Miguel Sena-Esteves

Bio: Miguel Sena-Esteves is an academic researcher from University of Massachusetts Medical School. The author has contributed to research in topics: Genetic enhancement & Gene delivery. The author has an hindex of 46, co-authored 151 publications receiving 10542 citations. Previous affiliations of Miguel Sena-Esteves include University of Cologne & University of Porto.

Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers

Abstract: Glioblastoma tumour cells release microvesicles (exosomes) containing mRNA, miRNA and angiogenic proteins. These microvesicles are taken up by normal host cells, such as brain microvascular endothelial cells. By incorporating an mRNA for a reporter protein into these microvesicles, we demonstrate that messages delivered by microvesicles are translated by recipient cells. These microvesicles are also enriched in angiogenic proteins and stimulate tubule formation by endothelial cells. Tumour-derived microvesicles therefore serve as a means of delivering genetic information and proteins to recipient cells in the tumour environment. Glioblastoma microvesicles also stimulated proliferation of a human glioma cell line, indicating a self-promoting aspect. Messenger RNA mutant/variants and miRNAs characteristic of gliomas could be detected in serum microvesicles of glioblastoma patients. The tumour-specific EGFRvIII was detected in serum microvesicles from 7 out of 25 glioblastoma patients. Thus, tumour-derived microvesicles may provide diagnostic information and aid in therapeutic decisions for cancer patients through a blood test.

Therapeutic silencing of mutant huntingtin with siRNA attenuates striatal and cortical neuropathology and behavioral deficits

Abstract: Huntington's disease (HD) is an autosomal dominant disease caused by a CAG repeat expansion in the Htt gene (1). Mutant Htt causes neuronal death, dementia, and movement dysfunction; there is no effective treatment. In an inducible transgenic mouse model of HD, turning off transgene expression reversed neuropathology and motor deficits (2). Lowering mutant Htt gene expression in brain may treat HD. In mice, viral vector delivery of short hairpin RNAs (shRNAs) against mutant Htt gene exon 1 or genes that cause other neurodegenerative disorders reduced neuropathology and motor deficits (3–10). Brain delivery of adeno-associated virus (AAV)-shRNA against mutant Htt improved signs of disease in HD transgenic models (7, 11). In the inaugural study on RNAi targeting Htt in vivo, shRNA against Htt in AAV2, delivered to the N171–82Q transgenic model of HD, improved ambulation at 4 months and rotarod performance at 10 and 18 weeks after injection (7). Five and one-half months after shRNA administration, quantitative RT-PCR revealed a 50% reduction in striatal Htt mRNA. Statistical changes in quantification of Htt protein reduction and inclusions were not reported. AAV5 delivery of shRNA against Htt in the R6/1 murine model of HD showed a 25% decrease in Htt protein and an 80% reduction in Htt mRNA 10 weeks after shRNA injection (10). The shRNA delayed onset of clasping by 2 weeks (20–22 weeks), and treated mice had fewer clasps. No difference in rotarod performance was detected. Inclusion size and number decreased in the striatum, but not in the cortex, compared with the corresponding contralateral brain regions. The authors provided an important caveat that one of the shRNAs had off-target effects; the cause of the off-target effects was not established. shRNA in AAV2 or AAV5 was used to target EGFP to knock down EGFP-Htt in another transgenic model of HD (11). shRNA reversed pathology after the onset of pathologic changes; however, behaviors were not studied. Administration of large amounts of siRNA against Htt in a Lipofectamine 2000 suspension into the lateral ventricle of newborn R6/2 transgenic mice (exon 1 of Htt) reduced whole-brain levels of mutant Htt in two mice and Htt mRNA up to 7 days posttreatment, delayed the onset of clasping, rotarod, and open-field phenotypes, and improved survival (12). Statistical quantification of neuropathology was not reported. Thus, prior studies examining RNAi against Htt provided the groundwork for therapeutic gene silencing in HD. Most of the studies used viral delivery of shRNA, and the study using siRNA required liposome delivery to newborns, with the potential liposome neuronal toxicity. Caveats attend the use of shRNAs, which can be toxic when integrated into the host genome (13, 14), in part because shRNA production is unregulated. Long siRNAs (>29 nt) and shRNAs are prone to activate off-target gene expression (15). For patient safety, shRNA will need to be able to be switched off, currently a hurdle in viral delivery systems. An alternative strategy for HD therapy is the use of small-interfering RNAs (siRNAs), ≈21-nt RNA duplexes. siRNA has been administered into cerebroventricles, vasculature, intrathecal space, and parenchyma (16–20). siRNAs were found effective and safe when introduced into mice and non-human primates (19, 21, 22). Several limitations impede progress in using siRNAs as a treatment for HD: entry and effectiveness in adult neurons without the use of potentially toxic transfection reagents; a clear demonstration that gene silencing reduces protein expression; and an improvement in behavioral deficits and neuropathology, especially neuron survival. Because bioactive molecules conjugated to cholesterol have improved cellular uptake in vitro (23), LDL receptors have been detected in brain (24), and cholesterol conjugation enhances siRNA uptake in cells outside of the central nervous system (16), we speculated that cholesterol-conjugated (cc) siRNA might enter neurons in vivo. An in vivo, rapid-onset model of HD would be optimal to test gene silencing in brain. The current rapid mouse model of HD shows mutant Htt-induced pathology after 2 months. Transgenic mice expressing exon 1 of mutant Htt [R6/2 (25)] develop nuclear inclusions throughout the brain at 2 months of age and exhibit a rapidly progressing, severe phenotype. Other transgenic or knock-in mice expressing mutant Htt exhibit late-onset, mild phenotypes, often after 6 months of age (26, 27), and lack prominent neuronal loss. Neither model is ideal to test transient effects of a single injection of siRNA against Htt introduced directly to the striatum. We therefore developed an acute, in vivo, HD mouse model tailored to addressing the efficacy of siRNA. Here, we used AAV to deliver a 1,395-nt cDNA fragment of human mutant Htt into mouse striatum to evaluate the effectiveness of an siRNA targeting human Htt.

