Search

The pathogenesis of amyotrophic lateral sclerosis (ALS), a lethal neurodegenerative disease, remains undisclosed. Mutations in ALS related genes have been identified, albeit the majority of cases are unmutated. Clinical pathology of ALS suggests a prion-like cell-to-cell diffusion of the disease possibly mediated by exosomes, small endocytic vesicles involved in the propagation of RNA molecules and proteins. In this pilot study, we focused on exosomal microRNAs (miRNAs), key regulators of many signaling pathways. We analyzed serum-derived exosomes from ALS patients in comparison with healthy donors. Exosomes were obtained by a commercial kit. Purification of miRNAs was performed using spin column chromatography and RNA was reverse transcribed into cDNA. All samples were run on the miRCURY LNATM Universal RT miRNA PCR Serum/Plasma Focus panel. An average of 29 miRNAs were detectable per sample. The supervised analysis did not identify any statistically significant difference among the groups indicating that none of the miRNA of our panel has a strong pathological role in ALS. However, selecting samples with the highest miRNA content, six biological processes shared across miRNAs through the intersection of the GO categories were identified. Our results, combined to those reported in the literature, indicated that further investigation is needed to elucidate the role of exosome-derived miRNA in ALS.

Background: Somatic mutations in the ubiquitin-specific protease 8 (USP8) gene have recently been shown to occur in ACTH-secreting pituitary adenomas, thus calling attention to the ubiquitin system in corticotrope adenomas. Objectives: Assess the consequences of USP8 mutations and establish the role of ubiquitin on ACTH turnover in human ACTH-secreting pituitary adenomas. Methods: USP8 mutation status was established in 126 ACTH-secreting adenomas. Differences in ACTH secretion and POMC expression from adenoma primary cultures and in microarray gene expression profiles from archival specimens were sought according to USP8 sequence. Ubiquitin/ACTH coimmunoprecipitation and incubation with MG132, a proteasome inhibitor, were performed in order to establish whether ubiquitin plays a role in POMC/ACTH degradation in corticotrope adenomas. Results: USP8 mutations were identified in 29 adenomas (23%). Adenomas presenting USP8 mutations secreted greater amounts of ACTH and expressed POMC at higher levels compared to USP wild-type specimens. USP8 mutant adenomas were also more sensitive to modulation by CRH and dexamethasone in vitro. At microarray analysis, genes associated with endosomal protein degradation and membrane components were downregulated in USP8 mutant adenomas as were AVPR1B, IL11RA, and PITX2. Inhibition of the ubiquitin-proteasome pathway increased ACTH secretion and POMC itself proved a target of ubiquitylation, independently of USP8 sequence status. Conclusions: Our study has shown that USP8 mutant ACTH-secreting adenomas present a more “typical” corticotrope phenotype and reduced expression of several genes associated with protein degradation. Further, ubiquitylation is directly involved in intracellular ACTH turnover, suggesting that the ubiquitin-proteasome system may represent a target for treatment of human ACTH-secreting adenomas.

Mechanisms underlying the resistance of acute lymphoblastic leukemia (ALL) blasts to l-asparaginase are still incompletely known. Here we demonstrate that human primary bone marrow mesenchymal stromal cells (MSCs) successfully adapt to l-asparaginase and markedly protect leukemic blasts from the enzyme-dependent cytotoxicity through an amino acid trade-off. ALL blasts synthesize and secrete glutamine, thus increasing extracellular glutamine availability for stromal cells. In turn, MSCs use glutamine, either synthesized through glutamine synthetase (GS) or imported, to produce asparagine, which is then extruded to sustain asparagine-auxotroph leukemic cells. GS inhibition prevents mesenchymal cells adaptation to l-asparaginase, lowers glutamine secretion by ALL blasts, and markedly hinders the protection exerted by MSCs on leukemic cells. The pro-survival amino acid exchange is hindered by the inhibition or silencing of the asparagine efflux transporter SNAT5, which is induced in mesenchymal cells by ALL blasts. Consistently, primary MSCs from ALL patients express higher levels of SNAT5 (P < .05), secrete more asparagine (P < .05), and protect leukemic blasts (P

