Publications by authors named "Maria Ericsson"

61 Publications

Plaque associated microglia hyper-secrete extracellular vesicles and accelerate tau propagation in a humanized APP mouse model.

Mol Neurodegener 2021 03 22;16(1):18. Epub 2021 Mar 22.

Departments of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Boston, MA, 02118, USA.

Background: Recent studies suggest that microglia contribute to tau pathology progression in Alzheimer's disease. Amyloid plaque accumulation transforms microglia, the primary innate immune cells in the brain, into neurodegenerative microglia (MGnD), which exhibit enhanced phagocytosis of plaques, apoptotic neurons and dystrophic neurites containing aggregated and phosphorylated tau (p-tau). It remains unclear how microglia promote disease progression while actively phagocytosing pathological proteins, therefore ameliorating pathology.

Methods: Adeno-associated virus expressing P301L tau mutant (AAV-P301L-tau) was stereotaxically injected into the medial entorhinal cortex (MEC) in C57BL/6 (WT) and humanized APP mutant knock-in homozygote (App) mice at 5 months of age. Mice were fed either chow containing a colony stimulating factor-1 receptor inhibitor (PLX5622) or control chow from 4 to 6 months of age to test the effect of microglia depletion. Animals were tested at 6 months of age for immunofluorescence, biochemistry, and FACS of microglia. In order to monitor microglial extracellular vesicle secretion in vivo, a novel lentiviral EV reporter system was engineered to express mEmerald-CD9 (mE-CD9) specifically in microglia, which was injected into the same region of MEC.

Results: Expressing P301L tau mutant in the MEC induced tau propagation to the granule cell layer of the hippocampal dentate gyrus, which was significantly exacerbated in App mice compared to WT control mice. Administration of PLX5622 depleted nearly all microglia in mouse brains and dramatically reduced propagation of p-tau in WT and to a greater extent in App mice, although it increased plaque burden and plaque-associated p-tau dystrophic neurites. Plaque-associated MGnD microglia strongly expressed an EV marker, tumor susceptibility gene 101, indicative of heightened synthesis of EVs. Intracortical injection of mE-CD9 lentivirus successfully induced microglia-specific expression of mE-CD9 EV particles, which were significantly enhanced in Mac2 MGnD microglia compared to Mac2 homeostatic microglia. Finally, consecutive intracortical injection of mE-CD9 lentivirus and AAV-P301L-tau into App mice revealed encapsulation of p-tau in microglia-specific mE-CD9 EVs as determined by super-resolution microscopy and immuno-electron microscopy.

Discussion: Our findings suggest that MGnD microglia hyper-secrete p-tau EVs while compacting Aβ plaques and clearing NP tau, which we propose as a novel mechanistic link between amyloid plaque deposition and exacerbation of tau propagation in App mice.
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http://dx.doi.org/10.1186/s13024-021-00440-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7986521PMC
March 2021

Crowded organelles, lipid accumulation, and abnormal membrane tubulation in cellular models of enhanced α-synuclein membrane interaction.

Brain Res 2021 May 9;1758:147349. Epub 2021 Feb 9.

Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Previous work from our group showed that certain engineered missense mutations to the α-synuclein (αS) KTKEGV repeat motifs abrogate the protein's ability to form native multimers. The resultant excess monomers accumulate in lipid-membrane-rich inclusions associated with neurotoxicity exceeding that of natural familial Parkinson's disease mutants such as E46K. We presented an initial characterization of the lipid-rich inclusions and found similarities to the αS- and vesicle-rich inclusions that form in baker's yeast when αS is expressed. We also discussed, with some caution, a possible role of membrane-rich inclusions as precursors to filamentous Lewy bodies, the widely accepted hallmark pathology of Parkinson's disease and other synucleinopathies. In the meantime, advances in the microscopic characterization of Lewy bodies have highlighted the presence of crowded organelles and lipid membranes in addition to αS accumulation. This prompted us to revisit the αS inclusions caused by our repeat motif variants in neuroblastoma cells. In addition to our previous characterization, we found that these inclusions can often be seen by brightfield microscopy, overlap with endogenous vesicle markers in immunofluorescence experiments, stain positive for lipid dyes, and can be found to be closely associated with mitochondria. We also observed abnormal tubulation of membranes, which was subtle in inducible lines and pronounced in cells that transiently expressed high amounts of the highly disruptive KTKEGV motif mutant "KLKEGV". Membrane tubulation had been reported before as an αS activity in reductionist systems. Our in-cellulo demonstration now suggests that this mechanism could possibly be a relevant aspect of aberrant αS behavior in cells.
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http://dx.doi.org/10.1016/j.brainres.2021.147349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7988302PMC
May 2021

Alzheimer's disease brain-derived extracellular vesicles spread tau pathology in interneurons.

Brain 2021 02;144(1):288-309

Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Boston, MA 02118, USA.

Extracellular vesicles are highly transmissible and play critical roles in the propagation of tau pathology, although the underlying mechanism remains elusive. Here, for the first time, we comprehensively characterized the physicochemical structure and pathogenic function of human brain-derived extracellular vesicles isolated from Alzheimer's disease, prodromal Alzheimer's disease, and non-demented control cases. Alzheimer's disease extracellular vesicles were significantly enriched in epitope-specific tau oligomers in comparison to prodromal Alzheimer's disease or control extracellular vesicles as determined by dot blot and atomic force microscopy. Alzheimer's disease extracellular vesicles were more efficiently internalized by murine cortical neurons, as well as more efficient in transferring and misfolding tau, than prodromal Alzheimer's disease and control extracellular vesicles in vitro. Strikingly, the inoculation of Alzheimer's disease or prodromal Alzheimer's disease extracellular vesicles containing only 300 pg of tau into the outer molecular layer of the dentate gyrus of 18-month-old C57BL/6 mice resulted in the accumulation of abnormally phosphorylated tau throughout the hippocampus by 4.5 months, whereas inoculation of an equal amount of tau from control extracellular vesicles, isolated tau oligomers, or fibrils from the same Alzheimer's disease donor showed little tau pathology. Furthermore, Alzheimer's disease extracellular vesicles induced misfolding of endogenous tau in both oligomeric and sarkosyl-insoluble forms in the hippocampal region. Unexpectedly, phosphorylated tau was primarily accumulated in glutamic acid decarboxylase 67 (GAD67) GABAergic interneurons and, to a lesser extent, glutamate receptor 2/3-positive excitatory mossy cells, showing preferential extracellular vesicle-mediated GABAergic interneuronal tau propagation. Whole-cell patch clamp recordings of CA1 pyramidal cells showed significant reduction in the amplitude of spontaneous inhibitory post-synaptic currents. This was accompanied by reductions in c-fos+ GAD67+ neurons and GAD67+ neuronal puncta surrounding pyramidal neurons in the CA1 region, confirming reduced GABAergic transmission in this region. Our study posits a novel mechanism for the spread of tau in hippocampal GABAergic interneurons via brain-derived extracellular vesicles and their subsequent neuronal dysfunction.
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http://dx.doi.org/10.1093/brain/awaa376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880668PMC
February 2021

Multiresolution Imaging Using Bioluminescence Resonance Energy Transfer Identifies Distinct Biodistribution Profiles of Extracellular Vesicles and Exomeres with Redirected Tropism.

