Publications by authors named "Alice Braga"

8 Publications

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Neural stem cells traffic functional mitochondria via extracellular vesicles.

PLoS Biol 2021 Apr 7;19(4):e3001166. Epub 2021 Apr 7.

Department of Clinical Neurosciences and NIHR Biomedical Research Centre, University of Cambridge, United Kingdom.

Neural stem cell (NSC) transplantation induces recovery in animal models of central nervous system (CNS) diseases. Although the replacement of lost endogenous cells was originally proposed as the primary healing mechanism of NSC grafts, it is now clear that transplanted NSCs operate via multiple mechanisms, including the horizontal exchange of therapeutic cargoes to host cells via extracellular vesicles (EVs). EVs are membrane particles trafficking nucleic acids, proteins, metabolites and metabolic enzymes, lipids, and entire organelles. However, the function and the contribution of these cargoes to the broad therapeutic effects of NSCs are yet to be fully understood. Mitochondrial dysfunction is an established feature of several inflammatory and degenerative CNS disorders, most of which are potentially treatable with exogenous stem cell therapeutics. Herein, we investigated the hypothesis that NSCs release and traffic functional mitochondria via EVs to restore mitochondrial function in target cells. Untargeted proteomics revealed a significant enrichment of mitochondrial proteins spontaneously released by NSCs in EVs. Morphological and functional analyses confirmed the presence of ultrastructurally intact mitochondria within EVs with conserved membrane potential and respiration. We found that the transfer of these mitochondria from EVs to mtDNA-deficient L929 Rho0 cells rescued mitochondrial function and increased Rho0 cell survival. Furthermore, the incorporation of mitochondria from EVs into inflammatory mononuclear phagocytes restored normal mitochondrial dynamics and cellular metabolism and reduced the expression of pro-inflammatory markers in target cells. When transplanted in an animal model of multiple sclerosis, exogenous NSCs actively transferred mitochondria to mononuclear phagocytes and induced a significant amelioration of clinical deficits. Our data provide the first evidence that NSCs deliver functional mitochondria to target cells via EVs, paving the way for the development of novel (a)cellular approaches aimed at restoring mitochondrial dysfunction not only in multiple sclerosis, but also in degenerative neurological diseases.
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http://dx.doi.org/10.1371/journal.pbio.3001166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8055036PMC
April 2021

Mechanosensory Signaling in Astrocytes.

J Neurosci 2020 12 29;40(49):9364-9371. Epub 2020 Oct 29.

Centre for Cardiovascular and Metabolic Neuroscience, Department of Neuroscience, Physiology & Pharmacology, University College London, London WC1E 6BT, United Kingdom

Mechanosensitivity is a well-known feature of astrocytes, however, its underlying mechanisms and functional significance remain unclear. There is evidence that astrocytes are acutely sensitive to decreases in cerebral perfusion pressure and may function as intracranial baroreceptors, tuned to monitor brain blood flow. This study investigated the mechanosensory signaling in brainstem astrocytes, as these cells reside alongside the cardiovascular control circuits and mediate increases in blood pressure and heart rate induced by falls in brain perfusion. It was found that mechanical stimulation-evoked Ca responses in astrocytes of the rat brainstem were blocked by (1) antagonists of connexin channels, connexin 43 (Cx43) blocking peptide Gap26, or Cx43 gene knock-down; (2) antagonists of TRPV4 channels; (3) antagonist of P2Y receptors for ATP; and (4) inhibitors of phospholipase C or IP3 receptors. Proximity ligation assay demonstrated interaction between TRPV4 and Cx43 channels in astrocytes. Dye loading experiments showed that mechanical stimulation increased open probability of carboxyfluorescein-permeable membrane channels. These data suggest that mechanosensory Ca responses in astrocytes are mediated by interaction between TRPV4 and Cx43 channels, leading to Cx43-mediated release of ATP which propagates/amplifies Ca signals via P2Y receptors and Ca recruitment from the intracellular stores. In astrocyte-specific Cx43 knock-out mice the magnitude of heart rate responses to acute increases in intracranial pressure was not affected by Cx43 deficiency. However, these animals displayed lower heart rates at different levels of cerebral perfusion, supporting the hypothesis of connexin hemichannel-mediated release of signaling molecules by astrocytes having an excitatory action on the CNS sympathetic control circuits. There is evidence suggesting that astrocytes may function as intracranial baroreceptors that play an important role in the control of systemic and cerebral circulation. To function as intracranial baroreceptors, astrocytes must possess a specialized membrane mechanism that makes them exquisitely sensitive to mechanical stimuli. This study shows that opening of connexin 43 (Cx43) hemichannels leading to the release of ATP is the key central event underlying mechanosensory Ca responses in astrocytes. This astroglial mechanism plays an important role in the autonomic control of heart rate. These data add to the growing body of evidence suggesting that astrocytes function as versatile surveyors of the CNS metabolic milieu, tuned to detect conditions of potential metabolic threat, such as hypoxia, hypercapnia, and reduced perfusion.
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http://dx.doi.org/10.1523/JNEUROSCI.1249-20.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724146PMC
December 2020

Combination of In Situ Lcn2 pRNA-RNAi Nanotherapeutics and iNSC Transplantation Ameliorates Experimental SCI in Mice.