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative diseases characterized by the presence of neuropathological aggregates of phosphorylated TDP-43 (P-TDP-43) protein. The RNA-binding protein TDP-43 participates also to cell stress response by forming stress granules (SG) in the cytoplasm to temporarily arrest translation. The hypothesis that TDP-43 pathology directly arises from SG has been proposed but is still under debate because only sub-lethal stress conditions have been tested experimentally so far. In this study we reproduced a mild and chronic oxidative stress by sodium arsenite to better mimic the persistent and subtle alterations occurring during the neurodegenerative process in primary fibroblasts and induced pluripotent stem cell-derived motoneurons (iPSC-MN) from ALS patients carrying mutations in TARDBP and C9ORF72 genes. We found that not only the acute sub-lethal stress usually used in literature, but also the chronic oxidative insult was able to induce SG formation in both primary fibroblasts and iPSC-MN. We also observed the recruitment of TDP-43 into SG only upon chronic stress in association to the formation of distinct cytoplasmic P-TDP-43 aggregates and a significant increase of the autophagy marker p62. A quantitative analysis revealed differences in both the number of cells forming SG in mutant ALS and healthy control fibroblasts, suggesting a specific genetic contribution to cell stress response, and in SG size, suggesting a different composition of these cytoplasmic foci in the two stress conditions. Upon removal of arsenite, the recovery from chronic stress was complete for SG and P-TDP-43 aggregates at 72 h with the exception of p62, which was reduced but still persistent, supporting the hypothesis that autophagy impairment may drive pathological TDP-43 aggregates formation. The gene-specific differences observed in fibroblasts in response to oxidative stress were not present in iPSC-MN, which showed a similar formation of SG and P-TDP-43 aggregates regardless their genotype. Our results show that SG and P-TDP-43 aggregates may be recapitulated in patient-derived neuronal and non-neuronal cells exposed to prolonged oxidative stress, which may be therefore exploited to study TDP-43 pathology and to develop individualized therapeutic strategies for ALS/FTD.

Silibinin, a milk thistle extract with known hepatoprotective effects, has recently been shown to act upon tumoral corticotropes and revert the Cushingoid phenotype in an allograft mouse model (Riebold et al 2015). Silbinin is known to inhibit HSP90 -a chaperone to the glucocorticoid receptor- thereby restoring sensitivity to glucocorticoid negative feedback in tumoral corticotropes. Aim of the present study was to assess the effect of silibinin on ACTH synthesis and secretion by human corticotrope adenomas in vitro. Methods: Eight human ACTH-secreting pituitary adenomas were collected during surgery and established in culture as per our protocol (Pecori Giraldi et al 2011). Specimens were treated with 10 - 50 µM silibinin for up to 72 hours. ACTH medium levels were measured by Elisa; POMC expression was assessed by RT-PCR (Cassarino et al 2017). Results. Silibinin reduced spontaneous ACTH secretion to a variable extent in individual adenomas: from 32 to 79% of baseline at 4h, and 54 - 85 % of baseline at 48 and 72h. Silibinin was also effective in reinstating or enhancing sensitivity to steroid negative feedback: ACTH decreases during 10–50 µM silibinin incubation ranged from 10 to 63% of dexamethasone-treated wells at 4 hours, 70 -80% at 48 hours and 36 to 80% at 72 hours, indicating long-lasting effect on glucocorticoid sensitivity. Silibinin induced a variable decrease in POMC expression, both as regards expression in control and dexamethasone-treated wells; some specimens exhibited a marked sensitivity to the inhibitory effect, with POMC expression decreasing to less than 50% of control. Conclusions:, this data suggests that silibinin can inhibit ACTH secretion and POMC synthesis and restore sensitivity to negative glucocorticoid feedback. References: Cassarino et al (2017) Endocrine55: 853–860. Pecori Giraldi et al (2011) Journal of Neuroendocrinology23:1214–21. Riebold et al (2015) Nature Medicine21:276–280.

