Repurposing of tamoxifen ameliorates CLN3 and CLN7 disease phenotype

Batten diseases (BDs) are a group of lysosomal storage disorders characterized by seizure, visual loss, and cognitive and motor deterioration. We discovered increased levels of globotriaosylceramide (Gb3) in cellular and murine models of CLN3 and CLN7 diseases and used fluorescent-conjugated bacterial toxins to label Gb3 to develop a cell-based high content imaging (HCI) screening assay for the repurposing of FDA-approved compounds able to reduce this accumulation within BD cells. We found that tamoxifen reduced the lysosomal accumulation of Gb3 in CLN3 and CLN7 cell models, including neuronal progenitor cells (NPCs) from CLN7 patient-derived induced pluripotent stem cells (iPSC). Here, tamoxifen exerts its action through a mechanism that involves activation of the transcription factor EB (TFEB), a master gene of lysosomal function and autophagy. In vivo administration of tamoxifen to the CLN7Δex2 mouse model reduced the accumulation of Gb3 and SCMAS, decreased neuroinflammation, and improved motor coordination. These data strongly suggest that tamoxifen may be a suitable drug to treat some types of Batten disease.This work was funded by the European Union’s Horizon 2020 research and innovation programme (BATCure, grant No. 666918 to DLM, JPB, SEM, TB and SS). JPB is funded by the Agencia Estatal de Investigación (PID2019-105699RB-I00/ AEI / 10.13039/501100011033 and RED2018-102576-T), Plan Nacional sobre Drogas (2020I028), Junta de Castilla y León (Escalera de Excelencia CLU-2017-03), Ayudas Equipos Investigación Biomedicina 2017 Fundación BBVA and Fundación Ramón Areces. SS was funded by a grant from the Mila’s Miracle Foundation. TB was supported by German Research Council (DFG) grant FOR2625. SM benefits from MRC funding to the MRC Laboratory for Molecular Cell Biology University Unit at UCL (award code MC_U12266B) towards laboratory and office space. We acknowledge Marcella Cesana for providing the TFEB virus. Graphical abstract was created using BioRender.com

Involvement of the exomer complex in the polarized transport of Ena1 required for Saccharomyces cerevisiae survival against toxic cations

[EN] Exomer is an adaptor complex required for the direct transport of a selected number of cargoes from the trans-Golgi network (TGN) to the plasma membrane in Saccharomyces cerevisiae However, exomer mutants are highly sensitive to increased concentrations of alkali metal cations, a situation that remains unexplained by the lack of transport of any known cargoes. Here we identify several HAL genes that act as multicopy suppressors of this sensitivity and are connected to the reduced function of the sodium ATPase Ena1. Furthermore, we find that Ena1 is dependent on exomer function. Even though Ena1 can reach the plasma membrane independently of exomer, polarized delivery of Ena1 to the bud requires functional exomer. Moreover, exomer is required for full induction of Ena1 expression after cationic stress by facilitating the plasma membrane recruitment of the molecular machinery involved in Rim101 processing and activation of the RIM101 pathway in response to stress. Both the defective localization and the reduced levels of Ena1 contribute to the sensitivity of exomer mutants to alkali metal cations. Our work thus expands the spectrum of exomer-dependent proteins and provides a link to a more general role of exomer in TGN organization.We acknowledge Emma Keck for English language revision. We also thank members of the Translucent group, J. Arino, J. Ramos, and L. Yenush, for many useful discussions throughout this work and especially L. Yenush for her generous gift of strains and reagents. The help of O. Vincent was essential for developing the work involving RIM101. We also thank R. Valle for her technical assistance at the CR Laboratory. M. Trautwein is acknowledged for data acquisition and discussions during the early stages of the project. C.A. is supported by a USAL predoctoral fellowship. Work at the Spang laboratory was supported by the University of Basel and the Swiss National Science Foundation (31003A-141207 and 310030B-163480). C.R. was supported by grant SA073U14 from the Regional Government of Castilla y Leon and by grant BFU2013-48582-C2-1-P from the CICYT/FEDER Spanish program. J.M.M. acknowledges the financial support from Universitat Politecnica de Valencia project PAID-06-10-1496.Anton, C.; Zanolari, B.; Arcones, I.; Wang, C.; Mulet, JM.; Spang, A.; Roncero, C. (2017). Involvement of the exomer complex in the polarized transport of Ena1 required for Saccharomyces cerevisiae survival against toxic cations. Molecular Biology of the Cell. 28(25):3672-3685. https://doi.org/10.1091/mbc.E17-09-0549S367236852825Ariño, J., Ramos, J., & Sychrová, H. (2010). Alkali Metal Cation Transport and Homeostasis in Yeasts. Microbiology and Molecular Biology Reviews, 74(1), 95-120. doi:10.1128/mmbr.00042-09Bard, F., & Malhotra, V. (2006). The Formation of TGN-to-Plasma-Membrane Transport Carriers. Annual Review of Cell and Developmental Biology, 22(1), 439-455. doi:10.1146/annurev.cellbio.21.012704.133126Barfield, R. 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Relapsing Campylobacter jejuni Systemic Infections in a Child with X-Linked Agammaglobulinemia