Adv Sci (Weinh) 2020 Oct 16;7(19):2001467. Epub 2020 Aug 16.

Institute of Atomic and Molecular Sciences Academia Sinica Taipei 10617 Taiwan.

Extracellular particles (EPs) including extracellular vesicles (EVs) and exomeres play significant roles in diseases and therapeutic applications. However, their spatiotemporal dynamics in vivo have remained largely unresolved in detail due to the lack of a suitable method. Therefore, a bioluminescence resonance energy transfer (BRET)-based reporter, PalmGRET, is created to enable pan-EP labeling ranging from exomeres (<50 nm) to small (<200 nm) and medium and large (>200 nm) EVs. PalmGRET emits robust, sustained signals and allows the visualization, tracking, and quantification of the EPs from whole animal to nanoscopic resolutions under different imaging modalities, including bioluminescence, BRET, and fluorescence. Using PalmGRET, it is shown that EPs released by lung metastatic hepatocellular carcinoma (HCC) exhibit lung tropism with varying distributions to other major organs in immunocompetent mice. It is further demonstrated that gene knockdown of lung-tropic membrane proteins, solute carrier organic anion transporter family member 2A1, alanine aminopeptidase/Cd13, and chloride intracellular channel 1 decreases HCC-EP distribution to the lungs and yields distinct biodistribution profiles. It is anticipated that EP-specific imaging, quantitative assays, and detailed in vivo characterization are a starting point for more accurate and comprehensive in vivo models of EP biology and therapeutic design.
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http://dx.doi.org/10.1002/advs.202001467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7539214PMC
October 2020

Mesenchymal stromal cell-derived small extracellular vesicles restore lung architecture and improve exercise capacity in a model of neonatal hyperoxia-induced lung injury.

J Extracell Vesicles 2020 Jul 13;9(1):1790874. Epub 2020 Jul 13.

Division of Newborn Medicine & Department of Pediatrics, Boston Children's Hospital, Boston, MA, USA.

Early administration of mesenchymal stromal cell (MSC)-derived small extracellular vesicles (MEx) has shown considerable promise in experimental models of bronchopulmonary dysplasia (BPD). However, the ability of MEx to reverse the long-term pulmonary complications associated with established BPD remains unknown. In this study, MEx were isolated from media conditioned by human Wharton's Jelly-derived MSC cultures. Newborn mice (FVB strain) were exposed to hyperoxia (HYRX (75% O2)) before returning to room air at postnatal day 14 (PN14). Following prolonged HYRX-exposure, animals received a single MEx dose at PN18 or serial MEx treatments at PN18-39 ("late" intervention). This group was compared to animals that received an early single MEx dose at PN4 ("early" intervention). Animals were harvested at PN28 or 60 for assessment of pulmonary parameters. We found that early and late MEx interventions effectively ameliorated core features of HYRX-induced neonatal lung injury, improving alveolar simplification, pulmonary fibrosis, vascular remodelling and blood vessel loss. Exercise capacity testing and assessment of pulmonary hypertension (PH) showed functional improvements following both early and late MEx interventions. In conclusion, delivery of MEx following prolonged HYRX-exposure improves core features of experimental BPD, restoring lung architecture, decreasing pulmonary fibrosis and vascular muscularization, ameliorating PH and improving exercise capacity. Taken together, delivery of MEx may not only be effective in the immediate neonatal period to prevent the development of BPD but may provide beneficial effects for the management and potentially the reversal of cardiorespiratory complications in infants and children with established BPD.
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http://dx.doi.org/10.1080/20013078.2020.1790874DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7480622PMC
July 2020

The Human and Mouse Enteric Nervous System at Single-Cell Resolution.

Cell 2020 09 3;182(6):1606-1622.e23. Epub 2020 Sep 3.

Klarman Cell Observatory, Broad Institute of MIT and Harvard, Cambridge, MA, USA; Howard Hughes Medical Institute and Koch Institute for Integrative Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA. Electronic address:

The enteric nervous system (ENS) coordinates diverse functions in the intestine but has eluded comprehensive molecular characterization because of the rarity and diversity of cells. Here we develop two methods to profile the ENS of adult mice and humans at single-cell resolution: RAISIN RNA-seq for profiling intact nuclei with ribosome-bound mRNA and MIRACL-seq for label-free enrichment of rare cell types by droplet-based profiling. The 1,187,535 nuclei in our mouse atlas include 5,068 neurons from the ileum and colon, revealing extraordinary neuron diversity. We highlight circadian expression changes in enteric neurons, show that disease-related genes are dysregulated with aging, and identify differences between the ileum and proximal/distal colon. In humans, we profile 436,202 nuclei, recovering 1,445 neurons, and identify conserved and species-specific transcriptional programs and putative neuro-epithelial, neuro-stromal, and neuro-immune interactions. The human ENS expresses risk genes for neuropathic, inflammatory, and extra-intestinal diseases, suggesting neuronal contributions to disease.
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http://dx.doi.org/10.1016/j.cell.2020.08.003DOI Listing
September 2020

Increased lysosomal biomass is responsible for the resistance of triple-negative breast cancers to CDK4/6 inhibition.

Sci Adv 2020 Jun 17;6(25):eabb2210. Epub 2020 Jun 17.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA.

Inhibitors of cyclin-dependent kinases CDK4 and CDK6 have been approved for treatment of hormone receptor-positive breast cancers. In contrast, triple-negative breast cancers (TNBCs) are resistant to CDK4/6 inhibition. Here, we demonstrate that a subset of TNBC critically requires CDK4/6 for proliferation, and yet, these TNBC are resistant to CDK4/6 inhibition due to sequestration of CDK4/6 inhibitors into tumor cell lysosomes. This sequestration is caused by enhanced lysosomal biogenesis and increased lysosomal numbers in TNBC cells. We developed new CDK4/6 inhibitor compounds that evade the lysosomal sequestration and are efficacious against resistant TNBC. We also show that coadministration of lysosomotropic or lysosome-destabilizing compounds (an antibiotic azithromycin, an antidepressant siramesine, an antimalaria compound chloroquine) renders resistant tumor cells sensitive to currently used CDK4/6 inhibitors. Lastly, coinhibition of CDK2 arrested proliferation of CDK4/6 inhibitor-resistant cells. These observations may extend the use of CDK4/6 inhibitors to TNBCs that are refractory to current anti-CDK4/6 therapies.
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http://dx.doi.org/10.1126/sciadv.abb2210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7360435PMC
June 2020

In Vivo Quasi-Elastic Light Scattering Eye Scanner Detects Molecular Aging in Humans.