As for the majority of neurodegenerative diseases, pathological mechanisms of amyotrophic lateral sclerosis (ALS) have been challenging to study due to the difficult access to alive patients' cells. Induced pluripotent stem cells (iPSCs) offer a useful in vitro system for modelling human diseases. iPSCs can be theoretically obtained by reprogramming any somatic tissue although fibroblasts (FB) remain the most used cells. However, reprogramming peripheral blood cells (PB) may offer significant advantages. In order to investigate whether the choice of starting cells may affect reprogramming and motor neuron (MNs) differentiation potential, we used both FB and PB from a same C9ORF72-mutated ALS patient to obtain iPSCs and compared several hallmarks of the pathology. We found that both iPSCs and MNs derived from the two tissues showed identical properties and features and can therefore be used interchangeably, giving the opportunity to easily obtain iPSCs from a more manageable source of cells, such as PB. The present raw data are related to the immunocytochemical analysis to identify the percentage of cells positive for the motor neuron specific marker HB9 as well as the percentage of TDP-43 mislocalization. In addition, the percentage of foci identified by FISH caused by the C9orf72 repeat expansion.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative diseases characterized by the presence of neuropathological aggregates of phosphorylated TDP-43 (P-TDP-43). The RNA-binding protein TDP-43 is also a component of stress granules (SG), cytoplasmic foci forming to arrest translation under sub-lethal stress conditions. Although commonly considered as distinct structures, a link between SG and pathological TDP-43 inclusions may occur despite evidence that TDP-43 pathology directly arises from SG is still under debate. Primary fibroblasts and iPSC-derived neurons (iPSC-N) from ALS patients carrying mutations in TARDBP (n=3) and C9ORF72 (n=3) genes and from healthy controls (n=3) were exposed to oxidative stress by sodium arsenite. SG formation and cell response to stress was evaluated and quantified by immunofluorescence and electron microscopy analyses. We found that, not only an acute, but also a chronic oxidative insult, better mimicking a persistent condition of stress as in neurodegeneration, is able to induce SG formation in primary fibroblasts and iPSC-N. Importantly, only upon chronic stress, we observed TDP-43 recruitment into SG and the formation of distinct P-TDP-43 aggregates, very similar to the abnormal inclusions observed in ALS/FTD autoptic brains. Moreover, in fibroblasts, cell response to stress was different in control compared with mutant ALS cells, probably due to their different vulnerability. A quantitative analysis revealed also differences in terms of number of SG-forming cells and SG size, suggesting a different composition of foci in acute and chronic stress. In condition of prolonged stress, SG and P-TDP-43 aggregate formation was concomitant with p62 increase and autophagy dysregulation in both ALS fibroblasts and iPSC-N, as confirmed by immunofluorescence and ultrastructural analyses. We found that exposure to a chronic oxidative insult promotes the formation of both SG and P-TDP-43 aggregates in patient-derived cells, reinforcing the idea that SG fail to properly disassemble, interfering with the protein quality control system. Moreover, we obtained a disease cell model recapitulating ALS/FTD P-TDP-43 aggregates, which represents an invaluable bioassay to study TDP-43 pathology and develop therapeutic strategies aimed at disaggregating or preventing the formation of pathological inclusions.

Neural stem cell (NSC) neuronal differentiation requires a metabolic shift towards oxidative phosphorylation. We now show that a branched-chain amino acids-driven, persistent metabolic shift toward energy metabolism is required for full neuronal maturation. We increased energy metabolism of differentiating neurons derived both from murine NSCs and human induced pluripotent stem cells (iPSCs) by supplementing the cell culture medium with a mixture composed of branched-chain amino acids, essential amino acids, TCA cycle precursors and co-factors. We found that treated differentiating neuronal cells with enhanced energy metabolism increased: i) total dendritic length; ii) the mean number of branches and iii) the number and maturation of the dendritic spines. Furthermore, neuronal spines in treated neurons appeared more stable with stubby and mushroom phenotype and with increased expression of molecules involved in synapse formation. Treated neurons modified their mitochondrial dynamics increasing the mitochondrial fusion and, consistently with the increase of cellular ATP content, they activated cellular mTORC1 dependent p70S6 K1 anabolism. Global transcriptomic analysis further revealed that treated neurons induce Nrf2 mediated gene expression. This was correlated with a functional increase in the Reactive Oxygen Species (ROS) scavenging mechanisms. In conclusion, persistent branched-chain amino acids-driven metabolic shift toward energy metabolism enhanced neuronal differentiation and antioxidant defences. These findings offer new opportunities to pharmacologically modulate NSC neuronal differentiation and to develop effective strategies for treating neurodegenerative diseases.

Shwachman-Diamond syndrome (SDS) is a rare multi-organ recessive disease mainly characterised by pancreatic insufficiency, skeletal defects, short stature and bone marrow failure (BMF). As in many other BMF syndromes, SDS patients are predisposed to develop a number of haematopoietic malignancies, particularly myelodysplastic syndrome and acute myeloid leukaemia. However, the mechanism of cancer predisposition in SDS patients is only partially understood. In light of the emerging role of mesenchymal stromal cells (MSCs) in the regulation of bone marrow homeostasis, we assessed the ability of MSCs derived from SDS patients (SDS-MSCs) to recreate a functional bone marrow niche, taking advantage of a murine heterotopic MSC transplant model. We show that the ability of semi-cartilaginous pellets (SCPs) derived from SDS-MSCs to generate complete heterotopic ossicles in vivo is severely impaired in comparison with HD-MSC-derived SCPs. Specifically, after in vitro angiogenic stimuli, SDS-MSCs showed a defective ability to form correct networks, capillary tubes and vessels and displayed a marked decrease in VEGFA expression. Altogether, these findings unveil a novel mechanism of SDS-mediated haematopoietic dysfunction based on hampered ability of SDS-MSCs to support angiogenesis. Overall, MSCs could represent a new appealing therapeutic target to treat dysfunctional haematopoiesis in paediatric SDS patients.