X-linked agammaglobulinemia (XLA) is a primary immunodeficiency of the humoral compartment, due to a mutation in the Bruton tyrosine kinase (BTK) gene, characterized by a severe defect of circulating B cells and serum immunoglobulins. Recurrent infections are the main clinical manifestations; although they are especially due to encapsulated bacteria, a specific association with Campylobacter species has been reported. Here, we report the case of a boy with XLA who presented with relapsing Campylobacter jejuni systemic infections. His clinical history supports the hypothesis of the persistence of C. jejuni in his intestinal tract. Indeed, as previously reported, XLA patients may become chronic intestinal carriers of Campylobacter, even in absence of symptoms, with an increased risk of relapsing bacteraemia. The humoral defect is considered to be crucial for this phenomenon, as well as the difficulties to eradicate the pathogen with an appropriate antibiotic therapy; drug resistance is raising in Campylobacter species, and the appropriate duration of treatment has not been established. C. jejuni should always be suspected in XLA patients with signs and symptoms of systemic infection, and treatment should be based on antibiogram to assure the eradication of the pathogen

Inhibition of p38 MAPK in the brain through nasal administration of p38 inhibitor loaded in chitosan nanocapsules

[Aim]: To determine whether a p38 MAPK inhibitor incorporated into nanoemulsion-based chitosan nanocapsules can reduce the activity of this kinase in the brain through their nasal administration in mice.[Materials & methods]: We selected the p38 MAPK inhibitor PH797804, an ATP-competitive inhibitor of p38α encapsulated in nanoemulsion-based chitosan nanocapsules. Biological effect was evaluated in microglial and neuronal cells in vitro and in ex vivo and in vivo systems, in a mouse model of Alzheimer’s disease.[Results]: Encapsulated inhibitor retains enzymatic inhibitory activity and tissue penetration capacity in vitro, ex vivo and in vivo.[Conclusion]: Nasal administration of chitosan nanocapsules can be an effective approach for brain-restricted reduction of p38 MAPK activity, thus reducing the side effects of systemic administration.Peer reviewe

Ultra-Deep Sequencing (UDS) Allows More Sensitive Detection of the D816V and Other Kit Gene Mutations in Systemic Mastocytosis

Objectives and background: According to the World Health Organization (WHO) classification, the diagnosis of Systemic Mastocytosis (SM) relies on bone marrow (BM) examination and is based on a major and four minor criteria. The somatic ‘autoactivating’ point mutation D816V in the KIT receptor gene is one of the minor criteria, founded in the great majority of patients (90%) and it plays a central role in the pathogenesis of the disease. Indolent Systemic Mastocytosis (ISM) is the most common variant of SM, characterized by a very low MC burden and associated with very different clinical pictures. A highly sensitive diagnostic methods for D816V detection are required to assure an appropriate diagnosis and to reduce false-negative results. The recent development of “ultra-deep amplicon sequencing” (UDS) technologies has opened the way to a more accurate characterization of molecular aberrations with higher sensitivity of screening for known and unknown mutations. Our aims were: i) to set-up and optimize a UDS-based mutation screening strategy of the KIT gene on the Roche GS Junior Instrument; ii) to test the sensitivity of our UDS assay to detect the D816V mutation; iii) to investigate the presence of additional KIT mutations in SM. Methods:We decided to take advantage of a next generation sequencing approach to perform an UDS KIT gene mutation analysis on 20 bone marrow (BM) samples from patients whit ISM that were negative for the D816V mutation by Sanger Sequencing which has a sensitivity of 20%. Fusion primers were designed to generate ten partially overlapping amplicon covering the whole KIT transcript (exons 1-21) by RT-PCR. To determine the lower detection limit of our UDS-assay, serial dilutions of the HMC-1 cell line (harboring the D816V mutation) into an unmutated K562 cell line in ratios such as to simulate the following mutation loads were sequenced: 50%, 37.5%, 25%, 12.5%, 5%; 2.5%, 1.25%, 0.5%, 0.25%. Results and significance: UDS of cell line dilutions showed a high accuracy of D816V mutation detection and linearity of mutation calling over the entire range down to 0.25%. The UDS technology allowed to detected the D816V mutation, below the lower detection limit of Sanger Sequencing, with an abundance from 0.5% to 11%, in 12/20 ISM patients. Two additional sequence variations were detected in a large proportion of patients. These two variations included a 3bp in-frame deletion in exon 15 (GenBank X06182.1: c.2164_2166delAGC; p.S715del) found in 11/20 patients and a 12bp in frame-deletion in exon 9 in all patients, whit an abundance ranging from 83% to 97% (GenBank X06182.1: c.1550_1561delGTAACAACAAAG; p.G510_K513del). Previously published studies indicate that the KIT Gly-Asn-Asn-Lys510-513+/- alternatively spliced located immediately downstream to the extracellular KIT domain and KIT Ser715+/-, an interkinase KIT domain, are expressed in normal human hematopoietic cell, leukemic cell lines, acute myeloid leukemia blast and GISTs and represent rather a splice variant of KIT transcript. Interestingly our results showed the presence of the transmembrane domain M541L (GenBank X06182.1: c.1642A>C; p.Met541Leu) KIT-activating mutation in exon 10, with an abundance of 50%, in addition to D816V, in 2/20 ISM. This mutation is known to retain sensitivity to imatinib mesylate. Conclusions:Our preliminary results suggest that our-UDS based KIT gene mutation screening assay might be a reliable and sensitive alternative to conventional sequencing methods for the detection of the D816V. We are now planning to investigate whether the greater sensitivity of UDS allows to detect the D816V mutation in peripheral blood mononuclear cells from patients with a suspected clonal mast cell disorder. These results could represent a starting point to plan other extensive studies to better understand the exact role of KIT receptor alterations in SM