J Gerontol A Biol Sci Med Sci 2020 09;75(9):e53-e62

Molecular Aging & Development Laboratory, Boston University School of Medicine, Massachusetts.

The absence of clinical tools to evaluate individual variation in the pace of aging represents a major impediment to understanding aging and maximizing health throughout life. The human lens is an ideal tissue for quantitative assessment of molecular aging in vivo. Long-lived proteins in lens fiber cells are expressed during fetal life, do not undergo turnover, accumulate molecular alterations throughout life, and are optically accessible in vivo. We used quasi-elastic light scattering (QLS) to measure age-dependent signals in lenses of healthy human subjects. Age-dependent QLS signal changes detected in vivo recapitulated time-dependent changes in hydrodynamic radius, protein polydispersity, and supramolecular order of human lens proteins during long-term incubation (~1 year) and in response to sustained oxidation (~2.5 months) in vitro. Our findings demonstrate that QLS analysis of human lens proteins provides a practical technique for noninvasive assessment of molecular aging in vivo.
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http://dx.doi.org/10.1093/gerona/glaa121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494032PMC
September 2020

A nano-carrier platform for the targeted delivery of nature-inspired antimicrobials using Engineered Water Nanostructures for food safety applications.

Food Control 2019 Feb 29;96:365-374. Epub 2018 Sep 29.

Center for Nanotechnology and Nanotoxicology, Department of Environmental Health, Harvard T. H. Chan School of Public Health, Harvard University, Boston, MA 02115, USA.

Despite the progress in the area of food safety, foodborne diseases still represent a massive challenge to the public health systems worldwide, mainly due to the substantial inefficiencies across the farm-to-fork continuum. Here, we report the development of a nano-carrier platform, for the targeted and precise delivery of antimicrobials for the inactivation of microorganisms on surfaces using Engineered Water Nanostructures (EWNS). An aqueous suspension of an active ingredient (AI) was used to synthesize iEWNS, with the 'i' denoting the AI used in their synthesis, using a combined electrospray and ionization process. The iEWNS possess unique, active-ingredient-dependent physicochemical properties: i) they are engineered to have a tunable size in the nanoscale; ii) they have excessive electric surface charge, and iii) they contain both the reactive oxygen species (ROS) formed due to the ionization of deionized (DI) water, and the AI used in their synthesis. Their charge can be used in combination with an electric field to target them onto a surface of interest. In this approach, a number of nature-inspired antimicrobials, such as HO, lysozyme, citric acid, and their combination, were used to synthesize a variety of iEWNS-based nano-sanitizers. It was demonstrated through foodborne-pathogen-inactivation experiments that due to the targeted and precise delivery, and synergistic effects of AI and ROS incorporated in the iEWNS structure, a pico- to nanogram-level dose of the AI delivered to the surface using this nano-carrier platform is capable of achieving 5-log reductions in minutes of exposure time. This aerosol-based, yet 'dry' intervention approach using iEWNS nano-carrier platform offers advantages over current 'wet' techniques that are prevalent commercially, which require grams of the AI to achieve similar inactivation, leading to increased chemical risks and chemical waste byproducts. Such a targeted nano-carrier approach has the potential to revolutionize the delivery of antimicrobials for sterilization in the food industry.
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http://dx.doi.org/10.1016/j.foodcont.2018.09.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7055713PMC
February 2019

Understanding cellular signaling and systems biology with precision: A perspective from ultrastructure and organelle studies in the midgut.

Curr Opin Syst Biol 2018 Oct 20;11:24-31. Epub 2018 Jul 20.

Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.

One of the aims of systems biology is to model and discover properties of cells, tissues and organisms functioning as a system. In recent years, studies in the adult gut have provided a wealth of information on the cell types and their functions, and the signaling pathways involved in the complex interactions between proliferating and differentiated cells in the context of homeostasis and pathology. Here, we document and discuss how high-resolution ultrastructure studies of organelle morphology have much to contribute to our understanding of how the gut functions as an integrated system.
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http://dx.doi.org/10.1016/j.coisb.2018.07.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6781628PMC
October 2018

Female Sex and Brain-Selective Estrogen Benefit α-Synuclein Tetramerization and the PD-like Motor Syndrome in 3K Transgenic Mice.

J Neurosci 2019 09 12;39(38):7628-7640. Epub 2019 Aug 12.

Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, and

Many studies report a higher risk for Parkinson's disease (PD) and younger age of onset in men. This, and the fact that the neuropathological process underlying PD symptoms may begin before menopause, suggests that estrogen-based hormone therapy could modify this higher risk in males. However, the effects of female sex or estrogen on α-synuclein (αS) homeostasis and related PD neuropathology remain unknown. Here, we used an αS tetramer-abrogating mouse model of PD (3K) that amplifies the familial E46K PD mutation to investigate the effects of female sex and brain-selective estrogen treatment on αS tetramerization and solubility, formation of vesicle-rich αS aggregates, dopaminergic and cortical fiber integrity, and associated motor deficits. In male 3K mice, the motor phenotype became apparent at ∼10 weeks and increased to age 6 months, paralleled by PD-like neuropathology, whereas 3K females showed a significant delay in onset. At 6 months, this beneficial phenotypic effect in 3K females was associated with a higher αS tetramer-to-monomer ratio and less decrease in dopaminergic and cortical fiber length and quantity. Brain-selective estrogen treatment in symptomatic 3K mice significantly increased the tetramer-to-monomer ratio, turnover by autophagy of aggregate-prone monomers, and neurite complexity of surviving DAergic and cortical neurons, in parallel with benefits in motor performance. Our findings support an upstream role for αS tetramer loss in PD phenotypes and a role for estrogen in mitigating PD-like neuropathology Brain-selective estrogen therapy may be useful in delaying or reducing PD symptoms in men and postmenopausal women. The mechanisms responsible for the male-to-female preponderance in Parkinson's disease (PD) are not well understood yet important for treatment efficacy. We previously showed that abrogating native α-synuclein (αS) tetramers produces a close PD model, including dopaminergic and cortical fiber loss and a progressive motor disorder responsive to l-DOPA. Here, we analyzed sex and use 10b-17β-dihydroxyestra-1,4-dien-3-one treatment of symptomatic 3K males, and demonstrate that the beneficial effects of female sex on PD-like neuropathology can be reinstated by elevating estrogen in the male brain. The study provides evidence that 17β-estradiol restores the tetramer-to-monomer ratio by autophagy turnover of excess αS monomers, vesicle and fiber integrity in brain regions critically involved in motor behavior. These data provide the basis for understanding sex differences in αS homeostasis and the development of therapeutic approaches to treating men and postmenopausal women with PD.
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http://dx.doi.org/10.1523/JNEUROSCI.0313-19.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6750939PMC
September 2019

The late stage of COPI vesicle fission requires shorter forms of phosphatidic acid and diacylglycerol.