Mol Ther 2020 12 5;28(12):2677-2690. Epub 2020 Aug 5.

Department of Clinical Neurosciences and NIHR Biomedical Research Centre, University of Cambridge, CB2 0HA Cambridge, UK. Electronic address:

Spinal cord injury (SCI) is a debilitating neurological condition characterized by different cellular and molecular mechanisms that interplay in exacerbating the progression of the pathology. No fully restorative therapies are yet available, and it is thus becoming recognized that combinatorial approaches aimed at addressing different aspects of SCI will likely results in greater functional outcomes. Here we employed packaging RNA-mediated RNA interference (pRNA-RNAi) nanotherapeutics to downregulate in situ the expression of lipocalin 2 (Lcn2), a known mediator of neuroinflammation and autocrine mediator of reactive astrogliosis, and to create a more amenable niche for the subsequent transplantation of induced neural stem cells (iNSCs). To our knowledge, this is the first approach that takes advantage of the modular and multifunctional pRNA three-way junction platform in the SCI niche, while also exploiting the therapeutic potential of immune-compatible and feasible iNSC transplants. We show the combination of such treatments in a mouse model of contusion thoracic SCI leads to significant improvement of locomotor function, albeit not better than single pRNA-RNAi treatment, and results in synergistic histopathological effects, such as reduction of glial scar volume, diminished pro-inflammatory response, and promotion of neuronal survival. Our results provide evidence for a novel combinatorial approach for treating SCI.
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http://dx.doi.org/10.1016/j.ymthe.2020.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7704756PMC
December 2020

RNA Nanotherapeutics for the Amelioration of Astroglial Reactivity.

Mol Ther Nucleic Acids 2018 Mar 24;10:103-121. Epub 2017 Nov 24.

Department of Clinical Neurosciences, Division of Stem Cell Neurobiology, Wellcome Trust-Medical Research Council Stem Cell Institute and NIHR Biomedical Research Centre, University of Cambridge, Cambridge, UK. Electronic address:

In response to injuries to the CNS, astrocytes enter a reactive state known as astrogliosis, which is believed to be deleterious in some contexts. Activated astrocytes overexpress intermediate filaments including glial fibrillary acidic protein (GFAP) and vimentin (Vim), resulting in entangled cells that inhibit neurite growth and functional recovery. Reactive astrocytes also secrete inflammatory molecules such as Lipocalin 2 (Lcn2), which perpetuate reactivity and adversely affect other cells of the CNS. Herein, we report proof-of-concept use of the packaging RNA (pRNA)-derived three-way junction (3WJ) motif as a platform for the delivery of siRNAs to downregulate such reactivity-associated genes. In vitro, siRNA-3WJs induced a significant knockdown of Gfap, Vim, and Lcn2 in a model of astroglial activation, with a concomitant reduction in protein expression. Knockdown of Lcn2 also led to reduced protein secretion from reactive astroglial cells, significantly impeding the perpetuation of inflammation in otherwise quiescent astrocytes. Intralesional injection of anti-Lcn2-3WJs in mice with contusion spinal cord injury led to knockdown of Lcn2 at mRNA and protein levels in vivo. Our results provide evidence for siRNA-3WJs as a promising platform for ameliorating astroglial reactivity, with significant potential for further functionalization and adaptation for therapeutic applications in the CNS.
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http://dx.doi.org/10.1016/j.omtn.2017.11.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5738063PMC
March 2018

Macrophage-Derived Extracellular Succinate Licenses Neural Stem Cells to Suppress Chronic Neuroinflammation.

Cell Stem Cell 2018 03 22;22(3):355-368.e13. Epub 2018 Feb 22.

Department of Clinical Neurosciences and NIHR Biomedical Research Centre, University of Cambridge, Cambridge, UK. Electronic address:

Neural stem cell (NSC) transplantation can influence immune responses and suppress inflammation in the CNS. Metabolites, such as succinate, modulate the phenotype and function of immune cells, but whether and how NSCs are also activated by such immunometabolites to control immunoreactivity and inflammatory responses is unclear. Here, we show that transplanted somatic and directly induced NSCs ameliorate chronic CNS inflammation by reducing succinate levels in the cerebrospinal fluid, thereby decreasing mononuclear phagocyte (MP) infiltration and secondary CNS damage. Inflammatory MPs release succinate, which activates succinate receptor 1 (SUCNR1)/GPR91 on NSCs, leading them to secrete prostaglandin E2 and scavenge extracellular succinate with consequential anti-inflammatory effects. Thus, our work reveals an unexpected role for the succinate-SUCNR1 axis in somatic and directly induced NSCs, which controls the response of stem cells to inflammatory metabolic signals released by type 1 MPs in the chronically inflamed brain.
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http://dx.doi.org/10.1016/j.stem.2018.01.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5842147PMC
March 2018

High Yield of Adult Oligodendrocyte Lineage Cells Obtained from Meningeal Biopsy.