Selective Deletion of Bdnf in the Ventromedial and Dorsomedial Hypothalamus of Adult Mice Results in Hyperphagic Behavior and Obesity

Abstract: Brain-derived neurotrophic factor (BDNF) and its receptor TrkB are expressed in several hypothalamic and hindbrain nuclei involved in regulating energy homeostasis, developmentally and in the adult animal. Their depletion during the fetal or early postnatal periods when developmental processes are still ongoing elicits hyperphagic behavior and obesity in mice. Whether BDNF is a chief element in appetite control in the mature brain remains controversial. The required sources of this neurotrophin are also unknown. We show that glucose administration rapidly induced BDNF mRNA expression, mediated by Bdnf promoter 1, and TrkB transcription in the ventromedial hypothalamus (VMH) of adult mice, consistent with a role of this pathway in satiety. Using viral-mediated selective knock-down of BDNF in the VMH and dorsomedial hypothalamus (DMH) of adult mice, we were able to elucidate the physiological relevance of BDNF in energy balance regulation. Site-specific mutants exhibited hyperphagic behavior and obesity but normal energy expenditure. Furthermore, intracerebroventricular administration of BDNF triggered an immediate neuronal response in multiple hypothalamic nuclei in wild-type mice, suggesting that its anorexigenic actions involve short-term mechanisms. Locomotor, aggressive, and depressive-like behaviors, all of which are associated with neural circuits involving the VMH, were not altered in VMH/DMH-specific BDNF mutants. These findings demonstrate that BDNF is an integral component of central mechanisms mediating satiety in the adult mouse and, moreover, that its synthesis in the VMH and/or DMH is required for the suppression of appetite.

Extracellular vesicles: exosomes, microvesicles, and friends.

Abstract: Cells release into the extracellular environment diverse types of membrane vesicles of endosomal and plasma membrane origin called exosomes and microvesicles, respectively. These extracellular vesicles (EVs) represent an important mode of intercellular communication by serving as vehicles for transfer between cells of membrane and cytosolic proteins, lipids, and RNA. Deficiencies in our knowledge of the molecular mechanisms for EV formation and lack of methods to interfere with the packaging of cargo or with vesicle release, however, still hamper identification of their physiological relevance in vivo. In this review, we focus on the characterization of EVs and on currently proposed mechanisms for their formation, targeting, and function.

Minimal information for studies of extracellular vesicles 2018 (MISEV2018) : a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

Abstract: The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

Integrative analysis of 111 reference human epigenomes

Abstract: The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but epigenomic studies lack a similar reference. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection so far of human epigenomes for primary cells and tissues. Here we describe the integrative analysis of 111 reference human epigenomes generated as part of the programme, profiled for histone modification patterns, DNA accessibility, DNA methylation and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease- and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation and human disease.

Shedding light on the cell biology of extracellular vesicles.

Abstract: Extracellular vesicles are a heterogeneous group of cell-derived membranous structures comprising exosomes and microvesicles, which originate from the endosomal system or which are shed from the plasma membrane, respectively They are present in biological fluids and are involved in multiple physiological and pathological processes Extracellular vesicles are now considered as an additional mechanism for intercellular communication, allowing cells to exchange proteins, lipids and genetic material Knowledge of the cellular processes that govern extracellular vesicle biology is essential to shed light on the physiological and pathological functions of these vesicles as well as on clinical applications involving their use and/or analysis However, in this expanding field, much remains unknown regarding the origin, biogenesis, secretion, targeting and fate of these vesicles