Nat Commun 2019 07 30;10(1):3409. Epub 2019 Jul 30.

Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Harvard Medical School, 60 Fenwood Road, Boston, MA, 02115, USA.

Studies on vesicle formation by the Coat Protein I (COPI) complex have contributed to a basic understanding of how vesicular transport is initiated. Phosphatidic acid (PA) and diacylglycerol (DAG) have been found previously to be required for the fission stage of COPI vesicle formation. Here, we find that PA with varying lipid geometry can all promote early fission, but only PA with shortened acyl chains promotes late fission. Moreover, diacylglycerol (DAG) acts after PA in late fission, with this role of DAG also requiring shorter acyl chains. Further highlighting the importance of the short-chain lipid geometry for late fission, we find that shorter forms of PA and DAG promote the vesiculation ability of COPI fission factors. These findings advance a general understanding of how lipid geometry contributes to membrane deformation for vesicle fission, and also how proteins and lipids coordinate their actions in driving this process.
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http://dx.doi.org/10.1038/s41467-019-11324-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667475PMC
July 2019

6-Phosphogluconate Dehydrogenase Links Cytosolic Carbohydrate Metabolism to Protein Secretion via Modulation of Glutathione Levels.

Cell Chem Biol 2019 09 13;26(9):1306-1314.e5. Epub 2019 Jun 13.

Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA. Electronic address:

The proteinaceous extracellular matrix (ECM) is vital for the survival, proliferation, migration, and differentiation of many types of cancer. However, little is known regarding metabolic pathways required for ECM secretion. By using an unbiased computational approach, we searched for enzymes whose suppression may lead to disruptions in protein secretion. Here, we show that 6-phosphogluconate dehydrogenase (PGD), a cytosolic enzyme involved in carbohydrate metabolism, is required for ER structural integrity and protein secretion. Chemical inhibition or genetic suppression of PGD activity led to cell stress accompanied by significantly expanded ER volume and was rescued by compensating endogenous glutathione supplies. Our results also suggest that this characteristic ER-dilation phenotype may be a general marker indicating increased ECM protein congestion inside cells and decreased secretion. Thus, PGD serves as a link between cytosolic carbohydrate metabolism and protein secretion.
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http://dx.doi.org/10.1016/j.chembiol.2019.05.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6754299PMC
September 2019

Interaction between tele-nurses and callers with an evolving myocardial infarction: Consequences for level of directed care.

Eur J Cardiovasc Nurs 2019 10 9;18(7):545-553. Epub 2019 May 9.

Department of Cardiology and Department of Medical and Health Sciences, Linköping University, Linköping, Sweden.

Background: Rapid contact with emergency medical services is imperative to save the lives of acute myocardial infarction patients. However, many patients turn to a telehealth advisory nurse instead, where the delivery of urgent and safe care largely depends on how the interaction in the call is established.

Purpose: The purpose of this study was to explore the interaction between tele-nurses and callers with an evolving myocardial infarction after contacting a national telehealth advisory service number as their first medical contact.

Method: Twenty men and 10 women (aged 46-89 years) were included. Authentic calls were analysed using inductive content analysis.

Findings: One overall category, Movement towards directed level of care, labelled the whole interaction between the tele-nurse and the caller. Four categories conceptualised the different interactions: a distinct, reasoning, indecisive or irrational interaction. The interactions described how tele-nurses and callers assessed and elaborated on symptoms, context and actions. The interaction was pivotal for progress in the dialogue and affected the achievement of mutual understanding in the communicative process. An indecisive or irrational interaction could increase the risk of failing to recommend or call for acute care.

Conclusion: The interaction in the communication could either lead or mislead the level of care directed in the call. This study adds new perspectives to the communicative process in the acute setting in order to identify a myocardial infarction and the level of urgency from both individuals experiencing myocardial infarction and professionals in the health system.
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http://dx.doi.org/10.1177/1474515119848195DOI Listing
October 2019

A comparison of clinically relevant sources of mesenchymal stem cell-derived exosomes: Bone marrow and amniotic fluid.

J Pediatr Surg 2019 Jan 5;54(1):86-90. Epub 2018 Oct 5.

Department of Surgery, Boston Children's Hospital, Boston and Cambridge, MA. Electronic address:

Background/purpose: Exosomes may constitute a more practical alternative to live cells in select stem cell-based therapies. We sought to compare exosomes from two mesenchymal stem cell (MSC) sources relevant to perinatal and pediatric diseases.

Methods: Exosomes were isolated by reagent-enhanced centrifugation from cell culture media of banked human bone marrow (bm) and amniotic fluid (af) MSCs after serum starvation. Characterization was by flow exometry for tetraspanin markers CD9, CD63, and CD81, transmission electron microscopy for size and morphology, and tunable resistive pulse sensing for size distribution and concentration. Statistical comparisons of count data were made by Poisson regression modeling and Student's T-test.

Results: Exosomes of appropriate size and morphology were isolated with comparable expressions of CD9 (96% vs. 94%), CD63 (88% vs. 66%), and CD81 (71% vs. 63%) for bmMSC and afMSC, respectively. Total exosome yield (particles/mL) adjusted for number of cells was higher from afMSCs than bmMSCs by an estimated 25% (P < 0.001).

Conclusions: While bone marrow and amniotic fluid mesenchymal stem cells are comparable sources of exosomes in size distribution, morphology, and expression of typical surface markers, yield may be higher from amniotic fluid cells. The amniotic fluid appears to be a preferable source of exosomes for clinical applications.

Level Of Evidence: N/A (bench laboratory study).
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http://dx.doi.org/10.1016/j.jpedsurg.2018.10.020DOI Listing
January 2019

Abrogating Native α-Synuclein Tetramers in Mice Causes a L-DOPA-Responsive Motor Syndrome Closely Resembling Parkinson's Disease.

Neuron 2018 10;100(1):75-90.e5

Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA. Electronic address:

α-Synuclein (αS) regulates vesicle exocytosis but forms insoluble deposits in Parkinson's disease (PD). Developing disease-modifying therapies requires animal models that reproduce cardinal features of PD. We recently described a previously unrecognized physiological form of αS, α-helical tetramers, and showed that familial PD-causing missense mutations shift tetramers to aggregation-prone monomers. Here, we generated mice expressing the fPD E46K mutation plus 2 homologous E→K mutations in adjacent KTKEGV motifs. This tetramer-abrogating mutant causes phenotypes similar to PD. αS monomers accumulate at membranes and form vesicle-rich inclusions. αS becomes insoluble, proteinase K-resistant, Ser129-phosphorylated, and C-terminally truncated, as in PD. These changes affect regions controlling motor behavior, including a decrease in nigrostriatal dopaminergic neurons. The outcome is a progressive motor syndrome including tremor and gait and limb deficits partially responsive to L-DOPA. This fully penetrant phenotype indicates that tetramers are required for normal αS homeostasis and that chronically shifting tetramers to monomers may result in PD, with attendant therapeutic implications.
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http://dx.doi.org/10.1016/j.neuron.2018.09.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6211795PMC
October 2018

The pseudokinase MLKL activates PAD4-dependent NET formation in necroptotic neutrophils.