BACKGROUND: Somatic mutations in the ubiquitin-specific protease 8 (USP8) gene have recently been shown to occur in ACTH-secreting pituitary adenomas, thus calling attention to the ubiquitin system in corticotrope adenomas. OBJECTIVES: Assess the consequences of USP8 mutations and establish the role of ubiquitin on ACTH turnover in human ACTH-secreting pituitary adenomas. METHODS: USP8 mutation status was established in 126 ACTH-secreting adenomas. Differences in ACTH secretion and POMC expression from adenoma primary cultures and in microarray gene expression profiles from archival specimens were sought according to USP8 sequence. Ubiquitin/ACTH coimmunoprecipitation and incubation with MG132, a proteasome inhibitor, were performed in order to establish whether ubiquitin plays a role in POMC/ACTH degradation in corticotrope adenomas. RESULTS: USP8 mutations were identified in 29 adenomas (23%). Adenomas presenting USP8 mutations secreted greater amounts of ACTH and expressed POMC at higher levels compared to USP wild-type specimens. USP8 mutant adenomas were also more sensitive to modulation by CRH and dexamethasone in vitro. At microarray analysis, genes associated with endosomal protein degradation and membrane components were downregulated in USP8 mutant adenomas as were AVPR1B, IL11RA, and PITX2. Inhibition of the ubiquitin-proteasome pathway increased ACTH secretion and POMC itself proved a target of ubiquitylation, independently of USP8 sequence status. CONCLUSIONS: Our study has shown that USP8 mutant ACTH-secreting adenomas present a more “typical” corticotrope phenotype and reduced expression of several genes associated with protein degradation. Further, ubiquitylation is directly involved in intracellular ACTH turnover, suggesting that the ubiquitin-proteasome system may represent a target for treatment of human ACTH-secreting adenomas.

Shwachman-Diamond syndrome (SDS) is a rare inherited recessive disease mainly caused by mutations in the Shwachman-Bodian-Diamond syndrome (SBDS) gene, which encodes for the homonymous protein SBDS, whose function still remains to be fully established. SDS affects several organs causing bone marrow failure, exocrine pancreatic insufficiency, skeletal malformations, and cognitive disorders. About 15% of SDS patients develop myelodysplastic syndrome (MDS) and are at higher risk of developing acute myeloid leukemia (AML). Deficiency in SBDS expression has been associated with increased apoptosis and lack of myeloid differentiation in bone marrow hematopoietic progenitors. Importantly, most SDS patients carry nonsense mutations in SBDS. Since ataluren is a well-characterized small molecule inhibitor that can suppress nonsense mutations, here, we have assessed the efficacy of this drug in restoring SBDS expression in hematopoietic cells obtained from a cohort of SDS patients. Remarkably, we show that ataluren treatment readily restores SBDS protein expression in different cell types, particularly bone marrow stem cells. Furthermore, ataluren promotes myeloid differentiation in hematopoietic progenitors, reduces apoptotic rate in primary PBMCs, and brings mammalian target of rapamycin phosphorylation levels back to normal in both lymphoblasts and bone marrow mesenchymal stromal cells (BM-MSCs). Since a specific therapy against SDS is currently lacking, these results provide the rationale for ataluren repurposing clinical trials.