Ultra-deep sequencing (uds) allows more sensitive detection of the D816V and other kit gene mutations in systemic mastocytosis

none21no56th ASH Annual Meeting 9-6 December 2014, San Francisco (CA)nonenoneCaterina De Benedittis, Simona Soverini, Cristina Papayannidis, Michela Rondoni, Sabrina Colarossi, Francesca Dal Pero, Luca Zazzeroni, Roberta Zanotti, Giovanna De Matteis, Serena Merante, Chiara Elena, Federica Irene Grifoni, Massimiliano Bonifacio, Omar Perbellini, Giorgina Specchia, Livio Pagano, Domenica Gangemi, Patrizia Bonadonna, Lisa Pieri, Michele Cavo, Giovanni MartinelliCaterina De Benedittis, Simona Soverini, Cristina Papayannidis, Michela Rondoni, Sabrina Colarossi, Francesca Dal Pero, Luca Zazzeroni, Roberta Zanotti, Giovanna De Matteis, Serena Merante, Chiara Elena, Federica Irene Grifoni, Massimiliano Bonifacio, Omar Perbellini, Giorgina Specchia, Livio Pagano, Domenica Gangemi, Patrizia Bonadonna, Lisa Pieri, Michele Cavo, Giovanni Martinell

Follicular Lymphoma Microenvironment Traits Associated with Event-Free Survival

The majority of patients with Follicular Lymphoma (FL) experience subsequent phases of remission and relapse, making the disease “virtually” incurable. To predict the outcome of FL patients at diagnosis, various clinical-based prognostic scores have been proposed; nonetheless, they continue to fail for a subset of patients. Gene expression profiling has highlighted the pivotal role of the tumor microenvironment (TME) in the FL prognosis; nevertheless, there is still a need to standardize the assessment of immune-infiltrating cells for the prognostic classification of patients with early or late progressing disease. We studied a retrospective cohort of 49 FL lymph node biopsies at the time of the initial diagnosis using pathologist-guided analysis on whole slide images, and we characterized the immune repertoire for both quantity and distribution (intrafollicular, IF and extrafollicular, EF) of cell subsets in relation to clinical outcome. We looked for the natural killer (CD56), T lymphocyte (CD8, CD4, PD1) and macrophage (CD68, CD163, MA4A4A)-associated markers. High CD163/CD8 EF ratios and high CD56/MS4A4A EF ratios, according to Kaplan–Meier estimates were linked with shorter EFS (event-free survival), with the former being the only one associated with POD24. In contrast to IF CD68+ cells, which represent a more homogeneous population, higher in non-progressing patients, EF CD68+ macrophages did not stratify according to survival. We also identify distinctive MS4A4A+CD163-macrophage populations with different prognostic weights. Enlarging the macrophage characterization and combining it with a lymphoid marker in the rituximab era, in our opinion, may enable prognostic stratification for low-/high-grade FL patients beyond POD24. These findings warrant validation across larger FL cohorts

Misure critiche. N.s. A. 7-8, n. 1-2 (2008-2009)

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