Sci Signal 2018 09 4;11(546). Epub 2018 Sep 4.

Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA 02115, USA.

Neutrophil extracellular trap (NET) formation can generate short-term, functional anucleate cytoplasts and trigger loss of cell viability. We demonstrated that the necroptotic cell death effector mixed lineage kinase domain-like (MLKL) translocated from the cytoplasm to the plasma membrane and stimulated downstream NADPH oxidase-independent ROS production, loss of cytoplasmic granules, breakdown of the nuclear membrane, chromatin decondensation, histone hypercitrullination, and extrusion of bacteriostatic NETs. This process was coordinated by receptor-interacting protein kinase-1 (RIPK1), which activated the caspase-8-dependent apoptotic or RIPK3/MLKL-dependent necroptotic death of mouse and human neutrophils. Genetic deficiency of RIPK3 and MLKL prevented NET formation but did not prevent cell death, which was because of residual caspase-8-dependent activity. Peptidylarginine deiminase 4 (PAD4) was activated downstream of RIPK1/RIPK3/MLKL and was required for maximal histone hypercitrullination and NET extrusion. This work defines a distinct signaling network that activates PAD4-dependent NET release for the control of methicillin-resistant (MRSA) infection.
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http://dx.doi.org/10.1126/scisignal.aao1716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6301070PMC
September 2018

Gender disparities in first medical contact and delay in ST-elevation myocardial infarction: a prospective multicentre Swedish survey study.

BMJ Open 2018 05 3;8(5):e020211. Epub 2018 May 3.

Department of Cardiology and Department of Medical and Health Sciences, Linköping University, Linköping, Sweden.

Objectives: Compare gender disparities in ST-elevation myocardial infarction (STEMI) regarding first medical contact (FMC) and prehospital delay times and explore factors associated with prehospital delay in men and women separately.

Design: Cross-sectional study based on medical records and a validated questionnaire. Eligible patients were enrolled within 24 hours after admittance to hospital.

Setting: Patients were included from November 2012 to January 2014 from five Swedish hospitals with catheterisation facilities 24/7.

Participants: 340 men and 109 women aged between 31 and 95 years completed the survey.

Main Outcome Measures: FMC were divided into five possible contacts: primary healthcare centre by phone or directly, national advisory nurse by phone, emergency medical services (EMS) and emergency room directly. Two parts of prehospital delay times were studied: time from symptom onset to FMC and time from symptom onset to diagnostic ECG.

Results: Women more often called an advisory nurse as FMC (28% vs 18%, p=0.02). They had a longer delay until FMC, 90 (IQR 39-221) vs 66 (28-161) min, p=0.04 and until ECG, 146 (68-316) vs 103 (61-221) min, p=0.03. Men went to hospital because of believing they were stricken by an MI to a higher extent than women did (25% vs 15%, p=0.04) and were more often recommended to call EMS by bystanders (38% vs 22%, p<0.01). Hesitating about going to hospital and experiencing pain in the stomach/back/shoulders were factors associated with longer delays in women. Believing the symptoms would disappear or interpreting them as nothing serious were corresponding factors in men. In both genders bystanders acting by contacting EMS explained shorter prehospital delays.

Conclusions: In STEMI, women differed from men in FMC and they had longer delays. This was partly due to atypical symptoms and a longer decision time. Bystanders acted more promptly when men than when women fell ill. Public knowledge of MI symptoms, and how to act properly, still seems insufficient.
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http://dx.doi.org/10.1136/bmjopen-2017-020211DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5942442PMC
May 2018

Gender differences in symptom presentation of ST-elevation myocardial infarction - An observational multicenter survey study.

Int J Cardiol 2018 08 24;264:7-11. Epub 2018 Mar 24.

Department of Cardiology, Department of Medical and Health Sciences, Linköping University, Linköping, Sweden. Electronic address:

Background: Symptom presentation has been sparsely studied from a gender perspective restricting the inclusion to ST elevation myocardial infarction (STEMI) patients. Correct symptom recognition is vital in order to promptly seek care in STEMI where fast reperfusion therapy is of utmost importance. Female gender has been found associated with atypical presentation in studies on mixed MI populations but it is unclear whether this is valid also in STEMI.

Objectives: We assessed whether there are gender differences in symptoms and interpretation of these in STEMI, and if this is attributable to sociodemographic and clinical factors.

Methods: SymTime was a multicenter observational study including a validated questionnaire and data from medical records. Eligible STEMI patients (n = 532) were enrolled within 24 h after admittance at five Swedish hospitals.

Results: Women were older, more often single and had lower educational level. Chest pain was less prevalent in women (74 vs 93%, p < 0.001), whereas shoulder (33 vs 15%, p < 0.001), throat/neck (34 vs 18%, p < 0.001), back pain (29 versus 12%, p < 0.001) and nausea (49 vs 29%, p < 0.001) were more prevalent. Women less often interpreted their symptoms as of cardiac origin (60 vs 69%, p = 0.04). Female gender was the strongest independent predictor of non-chest pain presentation, odds ratio 5.29, 95% confidence interval 2.85-9.80.

Conclusions: A striking gender difference in STEMI symptoms was found. As women significantly misinterpreted their symptoms more often, it is vital when informing about MI to the society or to high risk individuals, to highlight also other symptoms than just chest pain.
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http://dx.doi.org/10.1016/j.ijcard.2018.03.084DOI Listing
August 2018

Mutant torsinA in the heterozygous DYT1 state compromises HSV propagation in infected neurons and fibroblasts.

Sci Rep 2018 02 2;8(1):2324. Epub 2018 Feb 2.

Molecular Neurogenetics Unit, Department of Neurology and Center for Molecular Imaging Research, Department of Radiology, Massachusetts General Hospital and Program in Neuroscience, Harvard Medical School, Boston, MA, 02114, USA.