Front Pharmacol 2017 12;8:703. Epub 2017 Oct 12.

Section of Pharmacology, Department of Diagnostics and Public Health, University of Verona, Verona, Italy.

Oligodendrocyte loss can lead to cognitive and motor deficits. Current remyelinating therapeutic strategies imply either modulation of endogenous oligodendrocyte precursors or transplantation of expanded oligodendrocytes. Cell therapy, however, still lacks identification of an adequate source of oligodendrocyte present in adulthood and able to efficiently produce transplantable cells. Recently, a neural stem cell-like population has been identified in meninges. We developed a protocol to obtain high yield of oligodendrocyte lineage cells from one single biopsy of adult rat meningeal tissue. From 1 cm of adult rat spinal cord meninges, we efficiently expanded a homogenous culture of 10 millions of meningeal-derived oligodendrocyte lineage cells in a short period of time (approximately 4 weeks). Meningeal-derived oligodendrocyte lineage cells show typical mature oligodendrocyte morphology and express specific oligodendrocyte markers, such as galactosylceramidase and myelin basic protein. Moreover, when transplanted in a chemically demyelinated spinal cord model, meningeal-derived oligodendrocyte lineage cells display -remyelinating potential. This oligodendrocyte lineage cell population derives from an accessible and adult source, being therefore a promising candidate for autologous cell therapy of demyelinating diseases. In addition, the described method to differentiate meningeal-derived neural stem cells into oligodendrocyte lineage cells may represent a valid model to dissect oligodendrocyte differentiation and to screen for drugs capable to promote oligodendrocyte regeneration.
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http://dx.doi.org/10.3389/fphar.2017.00703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5643910PMC
October 2017

Comparison of static postural balance between healthy subjects and those with low back pain.

Acta Ortop Bras 2012 ;20(4):210-2

Universidade Estadual do Oeste do Paraná (Unioeste) - Cascavel, PR, Brazil.

Objective: To compare the static postural balance between women suffering from chronic low back pain and healthy subjects, by moving the center of pressure.

Methods: The study included 15 women with low back pain (LBP group) and 15 healthy women (healthy group). They were instructed to remain in standing on the force platform for 30 seconds. We analyzed the area and the speed of displacement of center of pressure of both groups. Data analysis was performed using the Student's t-test, with significance of 5%.

Results: Individuals with chronic low back pain showed a larger area of displacement of the center of pressure relative to the healthy ones but there was no significant difference in the speed of displacement of the center of pressure.

Conclusion: Individuals with chronic low back pain had alterations in static balance with respect to healthy ones. Level of Evidence III, Prognostic Studies.
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http://dx.doi.org/10.1590/S1413-78522012000400003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3718398PMC
January 2014

Preventing venous thrombosis in critically ill children: what is the right approach?

Paediatr Anaesth 2011 Apr 7;21(4):435-40. Epub 2011 Feb 7.

Department of Anaesthesia, South West Paediatric Burns and Neuroscience Services, Frenchay Hospital, North Bristol NHS Trust, Bristol, UK.

Background: The incidence of venous thromboembolic (VTE) events in children has increased in recent years (J Neurosurg, 101, 2004, 32; J Thromb Haemost, 1, 2003, 1443) yet there is currently no consensus as to what VTE prophylaxis, if any, should be applied to the pediatric population.

Objectives/aims: Our aim was to audit current practice in pediatric VTE prophylaxis across England and Wales and to advocate simple measures for prevention. We illustrate the importance of the condition with a series of cases from the South West Paediatric Burns and Neurosurgical Services based in Bristol.

Methods: Every pediatric intensive care unit (PICU) and burns center admitting children in England and Wales was invited to participate in a structured telephone questionnaire designed to find out how VTE in children were being prevented. We performed a literature review of specific risk factors and management of these factors.

Results: Only one of the 24 units surveyed had written guidelines specific for children. Four other units used modified adult guidelines in older children. In the remaining 19 units that had no written guidelines, decisions regarding prophylaxis were based on individual cases and consultant-led.

Conclusion: There is no consensus in England and Wales as to which VTE prophylactic measures should be applied in patients <18 years of age. The National Institute for Health and Clinical Excellence (NICE) guidelines apply to adults only. Given the rarity of VTE events in children, it is unlikely that randomized controlled trials will provide the answer. We therefore propose that simple empirical measures be formally implemented in critically ill children to reduce the risk of developing this important but under-recognized condition.
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http://dx.doi.org/10.1111/j.1460-9592.2011.03520.xDOI Listing
April 2011
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