and on the other hand, to optimize the doses of the drugs that are usually used. One consideration to be taken into account in MB treatment, as it occurs in all brain tumors, is that the drug must be able to cross the blood-brain barrier, which makes the development of new therapeutic agents difficult. The WNT MBs present a good rate of cure compared with the other subgroups, since the WNT activation increases the tumor’s radiosensitivity . That isTwo specific therapies have been developed to target the WNT MBs. The first one, norcantharidin, has been shown to block the WNT signaling pathway, impairing the growth of the MB . The secMany specific treatments for SHH MB have been developed. Almost all of these treatments are focused on inhibiting SMO, a G protein type receptor that is implicated in the SHH signaling pathway. These treatments are based on the structure of cyclopamine, a naturally occurring plant alkaloid, the first SHH pathway inhibitor that was discovered with an anticancer effect. Cyclopamine inhibits the SMO protein, binding to its transmembrane domain and avoiding its change of conformation to the active form. However, cyclopamine has failed in clinical development, mainly because of its pharmacokinetic characteristics . TherefoThe most studied of all of these analogs is vismodegib, the first food and drug administration (FDA)-approved drug as a SHH signaling inhibitor for advanced and metastatic basal cell carcinoma . The patThe lack of an altered signaling pathway responsible for initiating the tumor makes it difficult to develop a targeted therapy for these subgroups of MBs. Among other alterations, some Group 3 MBs are characterized with MYC overexpression ,74. Two As a result of the importance of the PI3K/AKT/mTOR pathway in cancer progression, targeting this signaling pathway has become one of the most studied strategies. Rapamycin is the first mTOR inhibitor that has been used in anti-cancer therapy. This compound is an antifungal agent that binds to FK506 Binding Protein 12 (FKBP12), forming a complex that allosterically inhibits the FKB12-Rapamycin Binding (FRB) domain of mTORC1, leading to the dissociation of Raptor from mTORC1 . As rapaSome of these rapalogs have also been tested in MB, demonstrating promising results and leading to three clinical trials in phase I. These compounds are temsirolimus, sirolimus, and tesirolimus, in combination with perifpsine, an AKT inhibitor . The PI3K/AKT/mTOR signaling pathway is primarily involved in SHH MBs. In these types of tumors, not only the mutations in the SMO receptor or the aberrant activation of the SHH pathway are the cause of MB initiation; an increased activation of the PI3K signaling or the alternative activation of the RAS-MAPK pathway could also eventually cause drug resistance . Thus, cFinally, it has been shown that the PI3K/AKT/mTOR signaling pathway is implicated in CSCs, and specifically in MBSCs, demonstrating that the inhibition of this signaling pathway reduces the MBSC population in the primary tumor culture . TherefoMB is the most common malignant solid tumor in childhood, and even if the current therapies have improved the overall survival, the side effects that they generate are devastating for children. Improved knowledge of MB has given rise to a new and detailed classification of the subgroups of MB, which may lead, in the future, to a better stratification of the patients, based on the molecular characteristics of their tumor, moving towards a personalized therapy for each patient. To reach this goal, a deeper molecular profiling of each tumor is needed after the biopsy or surgery. Together with the new classification, new signaling pathways that are implicated in different subgroups of MB, have been identified. Signaling pathways such as the SHH or WNT signaling pathways, as well as PI3K/AKT/mTOR are therapeutic targets in MB.mTOR is a master signaling pathway that regulates organismal growth and homeostasis, as a result of its implication in protein and lipid synthesis, and in the control of the cell cycle and the cellular metabolism. Different studies have shown that it is also necessary in cerebellar development and stem cell pluripotency maintenance. Being an essential protein in the homeostasis of the cells, when it is deregulated, it is implicated in different tumors, including MB. Furthermore, it has a decisive role in MBSCs, which is demonstrated by the fact that when the PI3K/AKT/mTOR signaling pathway is inhibited, the number of MBSCs decreases. In this review we describe the targeting of mTOR as a promising therapeutic approach, mainly, but not only, for SHH-driven MB patients. Moreover, the combination of this approach with the current therapies could be a promising strategy, as the mTOR inhibition impairs the growth of MBSCs and increases the sensitivity to radiation-induced apoptosis in Nestin positive MBSCs, decreasing the possibility of tumor recurrence and therapy resistance .