Most cases of early onset torsion dystonia (DYT1) are caused by a 3-base pair deletion in one allele of the TOR1A gene causing loss of a glutamate in torsinA, a luminal protein in the nuclear envelope. This dominantly inherited neurologic disease has reduced penetrance and no other medical manifestations. It has been challenging to understand the neuronal abnormalities as cells and mouse models which are heterozygous (Het) for the mutant allele are quite similar to wild-type (WT) controls. Here we found that patient fibroblasts and mouse neurons Het for this mutation showed significant differences from WT cells in several parameters revealed by infection with herpes simplex virus type 1 (HSV) which replicates in the nucleus and egresses out through the nuclear envelope. Using a red fluorescent protein capsid to monitor HSV infection, patient fibroblasts showed decreased viral plaque formation as compared to controls. Mouse Het neurons had a decrease in cytoplasmic, but not nuclear HSV fluorescence, and reduced numbers of capsids entering axons as compared to infected WT neurons. These findings point to altered dynamics of the nuclear envelope in cells with the patient genotype, which can provide assays to screen for therapeutic agents that can normalize these cells.
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http://dx.doi.org/10.1038/s41598-018-19865-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5797141PMC
February 2018

Concussion, microvascular injury, and early tauopathy in young athletes after impact head injury and an impact concussion mouse model.

Brain 2018 02;141(2):422-458

Alzheimer's Disease Center, CTE Program, Boston University School of Medicine, Boston, MA 02118, USA.

The mechanisms underpinning concussion, traumatic brain injury, and chronic traumatic encephalopathy, and the relationships between these disorders, are poorly understood. We examined post-mortem brains from teenage athletes in the acute-subacute period after mild closed-head impact injury and found astrocytosis, myelinated axonopathy, microvascular injury, perivascular neuroinflammation, and phosphorylated tau protein pathology. To investigate causal mechanisms, we developed a mouse model of lateral closed-head impact injury that uses momentum transfer to induce traumatic head acceleration. Unanaesthetized mice subjected to unilateral impact exhibited abrupt onset, transient course, and rapid resolution of a concussion-like syndrome characterized by altered arousal, contralateral hemiparesis, truncal ataxia, locomotor and balance impairments, and neurobehavioural deficits. Experimental impact injury was associated with axonopathy, blood-brain barrier disruption, astrocytosis, microgliosis (with activation of triggering receptor expressed on myeloid cells, TREM2), monocyte infiltration, and phosphorylated tauopathy in cerebral cortex ipsilateral and subjacent to impact. Phosphorylated tauopathy was detected in ipsilateral axons by 24 h, bilateral axons and soma by 2 weeks, and distant cortex bilaterally at 5.5 months post-injury. Impact pathologies co-localized with serum albumin extravasation in the brain that was diagnostically detectable in living mice by dynamic contrast-enhanced MRI. These pathologies were also accompanied by early, persistent, and bilateral impairment in axonal conduction velocity in the hippocampus and defective long-term potentiation of synaptic neurotransmission in the medial prefrontal cortex, brain regions distant from acute brain injury. Surprisingly, acute neurobehavioural deficits at the time of injury did not correlate with blood-brain barrier disruption, microgliosis, neuroinflammation, phosphorylated tauopathy, or electrophysiological dysfunction. Furthermore, concussion-like deficits were observed after impact injury, but not after blast exposure under experimental conditions matched for head kinematics. Computational modelling showed that impact injury generated focal point loading on the head and seven-fold greater peak shear stress in the brain compared to blast exposure. Moreover, intracerebral shear stress peaked before onset of gross head motion. By comparison, blast induced distributed force loading on the head and diffuse, lower magnitude shear stress in the brain. We conclude that force loading mechanics at the time of injury shape acute neurobehavioural responses, structural brain damage, and neuropathological sequelae triggered by neurotrauma. These results indicate that closed-head impact injuries, independent of concussive signs, can induce traumatic brain injury as well as early pathologies and functional sequelae associated with chronic traumatic encephalopathy. These results also shed light on the origins of concussion and relationship to traumatic brain injury and its aftermath.awx350media15713427811001.
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http://dx.doi.org/10.1093/brain/awx350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5837414PMC
February 2018

A Role for Myosin Va in Human Cytomegalovirus Nuclear Egress.

J Virol 2018 03 26;92(6). Epub 2018 Feb 26.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA

Herpesviruses replicate and package their genomes into capsids in replication compartments within the nuclear interior. Capsids then move to the inner nuclear membrane for envelopment and release into the cytoplasm in a process called nuclear egress. We previously found that nuclear F-actin is induced upon infection with the betaherpesvirus human cytomegalovirus (HCMV) and is important for nuclear egress and capsid localization away from replication compartment-like inclusions toward the nuclear rim. Despite these and related findings, it has not been shown that any specific motor protein is involved in herpesvirus nuclear egress. In this study, we have investigated whether the host motor protein, myosin Va, could be fulfilling this role. Using immunofluorescence microscopy and coimmunoprecipitation, we observed associations between a nuclear population of myosin Va and the viral major capsid protein, with both concentrating at the periphery of replication compartments. Immunoelectron microscopy showed that nearly 40% of assembled nuclear capsids associate with myosin Va. We also found that myosin Va and major capsid protein colocalize with nuclear F-actin. Importantly, antagonism of myosin Va with RNA interference or a dominant negative mutant revealed that myosin Va is important for the efficient production of infectious virus, capsid accumulation in the cytoplasm, and capsid localization away from replication compartment-like inclusions toward the nuclear rim. Our results lead us to suggest a working model whereby human cytomegalovirus capsids associate with myosin Va for movement from replication compartments to the nuclear periphery during nuclear egress. Little is known regarding how newly assembled and packaged herpesvirus capsids move from the nuclear interior to the periphery during nuclear egress. While it has been proposed that an actomyosin-based mechanism facilitates intranuclear movement of alphaherpesvirus capsids, a functional role for any specific myosin in nuclear egress has not been reported. Furthermore, the notion that an actomyosin-based mechanism facilitates intranuclear capsid movement is controversial. Here we show that human cytomegalovirus capsids associate with nuclear myosin Va and F-actin and that antagonism of myosin Va impairs capsid localization toward the nuclear rim and nuclear egress. Together with our previous results showing that nuclear F-actin is induced upon HCMV infection and is also important for these processes, our results lend support to the hypothesis that nascent human cytomegalovirus capsids migrate to the nuclear periphery via actomyosin-based movement. These results shed light on a poorly understood viral process and the cellular machinery involved.
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http://dx.doi.org/10.1128/JVI.01849-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5827399PMC
March 2018

An integrated methodology for assessing the impact of food matrix and gastrointestinal effects on the biokinetics and cellular toxicity of ingested engineered nanomaterials.

Part Fibre Toxicol 2017 10 13;14(1):40. Epub 2017 Oct 13.

Center for Nanotechnology and Nanotoxicology, HSPH-NIEHS Nanosafety Research Center, Department of Environmental Health, Harvard School of Public Health, Boston, MA, 02115, USA.