Excitatory amino acid transporters (EAATs) are high-affinity Na +-dependent carriers of major importance in maintaining glutamate homeostasis in the central nervous system. EAAT3, the human counterpart of the rodent excitatory amino acid carrier 1 (EAAC1), is encoded by the SLC1A1 gene. EAAT3/EAAC1 is ubiquitously expressed in the brain, mostly in neurons but also in other cell types, such as oligodendrocyte precursors. While most of the glutamate released in the synapses is taken up by the "glial-type" EAATs, EAAT2 (GLT-1 in rodents) and EAAT1 (GLAST), the functional role of EAAT3/EAAC1 is related to the subtle regulation of glutamatergic transmission. Moreover, because it can also transport cysteine, EAAT3/EAAC1 is believed to be important for the synthesis of intracellular glutathione and subsequent protection from oxidative stress. In contrast to other EAATs, EAAT3/EAAC1 is mostly intracellular, and several mechanisms have been described for the rapid regulation of the membrane trafficking of the transporter. Moreover, the carrier interacts with several proteins, and this interaction modulates transport activity. Much less is known about the slow regulatory mechanisms acting on the expression of the transporter, although several recent reports have identified changes in EAAT3/EAAC1 protein level and activity related to modulation of its expression at the gene level. Moreover, EAAT3/EAAC1 expression is altered in pathological conditions, such as hypoxia/ischemia, multiple sclerosis, schizophrenia, and epilepsy. This review summarizes these results and provides an overall picture of changes in EAAT3/EAAC1 expression in health and disease. © 2013 Springer Basel.

Allogeneic haematopoietic-stem-cell transplantation (HSCT) is the treatment of choice for many malignant and non-malignant disorders. However, Graft-versus -Host Disease (GvHD), the major complication of allogeneic HSCT, limits its wider application. Among different sites that can be involved, gastrointestinal GvHD represents the major cause of patients morbidity and mortality. The infiltration of different cell subsets into target organs is an important step in GvHD pathogenesis, and the modulation of cell trafficking could represent a promising strategy for GvHD prophylaxis and treatment. Chemerin has been recently identified as a chemotactic protein, which is produced by several tissues during inflammation and binds the G protein-coupled receptor ChemR23, expressed by immature myeloid and plasmacytoid Dendritic Cells, macrophages and natural killer cells. The aim of this study was to evaluate the potential role of Chemerin/ChemR23 axis in the pathogenesis of GvHD, in order to identify disease-specific pathways exploitable for developing new potential therapeutic targets. For this purpose, lethally irradiated Balb/C recipient mice were transplanted with bone marrow cells and splenocytes obtained from ChemR23deficient (ChemR23-/-) C57BL6 mice. After transplantation, mice were monitored daily for survival and GvHD severity. Recipient mice were sacrificed at different time points to evaluate Chemerin production and leukocytes infiltration in skin, lung, liver, and gut by using different techniques, such as ELISA, histopathology, FACS and PCR. Starting from day +6 after transplantation, Chemerin plasma levels appeared significantly higher in both wild type (WT) and ChemR23-/- mice who developed GvHD (WT mean level=86.8 ng/ml; range=80.1-91.9; ChemR23-/- mean level=83.8 ng/ml; range=70.5-103.6, n=6, day+7) compared to syngeneic controls (mean level=62.8 ng/ml; range=49.2-82.5, n=6, day+7), p=0.02. Interestingly, ChemR23-/- mice developed a more severe GvHD compared to mice transplanted with WT cells. In particular, ChemR23-/- transplantedmice showed a higher mortality rate (at day +28 after HSCT: 85% mortality in ChemR23-/- vs 25% in WT, p=0.0004, n=45) (Fig. 1). Differences in GvHD score between ChemR23-/- and WT transplanted mice resulted by a significantly increase in weight loss, associated to severe diarrhea. In accordance, histopathologic analysis performed on GvHD target organs showed a significantly higher GvHD score in large intestine of ChemR23-/- transplanted mice, whereas no differences were found in other GvHD target organs. In addition, a deeper histological analysis on large intestine showed that tissue damage is characterized by crypt hyperplasia and atrophy, epithelium apoptosis and colitis. Moreover, FACS analysis of large intestine infiltrating leukocytes showed an higher neutrophils frequency in ChemR23-/- transplantedmice compared to WT (neutrophils=10.39%, range 6.5%-15.1% versus neutrophils=5.84%, range 2.6%-12.8%, respectively, p=0.001, n=10). This observation was also obtained by analyzing the mesenteric lymphnode. The higher neutrophils infiltration was also confirmed by immunohistochemistry evaluating the number of myeloperoxidase (MPO) positive cells and by quantitative PCR detecting MPO expression (ChemR23-/- mean 2-ΔΔCt =22.35, range 2.43-46.05; WT mean 2-ΔΔCt =6.42, range 0.20-19.23; p=0.04, n=8). All these findings suggest that the Chemerin/ChemR23 axis play a crucial role in intestinal GVHD. Further studies are needed to better understand the mechanisms underling the severe damage observed in the gastrointestinal tract of mice transplanted with ChemR23-/- cells. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.