Constitutive activation of the Hedgehog (Hh) signaling pathway is associated with increased risk of developing several malignancies. The biological and pathogenic importance of Hh signaling emphasizes the need to control its action tightly, both physiologically and therapeutically. Evidence of crosstalk between Hh and other signaling pathways is reported in many tumor types. Here, we provide an overview of the current knowledge about the communication between Hh and major signaling pathways, such as Notch, Wnt, and transforming growth factor β (TGF-β), which play critical roles in both embryonic and adult life. When these pathways are unbalanced, impaired crosstalk contributes to disease development. It is reported that more than one of these pathways are active in different type of tumors, at the same time. Therefore, starting from a plethora of stimuli that activate multiple signaling pathways, we describe the signals that preferentially converge on the Hh signaling cascade that influence its activity. Moreover, we highlight several connection points between Hh and Notch, Wnt, or TGF-β pathways, showing a reciprocal synergism that contributes to tumorigenesis, supporting a more malignant behavior by tumor cells, such as in leukemia and brain tumors. Understanding the importance of these molecular interlinking networks will provide a rational basis for combined anticancer drug development. Signaling pathways are networks of regulatory proteins and other gene products that act in a coordinated manner to control various biological processes inside the cell. Remarkably, mutation of a single gene encoding a component of a specific pathway is able to affect related signaling cascades, triggering unbalanced crosstalk that leads to cancer development. Of note, impaired regulation of primary signaling pathways can ultimately culminate in constitutive activation of signaling effectors in the nucleus, where they act out of control, sustaining the expression of pro-tumoral target genes. To date, it is known that tumor development is characterized by deregulation of at least two major signaling pathways at the same time, which crosstalk with each other, determining the acquisition of malignant phenotypes .Hedgehog (Hh) signaling is a critical pathway that mainly controls embryonic development, whereas in post-natal life, it is inactive or poorly active, playing a restricted role in stem cell maintenance and tissue homeostasis/repair . Since HOf note, recent studies show that Hh signaling elements talk to several other cofactors belonging to major pathways, such as Notch, Wnt, and transforming growth factor β (TGF-β), resulting in significant crosstalk between these signaling networks. The integration of several signaling pathways is a key step able to determine a more aggressive behavior of tumor cells and their resistance to pharmacological approaches. Interestingly, Notch, Wnt, and TGF-β pathways are able to promote/sustain oncogenesis through synergistic associations with Hh signaling in several types of tumors.Here, we review a global picture of intricate protein–protein interaction networks between key components of Hedgehog, Wnt, Notch, and TGF-β signaling pathways in an unbalanced/malignant context. We also describe how these main pathways can integrate with each other and ultimately affect Hh signaling output.Drosophila, Hedgehog signaling is an evolutionarily conserved pathway that plays a key role as a morphogenesis driver for embryonic and post-natal development. It regulates diverse cellular processes, including cell proliferation, tissue differentiation, and repair of normal tissues  (Recent studies identify Mastermind-like 1 (Maml1) as a novel positive regulator of Hh signaling. It physically interacts with Gli proteins (Gli1 and Gli2), working as a potent transcriptional coactivator , empowert genes) , and elet genes) , Hes1 (It genes) , Wnt sigt genes) , and TGFt genes)  (non-cant genes) Figure 1Ptch1 and SuFu) or gain of function , both associated with an aberrant activation of the Hh pathway  , also called nevoid basal cell carcinoma syndrome (nevoid BCC) or basal cell nevus syndrome, are predisposed to multiple cancers, including basal cell carcinoma (BCC), medulloblastoma (MB), and rhabdomyosarcoma . Gorlin Fu genes . Of noterequency . By contty (LOH) , are typty (LOH) . In patielopment . The co-patients . Finallyosarcoma , suggeste of LOH . In addie of LOH . GS patisome 17q . In MB,  or SUFU  or, conv and SHh . Of notesubgroup . All theSuFu mutation as the cause of multiple meningiomas in a single large Finnish family, suggesting that SuFu alterations predispose to meningiomas in addition to MBs  phenotype. , with an incidence that increases by 10% per year . As argurequency . Moreoveadic BCC . Couvè-Padic BCC . The curadic BCC . In addi of Gli1 . A globaand Gli3 , and Hh and Gli3 . Also, imutation . Recentlmutation , and losof T-ALL . In addil tumors . Lee andenotype. . Finally maxilla .Although tumors not GS-related present direct mutations in genes involved in the Hh pathway, it is not possible to identify Hh signaling as the driving force for cancer onset. In fact, activated Hh signaling is able to empower the severity of the tumors, but this pathway alone is not capable of driving cancer development. A major elucidation of the mechanisms leading to the genesis of malignancy through an aberrant activation of this pathway and/or unbalanced cross-talking with other signaling pathways could provide additional therapeutic options to limit tumor growth and relapse and to reduce drug resistance.in trans between Notch receptors, expressed on the surface of signal-receiving cells, and Notch ligands, expressed on the surface of adjacent signal-sending cells. Such an interaction renders Notch susceptible to two sequential proteolytic cleavages that involve two distinct enzymes: an A-disintegrin and metalloprotease (ADAM) and Presenilin (PS)/γ-secretase complex. These events end in the release of a soluble Notch intracellular domain (NICD) , which are activated upon interaction with different types of ligands [Serrate-like Jagged1 and 2 (JAG1–2) and Delta-like ], expressed by adjacent cells. The activation of Notch signaling requires the binding n (NICD) . In canon (NICD) , induces1/5, HEY , Myc  signal initiation . These ft models . Despite therapy .On the contrary, a reciprocal exclusion between Hh and Notch signaling pathways is already defined in the skin. In particular, Notch1 deficiency results in an increased expression of Gli2, along with tumor development in squamous cell carcinoma .Likewise, an inverted gradient between Hh and Notch is observed in normal colon crypt development . Very liHowever, the existence of a regulatory mechanism impinging on the crosstalk between Hh and Notch in controlling tumor onset and progression needs to be better understood.via the transcriptional coactivator β-catenin, and it is also implicated in adult tissue homeostasis and repair  , the scaWnt genes through Gli transcription factors   . Althougia (CML) , direct ia (CML) Figure 2stricto sensu, bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), glial-derived neurotrophic factors (GDNFs), Nodal, Lefty, and the Müllerian inhibitory substance/anti-Müllerian hormone (MIS/AMH)  . MembersMIS/AMH) .stricto sensu are identified in three isoforms: TGF-β1, TGF-β2, and TGF-β3. They signal via cell-surface receptors, which consist of specific hetero-oligomeric complex of type-I and type-II serine/threonine kinase receptors (TbR-I and TbR-II). Typically, TGF-β signaling initiates with the binding to and subsequently activation of TbR-I and TbR-II. Smad proteins are the initial responders and transduce the signal from the TGF-β receptor activation process. Smads comprise a family of structurally similar proteins with different functions: receptor-regulated (R-Smads), common mediator (Co-Smads), and inhibitory (I-Smads). After ligand binding, the TbR-II receptor phosphorylates TbR-I, which in turn phosphorylates and activates R-Smads . Then, the R-Smads bind to Co-Smads , and this complex translocates to the nucleus, where it regulates the transcription of TGF-β–responsive genes, involved in cytostatic and/or apoptotic events (p15 and p21) (Family members of TGF-β and p21) , prolifeand p21) , and theand p21) . On the and p21) . Severaland p21) Figure 2and p21) . Interesand p21) . Concomiand p21) . In addiand p21) . Thus, tDuring the development of the cerebellum, the GCPs express BMPs. It is reported that in GCPs, BMP‐2 and ‐4 antagonize the proliferative effects of Hh through SMAD5. In addition, BMPs downregulate Hh signaling proteins such as SMO and Gli1. Moreover, it is suggested that BMP‐2 inhibits proliferation and promotes cell differentiation of GCPs, by downregulating the oncogene N-Myc .It is well described that the architectural structure of the colon is controlled by a gradient of WNT, Hh, BMP, and Notch signaling . In partAlthough TGF-β is proposed to be a potent negative regulator of hematopoiesis  and the Biological processes such as stem cell self-renewal, cell growth, and differentiation are orchestrated by a number of major signaling pathways that integrate with each other to modulate cell outcomes in response to several intracellular signals.The picture in This complex scenario could open a huge window of opportunity for the development of new therapeutic drugs for multiple cancers.MP, SZ, and DB wrote the paper. MP, SZ, and FN prepared the figures. SC, IS, and DB reviewed drafts of the paper. All authors read and approved the final manuscript.This work was supported by the following grants: Sapienza University (Progetti H2020 collaborativi) #PH118164340087CF (to IS) and Sapienza University 2016 project #RP116154CE3DD739 (to DB).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.