Background: Engineered nanomaterials (ENMs) are increasingly added to foods to improve their quality, sensory appeal, safety and shelf-life. Human exposure to these ingested ENMs (iENMS) is inevitable, yet little is known of their hazards. To assess potential hazards, efficient in vitro methodologies are needed to evaluate particle biokinetics and toxicity. These methodologies must account for interactions and transformations of iENMs in foods (food matrix effect) and in the gastrointestinal tract (GIT) that are likely to determine nano-biointeractions. Here we report the development and application of an integrated methodology consisting of three interconnected stages: 1) assessment of iENM-food interactions (food matrix effect) using model foods; 2) assessment of gastrointestinal transformations of the nano-enabled model foods using a three-stage GIT simulator; 3) assessment of iENMs biokinetics and cellular toxicity after exposure to simulated GIT conditions using a triculture cell model. As a case study, a model food (corn oil-in-water emulsion) was infused with FeO (Iron(III) oxide or ferric oxide) ENMs and processed using this three-stage integrated platform to study the impact of food matrix and GIT effects on nanoparticle biokinetics and cytotoxicity .

Methods: A corn oil in phosphate buffer emulsion was prepared using a high speed blender and high pressure homogenizer. Iron oxide ENM was dispersed in water by sonication and combined with the food model. The resulting nano-enabled food was passed through a three stage (mouth, stomach and small intestine) GIT simulator. Size distributions of nano-enabled food model and digestae at each stage were analyzed by DLS and laser diffraction. TEM and confocal imaging were used to assess morphology of digestae at each phase. Dissolution of Fe2O3 ENM along the GIT was assessed by ICP-MS analysis of supernatants and pellets following centrifugation of digestae. An in vitro transwell triculture epithelial model was used to assess biokinetics and toxicity of ingested FeO ENM. Translocation of FeO ENM was determined by ICP-MS analysis of cell lysates and basolateral compartment fluid over time.

Results: It was demonstrated that the interactions of iENMs with food and GIT components influenced nanoparticle fate and transport, biokinetics and toxicological profile. Large differences in particle size, charge, and morphology were observed in the model food with and without FeO and among digestae from different stages of the simulated GIT (mouth, stomach, and small intestine). Immunoflorescence and TEM imaging of the cell culture model revealed markers and morphology of small intestinal epithelium including enterocytes, goblet cells and M cells. FeO was not toxic at concentrations tested in the digesta. In biokinetics studies, translocation of FeO after 4 h was <1% and ~2% for digesta with and without serum, respectively, suggesting that use of serum proteins alters iENMs biokinetics and raises concerns about commonly-used approaches that neglect iENM - food-GIT interactions or dilute digestae in serum-containing media.

Conclusions: We present a simple integrated methodology for studying the biokinetics and toxicology of iENMs, which takes into consideration nanoparticle-food-GIT interactions. The importance of food matrix and GIT effects on biointeractions was demonstrated, as well as the incorporation of these critical factors into a cellular toxicity screening model. Standardized food models still need to be developed and used to assess the effect of the food matrix effects on the fate and bioactivity of iENMs since commercial foods vary considerably in their compositions and structures.
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http://dx.doi.org/10.1186/s12989-017-0221-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640936PMC
October 2017

Loss of native α-synuclein multimerization by strategically mutating its amphipathic helix causes abnormal vesicle interactions in neuronal cells.

Hum Mol Genet 2017 09;26(18):3466-3481

Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.

α-Synuclein (αS) forms round cytoplasmic inclusions in Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Evidence suggests a physiological function of αS in vesicle trafficking and release. In contrast to earlier tenets, recent work indicates that αS normally exists in cells in a dynamic equilibrium between monomers and tetramers/multimers. We engineered αS mutants incapable of multimerization, leading to excess monomers at vesicle membranes. By EM, such mutants induced prominent vesicle clustering, leading to round cytoplasmic inclusions. Immunogold labeling revealed abundant αS intimately associated with vesicles of varied size. Fluorescence microscopy with marker proteins showed that the αS-associated vesicles were of diverse endocytic and secretory origin. An αS '3K' mutant (E35K + E46K + E61K) that amplifies the PD/DLB-causing E46K mutation induced αS-rich vesicle clusters resembling the vesicle-rich areas of Lewy bodies, supporting pathogenic relevance. Mechanistically, E46K can increase αS vesicle binding via membrane-induced amphipathic helix formation, and '3K' further enhances this effect. Another engineered αS variant added hydrophobicity to the hydrophobic half of αS helices, thereby stabilizing αS-membrane interactions. Importantly, substituting charged for uncharged residues within the hydrophobic half of the stabilized helix not only reversed the strong membrane interaction of the multimer-abolishing αS variant but also restored multimerization and prevented the aberrant vesicle interactions. Thus, reversible αS amphipathic helix formation and dynamic multimerization regulate a normal function of αS at vesicles, and abrogating multimers has pathogenic consequences.
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http://dx.doi.org/10.1093/hmg/ddx227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5884392PMC
September 2017

Mesenchymal Stromal Cell Exosomes Ameliorate Experimental Bronchopulmonary Dysplasia and Restore Lung Function through Macrophage Immunomodulation.

Am J Respir Crit Care Med 2018 01;197(1):104-116

1 Division of Newborn Medicine, Department of Medicine, and.

Rationale: Mesenchymal stem/stromal cell (MSC) therapies have shown promise in preclinical models of pathologies relevant to newborn medicine, such as bronchopulmonary dysplasia (BPD). We have reported that the therapeutic capacity of MSCs is comprised in their secretome, and demonstrated that the therapeutic vectors are exosomes produced by MSCs (MSC-exos).

Objectives: To assess efficacy of MSC-exo treatment in a preclinical model of BPD and to investigate mechanisms underlying MSC-exo therapeutic action.

Methods: Exosomes were isolated from media conditioned by human MSC cultures. Newborn mice were exposed to hyperoxia (HYRX; 75% O), treated with exosomes on Postnatal Day (PN) 4 and returned to room air on PN7. Treated animals and appropriate controls were harvested on PN7, -14, or -42 for assessment of pulmonary parameters.

Measurements And Main Results: HYRX-exposed mice presented with pronounced alveolar simplification, fibrosis, and pulmonary vascular remodeling, which was effectively ameliorated by MSC-exo treatment. Pulmonary function tests and assessment of pulmonary hypertension showed functional improvements after MSC-exo treatment. Lung mRNA sequencing demonstrated that MSC-exo treatment induced pleiotropic effects on gene expression associated with HYRX-induced inflammation and immune responses. MSC-exos modulate the macrophage phenotype fulcrum, suppressing the proinflammatory "M1" state and augmenting an antiinflammatory "M2-like" state, both in vitro and in vivo.

Conclusions: MSC-exo treatment blunts HYRX-associated inflammation and alters the hyperoxic lung transcriptome. This results in alleviation of HYRX-induced BPD, improvement of lung function, decrease in fibrosis and pulmonary vascular remodeling, and amelioration of pulmonary hypertension. The MSC-exo mechanism of action is associated with modulation of lung macrophage phenotype.
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http://dx.doi.org/10.1164/rccm.201705-0925OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5765387PMC
January 2018

Rescue of Hearing by Gene Delivery to Inner-Ear Hair Cells Using Exosome-Associated AAV.

Mol Ther 2017 02 9;25(2):379-391. Epub 2017 Jan 9.

Department of Neurology, Massachusetts General Hospital and NeuroDiscovery Center, Harvard Medical School, Building 149, Charlestown, Boston, MA 02129, USA. Electronic address:

Adeno-associated virus (AAV) is a safe and effective vector for gene therapy for retinal disorders. Gene therapy for hearing disorders is not as advanced, in part because gene delivery to sensory hair cells of the inner ear is inefficient. Although AAV transduces the inner hair cells of the mouse cochlea, outer hair cells remain refractory to transduction. Here, we demonstrate that a vector, exosome-associated AAV (exo-AAV), is a potent carrier of transgenes to all inner ear hair cells. Exo-AAV1-GFP is more efficient than conventional AAV1-GFP, both in mouse cochlear explants in vitro and with direct cochlear injection in vivo. Exo-AAV shows no toxicity in vivo, as assayed by tests of auditory and vestibular function. Finally, exo-AAV1 gene therapy partially rescues hearing in a mouse model of hereditary deafness (lipoma HMGIC fusion partner-like 5/tetraspan membrane protein of hair cell stereocilia [Lhfpl5/Tmhs]). Exo-AAV is a powerful gene delivery system for hair cell research and may be useful for gene therapy for deafness.
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http://dx.doi.org/10.1016/j.ymthe.2016.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5368844PMC
February 2017

Myocardial rescue with autologous mitochondrial transplantation in a porcine model of ischemia/reperfusion.

J Thorac Cardiovasc Surg 2017 04 15;153(4):934-943. Epub 2016 Nov 15.

Department of Cardiac Surgery, Boston Children's Hospital, Boston, Mass. Electronic address:

Objective: To demonstrate the clinical efficacy of autologous mitochondrial transplantation in preparation for translation to human application using an in vivo swine model.

Methods: A left mini-thoracotomy was performed on Yorkshire pigs. The pectoralis major was dissected, and skeletal muscle tissue was removed and used for the isolation of autologous mitochondria. The heart was subjected to regional ischemia (RI) by temporarily snaring the circumflex artery. After 24 minutes of RI, hearts received 8 × 0.1 mL injections of vehicle (vehicle-only group; n = 6) or vehicle containing mitochondria (mitochondria group; n = 6) into the area at risk (AAR), and the snare was released. The thoracotomy was closed, and the pigs were allowed to recover for 4 weeks.

Results: Levels of creatine kinase-MB isoenzyme and cardiac troponin I were significantly increased (P = .006) in the vehicle-only group compared with the mitochondria group. Immune, inflammatory, and cytokine activation markers showed no significant difference between groups. There was no significant between-group difference in the AAR (P = .48), but infarct size was significantly greater in the vehicle group (P = .004). Echocardiography showed no significant differences in global function. Histochemistry and transmission electron microscopy revealed damaged heart tissue in the vehicle group that was not apparent in the mitochondria group. T2-weighted magnetic resonance imaging and histology demonstrated that the injected mitochondria were present for 4 weeks.

Conclusions: Autologous mitochondrial transplantation provides a novel technique to significantly enhance myocardial cell viability following ischemia and reperfusion in the clinically relevant swine model.
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http://dx.doi.org/10.1016/j.jtcvs.2016.10.077DOI Listing
April 2017

Domestication and ontogeny effects on the stress response in young chickens (Gallus gallus).

Sci Rep 2016 10 26;6:35818. Epub 2016 Oct 26.

AVIAN Behavioural Physiology and Genomics Group, IFM Biology, Linköping University, Linköping, Sweden.

Domestication is thought to increase stress tolerance. The connection between stressor exposure, glucocorticoids and behavioural responses has been studied in adults, where domestication effects are evident. Early stress exposure may induce detrimental effects both in short-and long term. Previous research has reported a lack of glucocorticoid response in newly hatched chickens (Gallus gallus), whereas others have found opposite results. Hence it remains unclear whether the HPA-axis is functional from hatch, and if domestication has affected the early post-hatch ontogeny of the stress response. Our aims were to investigate the early ontogeny of the HPA-axis and characterize behavioural and hormonal stress responses in ancestral Red Junglefowl and in two domestic layer strains. Plasma corticosteone and behavioural responses before and after physical restraint was measured on day one, nine, 16 and 23 post hatch. The results showed significant increases of corticosterone after stress in all three breeds at all the different ages. The HPA-response decreased with age and was lower in Red Junglefowl. Behavioural responses also decreased with age, and tended to be stronger in Red Junglefowl. In summary, the HPA-axis is reactive from day one, and domestication may have affected its development and reactivity, alongside with related behaviour responses.
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http://dx.doi.org/10.1038/srep35818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5080622PMC
October 2016

Intracoronary Delivery of Mitochondria to the Ischemic Heart for Cardioprotection.

PLoS One 2016 8;11(8):e0160889. Epub 2016 Aug 8.

Department of Cardiac Surgery, Boston Children's Hospital and Harvard Medical School, Boston, MA, United States of America.

We have previously shown that transplantation of autologously derived, respiration-competent mitochondria by direct injection into the heart following transient ischemia and reperfusion enhances cell viability and contractile function. To increase the therapeutic potential of this approach, we investigated whether exogenous mitochondria can be effectively delivered through the coronary vasculature to protect the ischemic myocardium and studied the fate of these transplanted organelles in the heart. Langendorff-perfused rabbit hearts were subjected to 30 minutes of ischemia and then reperfused for 10 minutes. Mitochondria were labeled with 18F-rhodamine 6G and iron oxide nanoparticles. The labeled mitochondria were either directly injected into the ischemic region or delivered by vascular perfusion through the coronary arteries at the onset of reperfusion. These hearts were used for positron emission tomography, microcomputed tomography, and magnetic resonance imaging with subsequent microscopic analyses of tissue sections to confirm the uptake and distribution of exogenous mitochondria. Injected mitochondria were localized near the site of delivery; while, vascular perfusion of mitochondria resulted in rapid and extensive dispersal throughout the heart. Both injected and perfused mitochondria were observed in interstitial spaces and were associated with blood vessels and cardiomyocytes. To determine the efficacy of vascular perfusion of mitochondria, an additional group of rabbit hearts were subjected to 30 minutes of regional ischemia and reperfused for 120 minutes. Immediately following regional ischemia, the hearts received unlabeled, autologous mitochondria delivered through the coronary arteries. Autologous mitochondria perfused through the coronary vasculature significantly decreased infarct size and significantly enhanced post-ischemic myocardial function. In conclusion, the delivery of mitochondria through the coronary arteries resulted in their rapid integration and widespread distribution throughout the heart and provided cardioprotection from ischemia-reperfusion injury.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0160889PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4976938PMC
August 2017